Blood Mixing Upregulates Platelet Membrane-Bound CD40 Ligand Expression in vitro Independent of Abo Compatibility.

PubMed

Huang, Go-Shine; Hu, Mei-Hua; Lin, Tso-Chou; Lin, Yi-Chang; Tsai, Yi-Ting; Lin, Chih-Yuan; Ke, Hung-Yen; Zheng, Xu-Zhi; Tsai, Chien-Sung

2017-11-30

Platelets play a central role in the inflammation response via CD40 ligand (CD40L) expression, which may lead to transfusion reactions. The precise role of platelet CD40L-mediated inflammation in transfusion reactions is unclear. Therefore, we assessed the effects of in vitro blood mixing on platelet CD40L expression. In addition, we examined the effect of ABO compatibility on CD40L expression. Donor packed red blood cells were acquired from a blood bank, and recipient blood was obtained from patients undergoing cardiac surgery and prepared as washed platelets. Donor blood was mixed with suspended, washed recipient platelets to obtain a final mixing ratio of 1%, 5%, or 10% (vol/vol). The blood mixtures were divided into three groups: Group M, cross-matched blood-type mixing (n = 20); Group S, ABO type-specific uncross-matched blood (n = 20); and Group I, ABO incompatibility (not ABO type-specific blood and not process cross-matched) mixing (n = 20). The blood mixtures were used to detect platelet membrane-bound CD40L expression by flow cytometry. Blood mixing resulted in an increase in CD40L expression in Group M (P < 0.001), Group S (P < 0.001), and Group I (P < 0.001). CD40L expression following blood mixing potentially led to a transfusion reaction in each of the groups. There were no differences in CD40L expression among the three groups (P = 0.988) correlated with ABO compatibility or incompatibility. This indicates that the reactions between red blood cell surface antigens and plasma antibodies do not play a role in the induction of CD40L expression.

Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells.

PubMed Central

Tsujimura, A; Shida, K; Kitamura, M; Nomura, M; Takeda, J; Tanaka, H; Matsumoto, M; Matsumiya, K; Okuyama, A; Nishimune, Y; Okabe, M; Seya, T

1998-01-01

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation. PMID:9461505

Immune regulation by CD40-CD40-l interactions - 2; Y2K update.

PubMed

van Kooten, C

2000-11-01

CD40 is a cell surface receptor, which belongs to the TNF-R family, and which was first identified and functionally characterized on B lymphocytes. However, in recent years it has become clear that CD40 is expressed much broader, including expression on monocytes, dendritic cells, endothelial cells and epithelial cells. Therefore it is now thought that CD40 plays a more general role in immune regulation. The present paper reviews recent developments in this field of research, with main emphasis on CD40 signal transduction and on in vivo functions of CD40/CD40-L interactions.

Impact of CD40 expression by flowcytometry on outcome of patients with non-Hodgkin's lymphoma.

PubMed

Soliman, Mohamed A; Fathy, Amr Ahmed; Alkilani, Amira; Abd El-Bary, Naser; El-Bassal, Fathai

2009-01-01

Lymphoid malignancies represent a wide variety of disease entities characterized by malignant proliferation of lymphoid cells which have distinct clinical features, cellular morphology, immunophenotype, cytogenetic changes and histologic features. CD40 is a member of the tumor necrosis factor receptor super-family. It was first identified and characterized in B cell, signaling through the CD40 receptor was found to play an important role in multiple events in T-cell dependent antibody response including B-cell survival and proliferation, memory B-cell formation and immunoglobulin isotype switching. The aim of this study is to detect the expression of CD40 on B lymphocytes in patients suffering from Non-Hodgkin's Lymphoma and correlate the results with the patients' response to treatment protocols. This study was carried out on 114 patients, of them only 100 patients completed 4 cycles of chemotherapy and were valuable. Their age was ranged from 17 to 63 years old. Fifteen age and gender matched individuals were, also, selected as a control group. CD40 expression was measured on peripheral blood samples by flowcytometry at patient's presentation as well as after 4 cycles of chemotherapy. This study showed that there's significant decrease in the mean values of % of CD40 on B-cell in patients with NHL in all stages when compared with normal control group. Also the study showed that there's statistical significant correlation between percent of CD40 on B-lymphocytes and stage of lymphoma, i.e., the more advanced stage, the lower the % of CD40 on B-cell. After receiving a corresponding treatment, the CD40 expression is increased in significant correlation with the response to treatment. (This is a preliminary result after 4 cycles of CHOP treatment). We concluded that CD40 Lymphocyte development occurs in discrete functional steps that are defined by the onset of expression is highly expressed in healthy subjects and its expression on B-lymphocyte is decreased with

YKL-40 expression in CD14+ liver cells in acute and chronic injury

PubMed Central

Pizano-Martínez, Oscar; Yañez-Sánchez, Irinea; Alatorre-Carranza, Pilar; Miranda-Díaz, Alejandra; Ortiz-Lazareno, Pablo C; García-Iglesias, Trinidad; Daneri-Navarro, Adrian; Mercado, Mónica Vázquez-Del; Fafutis-Morris, Mary; Delgado-Rizo, Vidal

2011-01-01

AIM: To demonstrate that CD14+ cells are an important source of the growth factor YKL-40 in acute and chronic liver damage. METHODS: Rats were inoculated with one dose of CCl4 to induce acute damage. Liver biopsies were obtained at 0, 6, 12, 24, 48 and 72 h. For chronic damage, CCl4 was administered three days per week for 6 or 8 wk. Tissue samples were collected, and cellular populations were isolated by liver digestion and purified by cell sorting. YKL-40 mRNA and protein expression were evaluated by real-time polymerase chain reaction and western blot. RESULTS: Acute liver damage induced a rapid increase of YKL-40 mRNA beginning at 12 h. Expression peaked at 24 h, with a 26-fold increase over basal levels. By 72 h however, YKL-40 expression levels had nearly returned to control levels. On the other hand, chronic damage induced a sustained increase in YKL-40 expression, with 7- and 9-fold higher levels at 6 and 8 wk, respectively. The pattern of YKL-40 expression in different subpopulations showed that CD14+ cells, which include Kupffer cells, are a source of YKL-40 after acute damage at 72 h [0.09 relative expression units (REU)] as well as after chronic injury at 6 wk (0.11 REU). Hepatocytes, in turn, accounted for 0.06 and 0.01 REU after 72 h (acute) or 6 wk (chronic), respectively. The rest of the CD14- cells (including T lymphocytes, B lymphocytes, natural killer and natural killer T cells) yielded 0.07 and 0.15 REU at 72 h and 6 wk, respectively. YKL-40 protein expression in liver was detected at 72 h as well as 6 and 8 wk, with the highest expression relative to controls (11-fold; P ≤ 0.05) seen at 6 wk. Macrophages were stimulated by lipopolysaccharide. We demonstrate that under these conditions, these cells showed maximum expression of YKL-40 at 12 h, with P < 0.05 compared with controls. CONCLUSION: Hepatic CD14+ cells are an YKL-40 mRNA and protein source in acute and chronic liver injury, with expression patterns similar to growth factors implicated

Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis.

PubMed

Teruel, María; Simeon, Carmen P; Broen, Jasper; Vonk, Madelon C; Carreira, Patricia; Camps, Maria Teresa; García-Portales, Rosa; Delgado-Frías, Esmeralda; Gallego, Maria; Espinosa, Gerard; Beretta, Lorenzo; Airó, Paolo; Lunardi, Claudio; Riemekasten, Gabriela; Witte, Torsten; Krieg, Thomas; Kreuter, Alexander; Distler, Jörg H W; Hunzelmann, Nicolas; Koeleman, Bobby P; Voskuyl, Alexandre E; Schuerwegh, Annemie J; González-Gay, Miguel Angel; Radstake, Timothy R D J; Martin, Javier

2012-06-25

The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc). In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes. No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis. Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.

Atomic structure of the murine norovirus protruding domain and sCD300lf receptor complex.

PubMed

Kilic, Turgay; Koromyslova, Anna; Malak, Virginie; Hansman, Grant S

2018-03-21

, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case host-range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism. Copyright © 2018 American Society for Microbiology.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

PubMed

Goel, C; Kalra, N; Dwarakanath, B S; Gaur, S N; Arora, N

2015-05-01

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses. © 2014 British Society for Immunology.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

PubMed Central

Goel, C; Kalra, N; Dwarakanath, B S; Gaur, S N; Arora, N

2015-01-01

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4+ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses. PMID:25492061

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

PubMed

Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

2017-10-27

CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

CD40 Ligand Promotes Mac-1 Expression, Leukocyte Recruitment, and Neointima Formation after Vascular Injury

PubMed Central

Li, Guohong; Sanders, John M.; Bevard, Melissa H.; Sun, ZhiQi; Chumley, James W.; Galkina, Elena V.; Ley, Klaus; Sarembock, Ian J.

2008-01-01

High levels of circulating soluble CD40 ligand (sCD40L) are frequently found in patients with hypercholesterolemia, diabetes, ischemic stroke, or acute coronary syndromes, predicting an increased rate of atherosclerotic plaque rupture and restenosis after coronary/carotid interventions. Clinical restenosis is characterized in part by exaggerated neointima formation, but the underlying mechanism remains incompletely understood. This study investigated the role of elevated sCD40L in neointima formation in response to vascular injury in an atherogenic animal model and explored the molecular mechanisms involved. apoE−/− mice fed a Western diet developed severe hypercholesterolemia, significant hyperglycemia, and high levels of plasma sCD40L. Neointima formation after carotid denudation injury was exaggerated in the apoE−/− mice. In vivo, blocking CD40L with anti-CD40L monoclonal antibody attenuated the early accumulation of Ly-6G+ neutrophils and Gr-1+ monocytes (at 3 days) and the late accumulation of Mac-2+ macrophages (at 28 days) in the denudated arteries; it also reduced the exaggerated neointima formation at 28 days. In vitro, recombinant CD40L stimulated platelet P-selectin and neutrophil Mac-1 expression and platelet-neutrophil co-aggregation and adhesive interaction. These effects were abrogated by anti-CD40L or anti-Mac-1 monoclonal antibody. Moreover, recombinant CD40L stimulated neutrophil oxidative burst and release of matrix metalloproteinase-9 in vitro. We conclude that elevated sCD40L promotes platelet-leukocyte activation and recruitment and neointima formation after arterial injury, potentially through enhancement of platelet P-selectin and leukocyte Mac-1 expression and oxidative activity. PMID:18349125

Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ(9)-tetrahydrocannabinol in human CD4(+) T cells.

PubMed

Ngaotepprutaram, Thitirat; Kaplan, Barbara L F; Kaminski, Norbert E

2013-11-15

We have previously reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4(+) T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ(9)-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ(9)-THC attenuated CD40L expression in human CD4(+) T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ(9)-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ(9)-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ(9)-THC suppresses human T cell function. © 2013.

Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.

PubMed

Kochenderfer, James N; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A

2010-11-11

Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model.

Genetic Adjuvantation of Recombinant MVA with CD40L Potentiates CD8 T Cell Mediated Immunity

PubMed Central

Lauterbach, Henning; Pätzold, Juliane; Kassub, Ronny; Bathke, Barbara; Brinkmann, Kay; Chaplin, Paul; Suter, Mark; Hochrein, Hubertus

2013-01-01

Modified vaccinia Ankara (MVA) is a safe and promising viral vaccine vector that is currently investigated in several clinical and pre-clinical trials. In contrast to inactivated or sub-unit vaccines, MVA is able to induce strong humoral as well as cellular immune responses. In order to further improve its CD8 T cell inducing capacity, we genetically adjuvanted MVA with the coding sequence of murine CD40L, a member of the tumor necrosis factor superfamily. Immunization of mice with this new vector led to strongly enhanced primary and memory CD8 T cell responses. Concordant with the enhanced CD8 T cell response, we could detect stronger activation of dendritic cells and higher systemic levels of innate cytokines (including IL-12p70) early after immunization. Interestingly, acquisition of memory characteristics (i.e., IL-7R expression) was accelerated after immunization with MVA-CD40L in comparison to non-adjuvanted MVA. Furthermore, the generated cytotoxic T-lymphocytes (CTLs) also showed improved functionality as demonstrated by intracellular cytokine staining and in vivo killing activity. Importantly, the superior CTL response after a single MVA-CD40L immunization was able to protect B cell deficient mice against a fatal infection with ectromelia virus. Taken together, we show that genetic adjuvantation of MVA can change strength, quality, and functionality of innate and adaptive immune responses. These data should facilitate a rational vaccine design with a focus on rapid induction of large numbers of CD8 T cells able to protect against specific diseases. PMID:23986761

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

PubMed

Lecoanet-Henchoz, S; Plater-Zyberk, C; Graber, P; Gretener, D; Aubry, J P; Conrad, D H; Bonnefoy, J Y

1997-09-01

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Soluble CD40-ligand (sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection

PubMed Central

Jenabian, M-A; Patel, M; Kema, I; Vyboh, K; Kanagaratham, C; Radzioch, D; Thébault, P; Lapointe, R; Gilmore, N; Ancuta, P; Tremblay, C; Routy, J-P

2014-01-01

CD40/CD40-ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO-induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV-infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti-retroviral therapy (ART)-naive, ART-successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme-linked immunosorbent assay (ELISA). IDO-mRNA expression was quantified by reverse transcription–polymerase chain reaction (RT–PCR). The in-vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART-naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART-naive alone, sCD40L was correlated with T cell activation, IDO-mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV-infected patients. In-vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co-expression of CD38/human leucocyte antigen D-related (HLA-DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO-induced Trp catabolism and Treg expansion. PMID:24924152

Soluble CD40-ligand (sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection.

PubMed

Jenabian, M-A; Patel, M; Kema, I; Vyboh, K; Kanagaratham, C; Radzioch, D; Thébault, P; Lapointe, R; Gilmore, N; Ancuta, P; Tremblay, C; Routy, J-P

2014-10-01

CD40/CD40-ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO-induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg ) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV-infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti-retroviral therapy (ART)-naive, ART-successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme-linked immunosorbent assay (ELISA). IDO-mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The in-vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART-naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART-naive alone, sCD40L was correlated with T cell activation, IDO-mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV-infected patients. In-vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co-expression of CD38/human leucocyte antigen D-related (HLA-DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO-induced Trp catabolism and Treg expansion. © 2014 British Society for Immunology.

Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar

2010-01-08

CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thusmore » releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.« less

[Evaluation of percentage of lymphocytes B with expression of co-receptors CD 40, CD22 and CD72 in hypertrophied adenoid at children with otitis media with effusion].

PubMed

Wysocka, Jolanta; Zelazowska-Rutkowska, Beata; Ratomski, Karol; Skotnicka, Bozena; Hassmann-Poznańska, Elzbieta

2009-01-01

In hypertrophied adenoid lymphocytes B make up about 60% all lymphocytes. When the lymphocytes B come in interaction with antigens this membranes signal be passed through their receptor (BCR) to interior of cell. This signal affect modulation on gene expression, activation from which depends activation, anergy or apoptosis of lymphocyte B. Accompany BCR co-receptors regulate his functions influence stimulate or inhibitive. To the most important co-receptors stepping out on lymphocyte B belong: CD40, CD22, CD72. The aim of study was evaluation of lymphocytes B (CD19) with co-expression with CD72 and CD40 receptors in hypertrophied adenoid with at children with otitis media with effusion. An investigation was executed in hypertrophied adenoids with or without otitis media with effusion. By flow cytometry percentage of lymphocytes B with co-receptors CD 40, CD22 and CD72 in was analyzed. The percentages of CD19+CD72+ lymphocytes in the group of children with adenoid hypertrophy and exudative otitis media were lower as compared to the reference group. However, the percentages of CD19+CD22+, CD19+CD40+ in the study group was approximate to the reference group. The lower percentage of lymphocytes B CD72 + near approximate percentages of lymphocytes B CD40+ and BCD22+ at children with otitis media with effusion can be the cause of incorrect humoral response in hypertrophied adenoid at children. Maybe it is cause reduced spontaneous production IgA and IgG through lymphocyte at children with otitis media with effusion.

CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent.

PubMed

D'Souza, Lucas; Gupta, Sneh Lata; Bal, Vineeta; Rath, Satyajit; George, Anna

2017-12-01

B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM + cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73 + IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory. © 2017 John Wiley & Sons Ltd.

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

PubMed

Rodewald, H R; Awad, K; Moingeon, P; D'Adamio, L; Rabinowitz, D; Shinkai, Y; Alt, F W; Reinherz, E L

1993-04-01

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta

INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

EPA Science Inventory

CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

Organization of the murine Cd22 locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Law, Che-Leung; Torres, R.M.; Sundeberg, H.A.

1993-07-01

Murine CD22 (mCD22) is a B cell-associated adhesion protein with seven extracellular Ig-like domains that has 62% amino acid identify to its human homologue. Southern analysis on genomic DNA isolated from tissues and cell lines from several mouse strains using mCD22 cDNA demonstrated that the Cd22 locus encoding mCD22 is a single copy gene of [le]30 kb. Digestion of genomic DNA preparations with four restriction endonucleases revealed the presence of restriction fragment length polymorphisms (RFLP) in BALB/c, C57BL/6, and C3H strains vs DBA/2j, NZB, and NZC strains, suggesting the presence of two or more Cd22 alleles. Using a mCD22 cDNAmore » clone derived from the BALB/c strain, the authors isolated genomic clones from a DBA/2 genomic library that contained all the exons necessary to encode the full length mCD22 cDNA. Fifteen exons, including exon 3 that encodes the translation start codon, were identified. Each extracellular Ig-like domain of mCD22 is encoded by a single exon. A comparison between the nucleotide sequences of the BALB/c CD22 cDNA and the exons of the DBA/2j CD22 genomic clones revealed an 18-nucleotide deletion in exon 4 (encoding the most distal Ig-like domain 1 of mCD22) of the DBA/2j genomic sequence in addition to a number of substitutions, insertions, and deletions in other exons. These nucleotide differences were also present in a cDNA clone isolated from total RNA of LPS-activated DBA/2j splenocytes mosome 7, a region sytenic to human chromosome 19q, close to the previously reported loci, Lyb-8 and Mag (a homologue of Cd22). An antibody (CY34) against the Lyb-8.2 B cell marker reacted with a BHK transfectant expressing the full length mCd22 cDNA, thus demonstrating that Lyb-8 and Cd22 loci are identical. Furthermore, a rat anti-mCD22 mAb, NIM-R6, bound to slgM[sup +] DBA/2j B cells, confirming the expression of a CD22 protein by the Cd22[sup a]/lyb-8[sup a] allele. 63 refs., 7 figs., 1 tab.« less

Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ngaotepprutaram, Thitirat; Center for Integrative Toxicology, Michigan State University; Kaplan, Barbara L.F.

We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{supmore » 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.« less

CD40 engagement on dendritic cells induces cyclooxygenase-2 and EP2 receptor via p38 and ERK MAPKs.

PubMed

Harizi, Hedi; Limem, Ilef; Gualde, Norbert

2011-02-01

We have previously reported that cyclooxygenase (COX)-2-derived prostaglandin (PG)E2 critically regulates dendritic cell (DC) inflammatory phenotype and function through EP2/EP4 receptor subtypes. As genes activated by CD40 engagement are directly relevant to inflammation, we examined the effects of CD40 activation on inflammatory PGs in murine bone marrow-derived DC (mBM-DC). We showed for the first time that activation of mBM-DC with agonist anti-CD40 monoclonal antibody (anti-CD40 mAb) dose dependently induces the synthesis of significant amounts of PGE2 via inducible expression of COX-2 enzyme, as NS-398, a COX-2-selective inhibitor reduces this upregulation. In contrast to lipopolysaccharide, which upregulates mBM-DC surface levels of EP2 and EP4 receptors, CD40 crosslinking on mBM-DC increases EP2, but not EP4, receptor expression. Flow cytometry analysis and radioligand-binding assay showed that EP2 was the major EP receptor subtype, which binds to PGE2 at the surface of anti-CD40-activated mBM-DC. Upregulation of COX-2 and EP2 levels by CD40 engagement was accompanied by dose-dependent phosphorylation of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) and was abrogated by inhibitors of both pathways. Collectively, we demonstrated that CD40 engagement on mBM-DC upregulates COX-2 and EP2 receptor expression through activation of p38 and ERK1/2 MAPK signaling. Triggering the PGE2/EP2 pathway by anti-CD40 mAb resulted on the induction of Th2 immune response. Thus, CD40-induced production of PGE2 by mBM-DC could represent a negative feedback mechanism involving EP2 receptor and limiting the propagation of Th1 responses. Blocking CD40 pathway may represent a novel therapeutic pathway of inhibiting COX-2-derived prostanoids in chronically inflamed tissues (that is, arthritis).

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

PubMed Central

Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

2013-01-01

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

Adoptive cell therapy for lymphoma with CD4 T cells depleted of CD137-expressing regulatory T cells.

PubMed

Goldstein, Matthew J; Kohrt, Holbrook E; Houot, Roch; Varghese, Bindu; Lin, Jack T; Swanson, Erica; Levy, Ronald

2012-03-01

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression.

PubMed

Honey, K; Cobbold, S P; Waldmann, H

1999-11-01

The CD40-CD40 ligand (CD40L) interaction is a key event in the initiation of an adaptive immune response, and as such the therapeutic value of CD40L blockade has been studied in many experimental models of tissue transplantation and autoimmune disease. In rodents, transplantation of allogeneic tissues under the cover of anti-CD40L Abs has resulted in prolonged graft survival but not tolerance. In this report, we show that failure to induce tolerance probably results from the inability of anti-CD40L Abs to prevent graft rejection elicited by the CD8+ T cell subset. When the CD8+ T cell population is controlled independently, using anti-CD8 Abs, then tolerance is possible. Transplantation tolerance induced by anti-CD4 mAbs can often be associated with dominant regulation, manifested as infectious tolerance and linked suppression, both of which are mediated by CD4+ T cells. We show here that CD4+ T cells rendered tolerant using anti-CD40L therapy exhibit the same regulatory property of linked suppression, as demonstrated by their ability to accept grafts expressing third party Ags only if they are expressed in conjunction with the tolerated Ags. This observation of linked suppression reveals a hitherto undocumented consequence of CD40L blockade that suggests the tolerant state is maintained by a dominant regulatory mechanism. Our results suggest that, although anti-CD40L Abs are attractive clinical immunotherapeutic agents, additional therapies to control aggressive CD8+ T cell responses may be required.

Response to programmed cell death-1 blockade in a murine melanoma syngeneic model requires costimulation, CD4, and CD8 T cells

PubMed Central

Moreno, Blanca Homet; Zaretsky, Jesse M.; Garcia-Diaz, Angel; Tsoi, Jennifer; Parisi, Giulia; Robert, Lidia; Meeth, Katrina; Ndoye, Abibatou; Bosenberg, Marcus; Weeraratna, Ashani T.; Graeber, Thomas G.; Comin-Anduix, Begoña; Hu-Lieskovan, Siwen; Ribas, Antoni

2016-01-01

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN-/-). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor associated macrophages in the tumors after PD-1 blockade. Compared to YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression from chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. PMID:27589875

Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68

PubMed Central

Hu, Zhuting; Blackman, Marcia A.; Kaye, Kenneth M.; Usherwood, Edward J.

2015-01-01

CD4+ T cells are critical for the control of virus infections, T cell memory and immune surveillance. Here we studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4+ T cells using gp150-specific TCR transgenic mice. This allowed a more detailed study of the characteristics of the CD4+ T cell response than previously available approaches for this virus. Most gp150-specific CD4+ T cells expressed T-bet and produced IFN-γ, indicating MHV-68 infection triggered differentiation of CD4+ T cells largely into the Th1 subset, whereas some became TFH and Foxp3+ regulatory T cells. These CD4+ T cells were protective against MHV-68 infection, in the absence of CD8+ T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4+ T cells, based on Ly6C expression. Ly6C expression positively correlated with IFN-γ, TNF-α and granzyme B production, T-bet and KLRG1 expression, proliferation and CD4+ T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression and secondary expansion potential. Ly6C+ and Ly6C− gp150-specific CD4+ T cells were able to interconvert in a bidirectional manner upon secondary antigen exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4+ T cells, but inversely correlated with memory potential. Interconversion between Ly6C+ and Ly6C− cells may maintain a balance between the two antigen-specific CD4+ T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4+ T cells during persistent virus infection. PMID:25662997

Bromelain treatment reduces CD25 expression on activated CD4+ T cells in vitro✩

PubMed Central

Secor, Eric R.; Singh, Anurag; Guernsey, Linda A.; McNamara, Jeff T.; Zhan, Lijun; Maulik, Nilanjana; Thrall, Roger S.

2009-01-01

Bromelain (Br), an extract from pineapple stem with cysteine protease activity, exerts anti-inflammatory effects in a number of inflammatory models. We have previously shown that Br treatment decreased activated CD4+ T cells and has a therapeutic role in an ovalbumin-induced murine model of allergic airway disease. The current study was designed to determine the effect of Br on CD4+ T cell activation, specifically the expression of CD25 in vitro. CD25 is up regulated upon T cell activation, found as a soluble fraction (sCD25) and is a therapeutic target in inflammation, autoimmunity and allergy. Br treatment of anti-CD3 stimulated CD4+ T cells reduced CD25 expression in a dose and time dependent manner. This reduction of CD25 was dependent on the proteolytic action of Br as the addition of E64 (a cysteine protease inhibitor) abrogated this response. The concentration of sCD25 was increased in supernatants of Br treated activated CD4+ T cells as compared to control cells, suggesting that Br proteolytically cleaved cell-surface CD25. This novel mechanism of action identifies how Br may exert its therapeutic benefits in inflammatory conditions. PMID:19162239

Regulatory T Cells and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment Undergo Fas-Dependent Cell Death during IL-2/αCD40 Therapy

PubMed Central

Weiss, Jonathan M.; Subleski, Jeff J.; Back, Tim; Chen, Xin; Watkins, Stephanie K.; Yagita, Hideo; Sayers, Thomas J.; Murphy, William J.

2014-01-01

Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (αCD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/αCD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8+ T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/αCD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/αCD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer. PMID:24808361

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

PubMed Central

Salwe, Sukeshani; Kothari, Sweta; Chowdhary, Abhay; Deshmukh, Ranjana A.

2018-01-01

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population. PMID:29682352

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method.

PubMed

Gosavi, Rahul Ashok; Salwe, Sukeshani; Mukherjee, Sandeepan; Dahake, Ritwik; Kothari, Sweta; Patel, Vainav; Chowdhary, Abhay; Deshmukh, Ranjana A

2018-01-01

12-14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference ( P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

A novel monoclonal antibody, C41, reveals IL-13Ralpha1 expression by murine germinal center B cells and follicular dendritic cells.

PubMed

Poudrier, J; Graber, P; Herren, S; Berney, C; Gretener, D; Kosco-Vilbois, M H; Gauchat, J F

2000-11-01

Responsiveness to IL-13 involves at least two chains, IL-4Ralpha and IL-13Ralpha1. Although mouse B cells express IL-4Ralpha, little is known about their expression of IL-13Ralpha chains. To investigate this topic further, we have generated a monoclonal antibody (C41) specific for murine IL-13Ralpha1. Using C41, IL-13Ralpha1 expression was detected on germinal center (GC) B cells by flow cytometry and immunohistochemistry. In addition, IL-13Ralpha1 was observed on follicular dendritic cells, but not interdigitating dendritic cells in the T cell areas. Furthermore, resting B cells also expressed IL-13Ralpha1, and in the presence of IL-13 produced increased amounts of IgM in response to in vitro CD40 stimulation. However, C41 was unable to neutralize this bioactivity. The distribution of IL-13Ralpha1 on murine B cells and during GC reactions suggests a role for IL-13 during B cell differentiation.

The role of CD40 and CD40L in bone mineral density and in osteoporosis risk: A genetic and functional study.

PubMed

Panach, Layla; Pineda, Begoña; Mifsut, Damián; Tarín, Juan J; Cano, Antonio; García-Pérez, Miguel Ángel

2016-02-01

Compelling data are revealing that the CD40/CD40L system is involved in bone metabolism. Furthermore, we have previously demonstrated that polymorphisms in both genes are associated with bone phenotypes. The aim of this study is to further characterize this association and to identify the causal functional mechanism. We conducted an association study of BMD with 15 SNPs in CD40/CD40L genes in a population of 779 women. In addition, we assessed the functionality of this association through the study of the allele-dependent expression of CD40 and CD40L in peripheral blood leukocytes (PBLs) and in human osteoblasts (OBs) obtained from bone explants by qPCR and by sequencing. When an allelic imbalance (AI) was detected, studies on allele-dependent in vitro transcription rate and on CpG methylation in the gene promoter were also performed. Our results confirm the genetic association between SNP rs116535 (T>C) of CD40L gene with LS-BMD. Regarding CD40 gene, two SNPs showed nominal P-values<0.05 for FN- and LS-BMD (Z-scores), although the association was not significant after correcting for multiple testing. Homozygous TT women for SNP rs1883832 (C>T) of CD40 gene showed a trend to have lower levels of OPG (Q-value=0.059), especially when women of BMD-quartile ends were selected (P<0.05). Regarding functionality, we detected an AI for rs1883832 with the C allele the most expressed in OBs and in PBLs. Since the rs116535 of CD40L gene did not show AI, it was not further analyzed. Finally, we described a differential methylation of CpGs in the CD40 promoter among women of high in comparison to low BMD. Our results suggest that the CD40/CD40L system plays a role in regulating BMD. Effectively, our data suggest that a decreased production of OPG could be the cause of the lower BMD observed in TT women for rs1883832 of the CD40 gene and that the degree of methylation of CpGs in the CD40 promoter could contribute to the acquisition of BMD. One possibility that deserves further

Immune receptors CD40 and CD86 in oral keratinocytes and implications for oral lichen planus.

PubMed

Marshall, Alison; Celentano, Antonio; Cirillo, Nicola; Mirams, Michiko; McCullough, Michael; Porter, Stephen

2017-01-01

Lichen planus (LP) is a chronic T-cell-mediated mucocutaneous inflammatory disease that targets stratified epithelia, including those lining the oral cavity. The intraoral variant of LP (OLP) is associated with interferon (IFN)-γ production by infiltrating T lymphocytes; however, the role of epithelial cells in the etiopathogenesis OLP is not completely understood. There is however a growing body of evidence regarding the involvement of epithelial-derived cytokines, immune receptors, and costimulatory molecules in the pathobiological processes that promote and sustain OLP. In the present study, we used a reverse transcriptase-polymerase chain reaction assay to assess whether CD40-a receptor found mainly on antigen presenting cells-and the costimulatory molecule CD86 were expressed in oral keratinocytes (three strains of primary normal oral keratinocytes and the H357 cell line) in the presence or absence of IFN-γ. To further characterize the involvement of CD40 in OLP, expression and distribution of receptor and ligand (CD40/CD154) in tissues from OLP were evaluated by immunohistochemistry. The present results are the first to show that both CD40 and CD86 are constitutively expressed at low levels in oral keratinocytes and that their expression was enhanced by IFN-γ stimulation. The intensity of CD40 staining in OLP tissues was strong. Taken together, the results strongly suggest that CD40 and CD86 play a role in the pathophysiology of oral inflammatory diseases such as OLP.

PPARα agonist fenofibrate attenuates TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Weirong; Lin, Qinqin; Lin, Rong, E-mail: linrong63@yahoo.com.cn

2013-06-10

The ligand-activated transcription factor peroxisome proliferator-activated receptor-α (PPARα) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD{sup +}-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPARα in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-α (TNF-α)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPARαmore » antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-α-induced CD40 expression in adipocytes. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-α-stimulated adipocytes. Taken together, these findings indicate that PPARα agonist fenofibrate inhibits TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-α-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPARα. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-κB. • Fenofibrate increases SIRT1 expression through PPARα and AMPK in

CD40 expression in human thyroid tissue: evidence for involvement of multiple cell types in autoimmune and neoplastic diseases.

PubMed

Smith, T J; Sciaky, D; Phipps, R P; Jennings, T A

1999-08-01

CD40, a member of the tumor necrosis factor-alpha (TNF-alpha) receptor family of surface molecules, is expressed by a variety of cell types. It is a crucial activational molecule displayed by lymphocytes and other bone marrow-derived cells and recently has also been found on nonlymphoid cells such as fibroblasts, endothelia, and epithelial cells in culture. While its role in lymphocyte signaling and activation has been examined in great detail, the function of CD40 expression on nonlymphoid cells, especially in vivo, is not yet understood. Most of the studies thus far have been conducted in cell culture. In this article, we report that several cell types resident in thyroid tissue in vivo can display CD40 under pathological conditions. Sections from a total of 46 different cases were examined immunohistochemically and included nodular hyperplasia, chronic lymphocytic thyroiditis, diffuse hyperplasia, follicular neoplasia, papillary carcinoma, and medullary carcinoma. Thyroid epithelial cells, lymphocytes, macrophages, endothelial cells, and spindle-shape fibroblast-like cells were found to stain positively in the context of inflammation. The staining pattern observed in all cell types was entirely membranous. In general, epithelial staining was limited to that adjacent to lymphocytic infiltration except in 5 of 17 cases of neoplasia and in diffuse hyperplasia. Moreover, we were able to detect CD40 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in human thyroid tissue. These results constitute convincing evidence for expression of CD40 in nonlymphocytic elements of the human thyroid gland. Our findings suggest a potentially important pathway that might be of relevance to the pathogenesis of thyroid diseases. They imply the potential participation of the CD40/CD40 ligand bridge in the cross-talk between resident thyroid cells and bone marrow-derived cells recruited to the thyroid.

CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

PubMed

Afford, S C; Randhawa, S; Eliopoulos, A G; Hubscher, S G; Young, L S; Adams, D H

1999-01-18

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia.

PubMed

Wen, Ting; Mingler, Melissa K; Blanchard, Carine; Wahl, Benjamin; Pabst, Oliver; Rothenberg, Marc E

2012-02-01

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 mAb having been used against B cell leukemia. In this study, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. To confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity, and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 d after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild type control mice, whereas the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the "B cell-specific" inhibitory molecule CD22 on GI eosinophils.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

PubMed Central

Pierini, Antonio; Baker, Jeanette; Armstrong, Randall; Pan, Yuqiong; Leveson-Gower, Dennis; Negrin, Robert; Meyer, Everett

2015-01-01

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies. PMID:26693907

Levels of human platelet-derived soluble CD40 ligand depend on haplotypes of CD40LG-CD40-ITGA2

PubMed Central

Aloui, Chaker; Prigent, Antoine; Tariket, Sofiane; Sut, Caroline; Fagan, Jocelyne; Cognasse, Fabrice; Chakroun, Tahar; Garraud, Olivier; Laradi, Sandrine

2016-01-01

Increased circulating soluble CD40 ligand (sCD40L) is commonly associated with inflammatory disorders. We aimed to investigate whether gene polymorphisms in CD40LG, CD40 and ITGA2 are associated with a propensity to secrete sCD40L; thus, we examined this issue at the level of human platelets, the principal source of sCD40L. We performed single polymorphism and haplotype analyses to test for the effect of twelve polymorphisms across the CD40LG, CD40 and ITGA2 genes in blood donors. ITGA2 presented a positive association with rs1126643, with a significant modification in sCD40L secretion (carriers of C allele, P = 0.02), unlike the investigated CD40LG and CD40 polymorphisms. One CD40LG haplotype (TGGC) showing rs975379 (C/T), rs3092952 (A/G), rs3092933 (A/G) and rs3092929 (A/C) was associated with increased sCD40L levels (1.906 μg/L (95% CI: 1.060 to 2.751); P = 0.000009). The sCD40L level was associated with the inter-chromosomal CD40LG/CD40/ITGA2 haplotype (ATC), displaying rs3092952 (A/G), rs1883832 (C/T) and rs1126643 (C/T), with increased sCD40L levels (P = 0.0135). Our results help to decipher the genetic role of CD40LG, CD40 and ITGA2 with regard to sCD40L levels found in platelet components. Given the crucial role of sCD40L, this haplotype study in a transfusion model may be helpful to further determine the role of haplotypes in inflammatory clinical settings. PMID:27094978

Effect of bleaching agent extracts on murine macrophages.

PubMed

Fernandes, Aletéia M M; Vilela, Polyana G F; Valera, Marcia C; Bolay, Carola; Hiller, Karl Anton; Schweikl, Helmut; Schmalz, Gottfried

2018-05-01

The aim of this study was to evaluate the cytotoxicity and the influence of bleaching agents on immunologically cell surface antigens of murine macrophages in vitro. RAW 264.7 cells were exposed to bleaching gel extracts (40% hydrogen peroxide or 20% carbamide peroxide) and different H 2 O 2 concentrations after 1 and 24-h exposure periods and 1-h exposure and 23-h recovery. Tests were performed with and without N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO). Cell viability was determined by MTT assay. The expression of surface markers CD14, CD40, and CD54 with and without LPS stimulation was detected by flow cytometry, while the production of TNF-α was measured by ELISA. Statistical analysis was performed using the Mann-Whitney U test (α = 0.05). Extracts of bleaching agents were cytotoxic for cells after a 1-h exposure; cells could not recover after 24 h. This effect can be mitigated by the antioxidant NAC and increased by BSO, an inhibitor of glutathione (GSH) synthesis. LPS stimulated expression of all surface markers and TNF-α production. Exposure to bleaching agent extracts and H 2 O 2 leads to a reduction of TNF-α, CD14, and CD40 expression, while the expression of CD54 was upregulated at non-cytotoxic concentrations. Whereas NAC reduced this effect, it was increased in the presence of BSO. Extracts of bleaching agents were irreversibly cytotoxic to macrophages after a 1-h exposure. Only the expression of CD54 was upregulated. The reactions are mediated by the non-enzymatic antioxidant GSH. The addition of an antioxidant can downregulate unfavorable effects of dental bleaching.

The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia¶

PubMed Central

Wen, Ting; Mingler, Melissa K.; Blanchard, Carine; Wahl, Benjamin; Pabst, Oliver; Rothenberg, Marc E.

2011-01-01

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 monoclonal antibody having been used against B cell leukemia. Herein, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. In order to confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 days after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild-type control mice, while the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the “B cell-specific” inhibitory molecule CD22 on GI eosinophils. PMID:22190185

Metabolically active CD4+ T cells expressing Glut1 and OX40 preferentially harbor HIV during in vitro infection.

PubMed

Palmer, Clovis S; Duette, Gabriel A; Wagner, Marc C E; Henstridge, Darren C; Saleh, Suah; Pereira, Candida; Zhou, Jingling; Simar, David; Lewin, Sharon R; Ostrowski, Matias; McCune, Joseph M; Crowe, Suzanne M

2017-10-01

High glucose transporter 1 (Glut1) surface expression is associated with increased glycolytic activity in activated CD4+ T cells. Phosphatidylinositide 3-kinases (PI3K) activation measured by p-Akt and OX40 is elevated in CD4+Glut1+ T cells from HIV+ subjects. TCR engagement of CD4+Glut1+ T cells from HIV+ subjects demonstrates hyperresponsive PI3K-mammalian target of rapamycin signaling. High basal Glut1 and OX40 on CD4+ T cells from combination antiretroviral therapy (cART)-treated HIV+ patients represent a sufficiently metabolically active state permissive for HIV infection in vitro without external stimuli. The majority of CD4+OX40+ T cells express Glut1, thus OX40 rather than Glut1 itself may facilitate HIV infection. Furthermore, infection of CD4+ T cells is limited by p110γ PI3K inhibition. Modulating glucose metabolism may limit cellular activation and prevent residual HIV replication in 'virologically suppressed' cART-treated HIV+ persons. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

PubMed

Xie, Jeffrey X; Zhang, Shungang; Cui, Xiaoyu; Zhang, Jue; Yu, Hui; Khalaf, Fatimah K; Malhotra, Deepak; Kennedy, David J; Shapiro, Joseph I; Tian, Jiang; Haller, Steven T

2017-12-22

Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection

PubMed Central

Bizzell, Erica; Madan-Lala, Ranjna

2017-01-01

Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB. PMID:28767735

Gene Therapy for Liver Transplantation Using Adenoviral Vectors: CD40–CD154 Blockade by Gene Transfer of CD40Ig Protects Rat Livers from Cold Ischemia and Reperfusion Injury

PubMed Central

Ke, Bibo; Shen, Xiu-Da; Gao, Feng; Busuttil, Ronald W.; Löwenstein, Pedro R.; Castro, Maria G.; Kupiec-Weglinski, Jerzy W.

2010-01-01

Liver injury induced by ischemia/reperfusion (I/R) is the prime factor in delayed or loss graft function following transplantation. CD4+ T lymphocytes are key cellular mediators of antigen-independent inflammatory response triggered by I/R. We attempted to modulate rat liver I/R injury by targeted gene therapy with CD40Ig, which blocks the CD40–CD154 costimulation pathway. One hundred percent of Ad-CD40Ig-pretreated orthotopic liver transplants (OLTs) subjected to 24 h of cold (4°C) ischemia survived >14 days (vs 50% in untreated/Ad-β-gal groups). Ad-CD40Ig treatment decreased sGOT levels and depressed neutrophil infiltration, compared with controls. These functional data correlated with histological Suzuki’s grading of hepatic injury, which in untreated/Ad-β-gal groups showed severe necrosis (>60%) and moderate to severe sinusoidal congestion; the Ad-CD40Ig-pretreated group revealed minimal sinusoidal congestion/necrosis. Unlike in controls, OLT expression of mRNA coding for IL-2/IFN-γ remained depressed, whereas that of IL-4/IL-13 reciprocally increased in the Ad-CD40Ig group. Ad-CD40Ig reduced frequency of TUNEL+ cells and proapoptotic Caspase-3, but enhanced antioxidant HO-1 and antiapoptotic Bcl-2/Bcl-xl expression. Thus, prolonged blockade of CD40–CD154 by CD40Ig exerts potent cytoprotection against hepatic I/R injury. These results provide the rationale for a novel gene therapy approach to maximize the organ donor pool through the safer use of liver transplants exposed to prolonged cold ischemia. PMID:14741776

Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40(hi)CD5(+) regulatory B cells in vitro and in vivo.

PubMed

Kim, Hyuk Soon; Lee, Jun Ho; Han, Hee Dong; Kim, A-Ram; Nam, Seung Taek; Kim, Hyun Woo; Park, Young Hwan; Lee, Dajeong; Lee, Min Bum; Park, Yeong Min; Kim, Hyung Sik; Kim, Young Mi; You, Ji Chang; Choi, Wahn Soo

2015-01-01

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40(hi)CD5(+) B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40(hi) is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10(-/-)CD5(+)CD19(+) B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40(hi)CD5(+) Breg cells in mice. However, the population of CD40(hi)CD5(+) B cells was minimal in IL-10(-/-) mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40(hi)CD5(+) B cells and the autocrine effect of IL-10 is critical to the formation of CD40(hi)CD5(+) Breg cells.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

High Cell Surface Expression of CD4 Allows Distinction of CD4+CD25+ Antigen-specific Effector T Cells from CD4+CD25+ Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

PubMed Central

Li, Jinzhu; Ridgway, William; Fathman, C. Garrison; Tse, Harley Y.; Shaw, Michael K.

2008-01-01

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up regulate cell surface expression of CD4. The CD4high sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25+ sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses. PMID:17920698

Mast cells in Waldenstrom's macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling.

PubMed

Tournilhac, O; Santos, D D; Xu, L; Kutok, J; Tai, Y-T; Le Gouill, S; Catley, L; Hunter, Z; Branagan, A R; Boyce, J A; Munshi, N; Anderson, K C; Treon, S P

2006-08-01

Bone marrow (BM) mast cells (MC) are commonly found in association with lymphoplasmacytic cells (LPC) in patients with Waldenström's macroglobulinemia (WM). We therefore sought to clarify the role of MC in WM. Co-culture of sublethally irradiated HMC-1 MC, KU812 basophilic cells, or autologous BM MC along with BM LPC from WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. Furthermore, by immunohistochemistry, multicolor flow cytometry and/or RT-PCR analysis, CD40 ligand (CD154), a potent inducer of B-cell expansion, was expressed on BM MC from 32 of 34 (94%), 11 of 13 (85%), and 7 of 9 (78%) patients, respectively. In contrast, MC from five healthy donors did not express CD154. By multicolor flow cytometry, CD154 was expressed on BM LPC from 35 of 38 (92%) patients and functionality was confirmed by CD154 and CD40 agonistic antibody stimulation, which induced proliferation, support survival and/or pERK phosphorylation of LPC. Moreover, MC induced expansion of LPC from 3 of 5 patients was blocked in a dose dependent manner by use of a CD154 blocking protein. These studies demonstrate that in WM, MC may support tumor cell expansion through constitutive CD154-CD40 signaling and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

PubMed Central

Wykes, Michelle N; Beattie, Lynette; MacPherson, Gordon G; Hart, Derek N

2004-01-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b− DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs. PMID:15500618

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses.

PubMed

Wykes, Michelle N; Beattie, Lynette; Macpherson, Gordon G; Hart, Derek N

2004-11-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

An age-related numerical and functional deficit in CD19(+) CD24(hi) CD38(hi) B cells is associated with an increase in systemic autoimmunity.

PubMed

Duggal, Niharika A; Upton, Jane; Phillips, Anna C; Sapey, Elizabeth; Lord, Janet M

2013-10-01

Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19(+) CD24(hi) CD38(hi) phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19(+) CD24(hi) CD38(hi) cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll-like receptors was also impaired in CD19(+) CD24(hi) CD38(hi) B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19(+) CD24(hi) CD38(hi) B-cell function was compromised by age-related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19(+) CD24(hi) CD38(hi) B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age-associated change in expression of costimulatory molecules CD80 and CD86 on CD19(+) CD24(hi) CD38(hi) cells, suggesting IL10-dependent immune suppression is impaired, but contact-dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19(+) CD24(hi) CD38(hi) B-cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age-related decline in CD19(+) CD24(hi) CD38(hi) B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging. © 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Constitutive CD40L Expression on B Cells Prematurely Terminates Germinal Center Response and Leads to Augmented Plasma Cell Production in T Cell Areas

PubMed Central

Bolduc, Anna; Long, Eugene; Stapler, Dale; Cascalho, Marilia; Tsubata, Takeshi; Koni, Pandelakis A.; Shimoda, Michiko

2013-01-01

CD40/CD40L engagement is essential to T cell-dependent B cell proliferation and differentiation. However, the precise role of CD40 signaling through cognate T–B interaction in the generation of germinal center and memory B cells is still incompletely understood. To address this issue, a B cell-specific CD40L transgene (CD40LBTg) was introduced into mice with B cell-restricted MHC class II deficiency. Using this mouse model, we show that constitutive CD40L expression on B cells alone could not induce germinal center differentiation of MHC class II-deficient B cells after immunization with T cell-dependent Ag. Thus, some other MHC class II-dependent T cell-derived signals are essential for the generation of germinal center B cells in response to T cell-dependent Ag. In fact, CD40LBTg mice generated a complex Ag-specific IgG1 response, which was greatly enhanced in early, but reduced in late, primary response compared with control mice. We also found that the frequency of Ag-specific germinal center B cells in CD40LBTg mice was abruptly reduced 1 wk after immunization. As a result, the numbers of Ag-specific IgG1 long-lived plasma cells and memory B cells were reduced. By histology, large numbers of Ag-specific plasma cells were found in T cell areas adjacent to Ag-specific germinal centers of CD40LBTg mice, temporarily during the second week of primary response. These results indicate that CD40L expression on B cells prematurely terminated their ongoing germinal center response and produced plasma cells. Our results support the notion that CD40 signaling is an active termination signal for germinal center reaction. PMID:20505142

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

PubMed Central

Peres, Raphael Sanches; Liew, Foo Y.; Talbot, Jhimmy; Carregaro, Vanessa; Oliveira, Rene D.; Almeida, Sergio L.; França, Rafael F. O.; Donate, Paula B.; Pinto, Larissa G.; Ferreira, Flavia I. S.; Costa, Diego L.; Demarque, Daniel P.; Gouvea, Dayana Rubio; Lopes, Norberto P.; Queiroz, Regina Helena C.; Silva, Joao Santana; Figueiredo, Florencio; Alves-Filho, Jose Carlos; Cunha, Thiago M.; Ferreira, Sérgio H.; Louzada-Junior, Paulo; Cunha, Fernando Q.

2015-01-01

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients. PMID:25675517

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis.

PubMed

Peres, Raphael Sanches; Liew, Foo Y; Talbot, Jhimmy; Carregaro, Vanessa; Oliveira, Rene D; Almeida, Sergio L; França, Rafael F O; Donate, Paula B; Pinto, Larissa G; Ferreira, Flavia I S; Costa, Diego L; Demarque, Daniel P; Gouvea, Dayana Rubio; Lopes, Norberto P; Queiroz, Regina Helena C; Silva, Joao Santana; Figueiredo, Florencio; Alves-Filho, Jose Carlos; Cunha, Thiago M; Ferreira, Sérgio H; Louzada-Junior, Paulo; Cunha, Fernando Q

2015-02-24

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39(+)CD4(+)CD25(+)FoxP3(+) Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Apigenin: Selective CK2 inhibitor increases Ikaros expression and improves T cell homeostasis and function in murine pancreatic cancer

PubMed Central

Nelson, Nadine; Szekeres, Karoly; Iclozan, Cristina; Rivera, Ivannie Ortiz; McGill, Andrew; Johnson, Gbemisola; Nwogu, Onyekachi

2017-01-01

Pancreatic cancer (PC) evades immune destruction by favoring the development of regulatory T cells (Tregs) that inhibit effector T cells. The transcription factor Ikaros is critical for lymphocyte development, especially T cells. We have previously shown that downregulation of Ikaros occurs as a result of its protein degradation by the ubiquitin-proteasome system in our Panc02 tumor-bearing (TB) mouse model. Mechanistically, we observed a deregulation in the balance between Casein Kinase II (CK2) and protein phosphatase 1 (PP1), which suggested that increased CK2 activity is responsible for regulating Ikaros’ stability in our model. We also showed that this loss of Ikaros expression is associated with a significant decrease in CD4+ and CD8+ T cell percentages but increased CD4+CD25+ Tregs in TB mice. In this study, we evaluated the effects of the dietary flavonoid apigenin (API), on Ikaros expression and T cell immune responses. Treatment of splenocytes from naïve mice with (API) stabilized Ikaros expression and prevented Ikaros downregulation in the presence of murine Panc02 cells in vitro, similar to the proteasome inhibitor MG132. In vivo treatment of TB mice with apigenin (TB-API) improved survival, reduced tumor weights and prevented splenomegaly. API treatment also restored protein expression of some Ikaros isoforms, which may be attributed to its moderate inhibition of CK2 activity from splenocytes of TB-API mice. This partial restoration of Ikaros expression was accompanied by a significant increase in CD4+ and CD8+ T cell percentages and a reduction in Treg percentages in TB-API mice. In addition, CD8+ T cells from TB-API mice produced more IFN-γ and their splenocytes were better able to prime allogeneic CD8+ T cell responses compared to TB mice. These results provide further evidence that Ikaros is regulated by CK2 in our pancreatic cancer model. More importantly, our findings suggest that API may be a possible therapeutic agent for stabilizing Ikaros

Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells

PubMed Central

Vaitaitis, Gisela M.; Yussman, Martin G.; Waid, Dan M.; Wagner, David H.

2017-01-01

CD40-CD154 interaction is critically involved in autoimmune diseases, and CD4 T cells play a dominant role in the Experimental Autoimmune Encephalomyelitis (EAE) model of Multiple Sclerosis (MS). CD4 T cells expressing CD40 (Th40) are pathogenic in type I diabetes but have not been evaluated in EAE. We demonstrate here that Th40 cells drive a rapid, more severe EAE disease course than conventional CD4 T cells. Adoptively transferred Th40 cells are present in lesions in the CNS and are associated with wide spread demyelination. Primary Th40 cells from EAE-induced donors adoptively transfer EAE without further in-vitro expansion and without requiring the administration of the EAE induction regimen to the recipient animals. This has not been accomplished with primary, non-TCR-transgenic donor cells previously. If co-injection of Th40 donor cells with Freund’s adjuvant (CFA) in the recipient animals is done, the disease course is more severe. The CFA component of the EAE induction regimen causes generalized inflammation, promoting expansion of Th40 cells and infiltration of the CNS, while MOG-antigen shapes the antigen-specific TCR repertoire. Those events are both necessary to precipitate disease. In MS, viral infections or trauma may induce generalized inflammation in susceptible individuals with subsequent disease onset. It will be important to further understand the events leading up to disease onset and to elucidate the contributions of the Th40 T cell subset. Also, evaluating Th40 levels as predictors of disease onset would be highly useful because if either the generalized inflammation event or the TCR-honing can be interrupted, disease onset may be prevented. PMID:28192476

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

PubMed Central

Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

2001-01-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

PubMed

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

[Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

PubMed

Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

2016-09-01

Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis.

PubMed

Hueso, Miguel; De Ramon, Laura; Navarro, Estanislao; Ripoll, Elia; Cruzado, Josep M; Grinyo, Josep M; Torras, Joan

2016-12-01

CD40/CD40L signaling exerts a critical role in the development of atherosclerosis, and microRNAs (miRNAs) are key regulators in vascular inflammation and plaque formation. In this work, we investigated mRNA/miRNA expression during progression of atherosclerotic lesions through CD40 silencing. We silenced CD40 with a specific siRNA in ApoE -/- mice and compared expression of mRNA/miRNA in ascending aorta with scrambled treated mice. siRNA-CD40 treated mice significantly reduced the extension and severity of atherosclerotic lesions, as well as the number of F4/80 + , galectin-3 + macrophages and NF-κB + cells in the intima. Genome-wide mRNA/miRNA profiling allowed the identification of transcripts, which were significantly upregulated during atherosclerosis; among them, miR-125b and miR-30a, Xpr1, a regulator of macrophage differentiation, Taf3, a core transcription factor and the NF-κB activator Ikkβ, whereas, the NF-κB inhibitor Ikbα was downregulated during disease progression. All those changes were reversed upon CD40 silencing. Interestingly, TAF3, XPR1 and miR-125b were also overexpressed in human atherosclerotic plaques. Murine Taf3 and Xpr1 were detected in the perivascular adipose tissue (PVAT), and Taf3 also in intimal foam cells. Finally, expression of miR-125b was regulated by the CD40-NF-κB signaling axis in RAW264.7 macrophages. CD40 silencing with a specific siRNA ameliorates progression of experimental atherosclerosis in ApoE -/- mice, and evidences a role for NF-κB, Taf3, Xpr1, and miR-125b in the pathogenesis of atherosclerosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The Role of Soluble CD40L Ligand in Human Carcinogenesis.

PubMed

Angelou, Anastasios; Antoniou, Efstathios; Garmpis, Nikolaos; Damaskos, Christos; Theocharis, Stamatios; Margonis, Georgios-Antonios

2018-05-01

The role of CD40/CD40L in carcinogenesis is widely examined. The mechanisms linking the CD40/CD40L system and the soluble form of CD40 ligand (sCD40L) with neoplasia are nowadays a topic of intensive research. CD40L and sCD40L belong to the TNF superfamily and are molecules with a proinflammatory role. A variety of cells express CD40L such as the immune system cells, the endothelial cells and activated platelets. Although many medications such as statins have been shown to reduce sCD40L, it is still debated whether specific treatments targeting the CD40/CD40L system will prove to be effective against carcinogenesis in the near future. A comprehensive search of the Pubmed Database was conducted for English-language studies using a list of key words. At diagnosis, serum samples of patients with neoplasia contained higher levels of sCD40L than healthy controls, suggesting that sCD40L may play a predictive role in human carcinogenesis. Patients with neoplasia had higher circulating sCD40L levels and it is likely that sCD40L may have a predictive role. It is still unclear whether sCD40L can be used as a therapeutic target. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans.

PubMed

van der Voort, R; Keehnen, R M; Beuling, E A; Spaargaren, M; Pals, S T

2000-10-16

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.

CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice

PubMed Central

Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M.; Wiltrout, Robert H.; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A.; Manns, Michael P.; Wang, Ena; Marincola, Francesco M.; Korangy, Firouzeh; Greten, Tim F.

2015-01-01

Immunosuppressive CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) accumulate in the livers of tumor-bearing mice. We studied hepatic MDSC in two murine models of immune mediated hepatitis. Unexpectedly, treatment of tumor bearing mice with Concanavalin A or α-Galactosylceramide resulted in increased ALT and AST serum levels in comparison to tumor free mice. Adoptive transfer of hepatic MDSC into naïve mice exacerbated Concanavalin A induced liver damage. Hepatic CD11b+Gr-1+ cells revealed a polarized pro-inflammatory gene signature after Concanavalin A treatment. An interferon gamma- dependent up-regulation of CD40 on hepatic CD11b+Gr-1+ cells along with an up-regulation of CD80, CD86, and CD1d after Concanavalin A treatment was observed. Concanavalin A treatment resulted in a loss of suppressor function by tumor-induced CD11b+Gr-1+ MDSC as well as enhanced reactive oxygen species-mediated hepatotoxicity. CD40 knockdown in hepatic MDSC led to increased arginase activity upon Concanavalin A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased reactive oxygen species production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSC act as pro-inflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner. PMID:25616156

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4+ Vα24 natural killer T cells expressing CD40L

PubMed Central

Nieda, M; Kikuchi, A; Nicol, A; Koezuka, Y; Ando, Y; Ishihara, S; Lapteva, N; Yabe, T; Tokunaga, K; Tadokoro, K; Juji, T

2001-01-01

Human Vα24 natural killer T (Vα24NKT) cells are activated by α-glycosylceramide-pulsed dendritic cells (DCs) in a CD1d-dependent and T-cell receptor-mediated manner. There are two major subpopulations of Vα24NKT cells, CD4– CD8– Vα24NKT and CD4+ Vα24NKT cells. We have recently shown that activated CD4– CD8– Vα24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of Vα24NKT cells is currently limited. We aimed to investigate whether CD4+ Vα24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4+ Vα24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4+ Vα24NKT cells, but not with resting CD4+ Vα24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb. Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40–CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Vα24NKT cells. The apoptosis of DCs from normal donors, triggered by the CD40–CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4+ Vα24NKT cells by virtue of apoptosis of DCs. PMID:11260318

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

PubMed

Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

2017-08-01

Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner.

PubMed

Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M; Wiltrout, Robert H; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A; Manns, Michael P; Wang, Ena; Marincola, Francesco M; Korangy, Firouzeh; Greten, Tim F

2015-04-01

Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

CD8+T cells expressing both PD-1 and TIGIT but not CD226 are dysfunctional in acute myeloid leukemia (AML) patients.

PubMed

Wang, Mengjie; Bu, Jin; Zhou, Maohua; Sido, Jessica; Lin, Yu; Liu, Guanfang; Lin, Qiwen; Xu, Xiuzhang; Leavenworth, Jianmei W; Shen, Erxia

2018-05-01

Acute myeloid leukemia (AML) is one of the most common types of leukemia among adults with an overall poor prognosis and very limited treatment management. Immune checkpoint blockade of PD-1 alone or combined with other immune checkpoint blockade has gained impressive results in murine AML models by improving anti-leukemia CD8 + T cell function, which has greatly promoted the strategy to utilize combined immune checkpoint inhibitors to treat AML patients. However, the expression profiles of these immune checkpoint receptors, such as co-inhibitory receptors PD-1 and TIGIT and co-stimulatory receptor CD226, in T cells from AML patients have not been clearly defined. Here we have defined subsets of CD8 + and CD4 + T cells in the peripheral blood (PB) from newly diagnosed AML patients and healthy controls (HCs). We have observed increased frequencies of PD-1- and TIGIT- expressing CD8 + T cells but decreased occurrence of CD226-expressing CD8 + T cells in AML patients. Further analysis of these CD8 + T cells revealed a unique CD8 + T cell subset that expressed PD-1 and TIGIT but displayed lower levels of CD226 was associated with failure to achieve remission after induction chemotherapy and FLT3-ITD mutations which predict poor clinical prognosis in AML patients. Importantly, these PD-1 + TIGIT + CD226 - CD8 + T cells are dysfunctional with lower expression of intracellular IFN-γ and TNF-α than their counterparts in HCs. Therefore, our studies revealed that an increased frequency of a unique CD8 + T cell subset, PD-1 + TIGIT + CD226 - CD8 + T cells, is associated with CD8 + T cell dysfunction and poor clinical prognosis of AML patients, which may reveal critical diagnostic or prognostic biomarkers and direct more efficient therapeutic strategies. Copyright © 2017. Published by Elsevier Inc.

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response.

PubMed

Gri, Giorgia; Gallo, Elena; Di Carlo, Emma; Musiani, Piero; Colombo, Mario P

2003-01-01

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

Arteriogenesis in murine adipose tissue is contingent on CD68+/CD206+ macrophages

PubMed Central

Seaman, Scott A.; Cao, Yiqi; Campbell, Chris A.; Peirce, Shayn M.

2016-01-01

Objective The surgical transfer of skin, fat, and/or muscle from a donor site to a recipient site within the same patient, is a widely performed procedure in reconstructive surgeries. A surgical pre-treatment strategy that is intended to increase perfusion in the flap, termed “flap delay”, is a commonly employed technique by plastic surgeons prior to flap transplantation. Here, we explored whether CD68+/CD206+ macrophages are required for arteriogenesis within the flap by performing gain-of-function and loss-of-function studies in a previously published flap delay murine model. Methods and Results Local injection of M2-polarized macrophages into the flap resulted in an increase in collateral vessel diameter. Application of a thin biomaterial film loaded with a pharmacological agent (FTY720), which has been previously shown to recruit CD68+/CD206+ macrophages to remodeling tissue, increased CD68+/CD206+ cell recruitment and collateral vessel enlargement. Conversely, when local macrophage populations were depleted within the inguinal fat pad via clodronate liposome delivery, we observed fewer CD68+ cells accompanied by diminished collateral vessel enlargement. Conclusions Our study underscores the importance of macrophages during microvascular adaptations that are induced by flap delay. These studies suggest a mechanism for a translatable therapeutic target that may be used to enhance the clinical flap delay procedure. PMID:27976451

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Vitamin E attenuates myocardial ischemia-reperfusion injury in murine AIDS.

PubMed

Chen, Yinhong; Davis-Gorman, Grace; Watson, Ronald Ross; McDonagh, Paul F

2002-01-01

The incidence of myocardial infarction in patients who have the aquired immunodeficiency syndrome (AIDS) is increasing. However, no effective therapeutic agents have been discovered to reduce myocardial ischemia-reperfusion (I/R) injury in pathologies associated with AIDS. The aim of this study was to determine if infarct size is increased in murine AIDS after I/R injury and if I/R injury could be attenuated with vitamin E supplementation. Three groups of mice were studied: control, murine AIDS, and murine AIDS with vitamin E supplementation. Anesthetized mice were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. The hearts in mice that had murine AIDS had a larger infarct size compared to controls after I/R injury. Vitamin E supplementation significantly reduced infarct size and inhibited polymorphonuclear neutrophil (PMN) CD11b expression (p < 0.05). However, vitamin E supplementation did not affect PMN reactive oxygen species (ROS) production and platelet CD62p expression. These results suggest that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of PMN CD11b expression. Vitamin E may be a promising prophylactic agent for the reduction of the severity of myocardial I/R injury in patients who have AIDS.

CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.

PubMed

Portillo, Jose-Andres C; Lopez Corcino, Yalitza; Miao, Yanling; Tang, Jie; Sheibani, Nader; Kern, Timothy S; Dubyak, George R; Subauste, Carlos S

2017-02-01

Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40 + Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X 7 -dependent production of TNF-α and IL-1β by macrophages. P2X 7 -/- mice and mice treated with a P2X 7 inhibitor were protected from diabetes-induced TNF-α, IL-1β, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X 7 pathway. © 2017 by the American Diabetes Association.

Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice.

PubMed

Dory, Daniel; Echchannaoui, Hakim; Letiembre, Maryse; Ferracin, Fabrizia; Pieters, Jean; Adachi, Yoshiyuki; Akashi, Sachiko; Zimmerli, Werner; Landmann, Regine

2003-07-01

Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.

Human CD40 ligand-expressing type 3 innate lymphoid cells induce IL-10-producing immature transitional regulatory B cells.

PubMed

Komlósi, Zsolt I; Kovács, Nóra; van de Veen, Willem; Kirsch, Anna Isabella; Fahrner, Heinz Benedikt; Wawrzyniak, Marcin; Rebane, Ana; Stanic, Barbara; Palomares, Oscar; Rückert, Beate; Menz, Günter; Akdis, Mübeccel; Losonczy, György; Akdis, Cezmi A

2017-09-20

Type 3 innate lymphoid cells (ILC3s) are involved in maintenance of mucosal homeostasis; however, their role in immunoregulation has been unknown. Immature transitional regulatory B (itBreg) cells are innate-like B cells with immunosuppressive properties, and the in vivo mechanisms by which they are induced have not been fully clarified. We aimed to investigate the ILC3-B-cell interaction that probably takes place in human tonsils. ILC3s were isolated from peripheral blood and palatine tonsils, expanded, and cocultured with naive B cells. Tonsillar ILC3s and regulatory B cells were visualized with immunofluorescence histology. ILC3 frequencies were measured in tonsil tissue of allergic and nonallergic patients and in peripheral blood of allergic asthmatic patients and healthy control subjects. A mutually beneficial relationship was revealed between ILC3s and B cells: ILC3s induced IL-15 production in B cells through B cell-activating factor receptor, whereas IL-15, a potent growth factor for ILC3s, induced CD40 ligand (CD40L) expression on circulating and tonsillar ILC3s. IL-15-activated CD40L + ILC3s helped B-cell survival, proliferation, and differentiation of IL-10-secreting, PD-L1-expressing functional itBreg cells in a CD40L- and B cell-activating factor receptor-dependent manner. ILC3s and regulatory B cells were in close connection with each other in palatine tonsils. ILC3 frequency was reduced in tonsil tissue of allergic patients and in peripheral blood of allergic asthmatic patients. Human CD40L + ILC3s provide innate B-cell help and are involved in an innate immunoregulatory mechanism through induction of itBreg cell differentiation, which takes place in palatine tonsils in vivo. This mechanism, which can contribute to maintenance of immune tolerance, becomes insufficient in allergic diseases. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Murine CD103+ dendritic cells protect against steatosis progression towards steatohepatitis.

PubMed

Heier, Eva-Carina; Meier, Anna; Julich-Haertel, Henrike; Djudjaj, Sonja; Rau, Monica; Tschernig, Thomas; Geier, Andreas; Boor, Peter; Lammert, Frank; Lukacs-Kornek, Veronika

2017-06-01

Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can progress to non-alcoholic steatohepatitis (NASH). The identification of molecular and cellular factors that determine the progression of NASH and lead to irreversible hepatocellular damage are crucial. Dendritic cells (DCs) represent a heterogeneous cell population among which CD103 + DCs play a significant role in immunity and tolerance. We aimed to clarify the role of this DC subset in the pathomechanism of NASH. Steatosis progression towards steatohepatitis was analysed using multicolor FACS analyses, cytokine and qPCR array in high sucrose diet (HSD) and methionine and choline deficient diet (MCD) fed wild-type and basic leucine zipper transcription factor, ATF-Like-3 (Batf3) deficient animals, which lack CD103 + DCs (classical type-1 DC, cDC1s). Metabolic challenge of Batf3 -/- animals resulted in the progression of steatosis towards steatohepatitis, manifesting by an increased influx of inflammatory cells into the liver and elevated inflammatory cytokine production of myeloid cells upon innate stimuli. However, the lack of cDC1s did not affect cellular apoptosis and fibrosis progression but altered genes involved in lipid metabolism. The adoptive transfer of CD103 + cDC1s to Batf3 deficient animals reversed these observed changes and more importantly could attenuate cellular damage and inflammation in established murine steatohepatitis. Here, we have identified the murine CD103 + cDC1s as a protective DC subtype that influences the pro-anti-inflammatory balance and protects the liver from metabolic damage. As guardians of liver integrity, they play a key role in the inflammatory process during the development of steatohepatitis in mice. Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can lead to non-alcoholic steatohepatitis (NASH). The current study demonstrated that a specific murine dendritic cell subtype possesses a potent

Specific Mutation of a Gammaherpesvirus-Expressed Antigen in Response to CD8 T Cell Selection In Vivo

PubMed Central

Loh, Joy; Popkin, Daniel L.; Droit, Lindsay; Braaten, Douglas C.; Zhao, Guoyan; Zhang, Xin; Vachharajani, Punit; Myers, Nancy; Hansen, Ted H.

2012-01-01

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo. PMID:22171269

CD56 Expression in Odontogenic Cysts and Tumors.

PubMed

Jaafari-Ashkavandi, Zohreh; Dehghani-Nazhvani, Ali; Razmjouyi, Faranak

2014-01-01

Background and aims. Odontogenic cysts and tumors have a wide spectrum of clinical characteristics that lead to the different management strategies. Since definite diagnosis is difficult in some cases, it has been suggested that CD56 may be a candidate marker for definitive diagnosis of some odontogenic tumors. The present study was designed to examine CD56 expression in lesions with histopathological similarities. Materials and methods. In this cross-sectional, analytical study the subjects were 22 ameloblastomas, 13 dentigerous cysts, 10 keratocystic odontogenic tumors (KCOT), 4 adenomatoid odontogenic tumors (AOT), 3 orthokeratinized odonto-genic cysts, 3 calcifying odontogenic cysts (COC) and one glandular odontogenic cyst (GOC). All the samples were examined for CD56 immunoreactivity. Data were analyzed using chi-square test. Results. Twenty cases (91%) of ameloblastomas, 3 (75%) AOT, 4 (40%) KCOT and one case of GOC were positive for CD56. None of the dentigerous cysts, COC and orthokeratinized odontogenic cysts was CD56-positive. There was a significant difference in the CD56 expression between ameloblastoma and dentigerous cyst, as well as COC. Also, KCOT showed significantly higher expression than orthokeratinized odontogenic cyst. Conclusion. In this study CD56 expression was limited to the odontogenic tumors and more aggressive cystic lesions. This marker can be a useful aid for distinguishing cysts and tumors from similar lesions.

CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis

PubMed Central

Burt, Bryan M.; Plitas, George; Stableford, Jennifer A.; Nguyen, Hoang M.; Bamboat, Zubin M.; Pillarisetty, Venu G.; DeMatteo, Ronald P.

2008-01-01

The liver contains a unique repertoire of immune cells and a particular abundance of NK cells. We have found that CD11c defines a distinct subset of NK cells (NK1.1+CD3−) in the murine liver whose function was currently unknown. In naïve animals, CD11c+ liver NK cells displayed an activated phenotype and possessed enhanced effector functions when compared with CD11c− liver NK cells. During the innate response to adenovirus infection, CD11c+ NK cells were the more common IFN-γ-producing NK cells in the liver, demonstrated enhanced lytic capability, and gained a modest degree of APC function. The mechanism of IFN-γ production in vivo depended on TLR9 ligation as well as IL-12 and -18. Taken together, our findings demonstrate that CD11c+ NK cells are a unique subset of NK cells in the murine liver that contribute to the defense against adenoviral hepatitis. PMID:18664530

Altered expression of regulatory T and Th17 cells in murine bronchial asthma

PubMed Central

Zhu, Jianbo; Liu, Xiaoying; Wang, Wenxia; Ouyang, Xiuhe; Zheng, Wentao; Wang, Qingyuan

2017-01-01

Alteration of the careful balance of the ratio of Th1/Th2 cell subsets impacts immune function and plays an important role in the pathogenesis of asthma. There is little research on the impact of changes on the balance of the regulatory T (Treg)/Th17 subset ratio and its possible repercussions for asthma. This investigation used a murine model of asthma to measure the expression levels of Treg and Th17 cells and the levels of their transcription factors Foxp3 and retinoic acid receptor-related orphan nuclear receptor (ROR)γt in bronchial asthma while assessing indexes of airway inflammation. Thirty female SPF BALB/c mice were divided into three equally numbered groups: a normal control, an asthma and a dexamethasone treatment group. All the airway inflammation indexes measured were more prominent in the asthma group and less so in the control group. The percentage of the lymphocyte subset CD4+CD25+Foxp3+ cells in the CD4+ cells in the asthma group was significantly lower than that in the normal control group (P<0.01). The percentage of the lymphocyte subset CD4+IL-17+ cells in the CD4+ cells in the asthma group was significantly higher than that in the normal control group (P<0.01). The ratio of CD4+CD25+Foxp3+ cells/CD4+IL-17+ cells in the asthma group decreased compared with that in the normal control group (P<0.01). The expression level of Foxp3 of the mice in the asthma group was significantly lower than that in the control group (P<0.01). The expression intensity of RORγt in the asthma group was higher than that in the normal control group (P<0.01). Finally, the Foxp3/RORγt protein expression ratio in the asthma group was significantly lower than that in the normal control group (P<0.01). The Foxp3/RORγt protein expression ratio and the airway responsiveness were negatively correlated. The average levels of inflammation markers in the dexamethasone group were intermediate between the other groups. During the course of bronchial asthma the unbalanced

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

PubMed

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pagel, John M.; Matthews, Dana C.; Kenoyer, Aimee L.

2009-01-01

The efficacy of radioimmunotherapy (RIT) for treatment of patients with hematological malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by delivery of a biotinylated clearing agent and radiolabeled-DOTA-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the pretargeted RIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly-labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a largemore » amount of labeled antibody for a prolonged time. Therapy experiments demonstrated that 90Y-DOTA-biotin significantly prolonged survival of mice treated pretargeted with anti-hCD45 Ab-SA compared to mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at the maximally tolerated dose (400 µCi). Since human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and pretargeted RIT using an anti-murine (m)CD45 Ab (A20 ) in a model where the target antigen is present on normal hematopoietic tissues. After 24 hours, 27.3 ± 2.8% of the injected dose of radionuclide was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin (p value). These data suggest that multi-step pretargeted methods for delivering RIT are superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.« less

OX40 signaling is involved in the autoactivation of CD4+CD28- T cells and contributes to the pathogenesis of autoimmune arthritis.

PubMed

Jiang, Juean; Liu, Cuiping; Liu, Mi; Shen, Yu; Hu, Xiaohan; Wang, Qin; Wu, Jian; Wu, Min; Fang, Qi; Zhang, Xueguang

2017-03-21

CD4 + CD28 - T cells exhibit autoreactive potential in autoimmune disorders, including rheumatoid arthritis (RA). It is not well known which costimulator functions as an alternative second signal in the activation of this subset after CD28 expression is downregulated. Tumor necrosis factor receptor superfamily member OX40 is a key costimulator in the activation of T cells. The aim of this study was to investigate the costimulatory effects of OX40 on CD4 + CD28 - T cells in autoimmune arthritis. Clinical samples were collected from patients with RA and control subjects. Collagen-induced arthritis (CIA) was induced with collagen type II (CII) in DBA/1 mice. The CD4 + CD28 - OX40 + T-cell subset and its cytokine production were detected by flow cytometry. After T-cell purification, adoptive transfer was performed in CIA mice. The regulatory role of OX40 was determined by blocking experiments in vitro and in vivo. OX40 and OX40L were abnormally expressed in patients with RA and CIA mice. Further analysis showed that CD4 + CD28 - OX40 + T cells accumulated in patients with RA and in animal models. These cells produced higher levels of proinflammatory cytokines and were closely correlated with the clinicopathological features of the affected individuals. Adoptive transfer of CII-specific CD4 + CD28 - OX40 + T cells remarkably aggravated arthritic development and joint pathology in CIA mice. Moreover, OX40 blockade significantly reduced the proinflammatory responses and ameliorated arthritis development. OX40 acts as an alternative costimulator of CD4 + CD28 - T cells and plays a pathogenic role in autoimmune arthritic development, suggesting that it is a potential target for immunomodulatory therapy of RA.

Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).

PubMed

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Lokensgard, James R

2018-06-01

Previous work from our laboratory has demonstrated in vivo persistence of CD103 + CD69 + brain resident memory CD8 + T-cells (bT RM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T RM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 + T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT RM . In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8 + T-cells promote development of CD103 + CD69 + CD8 + T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8 + T-cells obtained from either WT or PD-1 KO mice. We found that αCD3 Ab-stimulated CD8 + T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8 + T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69 + CD8 + T-cells, which promotes development of T RM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69 + CD8 + cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69 + CD8 + T-cells. Taken together, these results demonstrate a role for activated glia in promoting development of bT RM through the PD-1: PD-L1 pathway. © 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation.

PubMed

Kawabe, T; Naka, T; Yoshida, K; Tanaka, T; Fujiwara, H; Suematsu, S; Yoshida, N; Kishimoto, T; Kikutani, H

1994-06-01

An engagement of CD40 with CD40 ligand (CD40L) expressed on activated T cells is known to provide an essential costimulatory signal to B cells in vitro. To investigate the role of CD40 in in vivo immune responses, CD40-deficient mice were generated by gene targeting. The significant reduction of CD23 expression on mature B cells and relatively decreased number of IgM bright and IgD dull B cells were observed in the mutant mice. The mutant mice mounted IgM responses but no IgG, IgA, and IgE responses to thymus-dependent (TD) antigens. However, IgG as well as IgM responses to thymus-independent (TI) antigens were normal. Furthermore, the germinal center formation was defective in the mutant mice. These results suggest that CD40 is essential for T cell-dependent immunoglobulin class switching and germinal center formation, but not for in vivo T cell-dependent IgM responses and T cell-independent antibody responses.

Generation and function of immunosuppressive human and murine CD8+ T cells by transforming growth factor-β and retinoic acid

PubMed Central

Fleissner, Diana; Frede, Annika; Knott, Markus; Knuschke, Torben; Geffers, Robert; Hansen, Wiebke; Dobos, Gustav; Langhorst, Jost; Buer, Jan; Westendorf, Astrid M

2011-01-01

The intestinal immune system is constantly challenged by foreign antigens and commensal bacteria. Therefore, proper control of the intestinal microenvironment is required. One important arm of this regulatory network consists of regulatory T cells. In contrast to CD4+ Foxp3+ regulatory T cells, which have been well characterized, immunomodulatory CD8+ T cells that express Foxp3 are less well defined in terms of their generation and function. Failures of these regulatory mechanisms contribute to the development of inflammatory bowel disease. In this study we demonstrate that the frequency of CD8+ Foxp3+ T cells is reduced in the peripheral blood of patients with ulcerative colitis. As these cells might play a currently underestimated role in the maintenance of intestinal homeostasis, we have investigated human and murine CD8+ Foxp3+ T cells generated by stimulating naive CD8+ T cells in the presence of transforming growth factor-β and retinoic acid, mediators that are abundantly produced in the intestinal mucosa. These CD8+ Foxp3+ fully competent regulatory T cells show strong expression of regulatory molecules CD25, Gpr83 and CTLA-4 and exhibit cell–cell contact-dependent immunosuppressive activity in vitro. Our study illustrates a previously unappreciated critical role of CD8+ Foxp3+ T cells in controlling potentially dangerous T cells and in the maintenance of intestinal homeostasis. PMID:21711349

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

PubMed

Bristol, J A; Schlom, J; Abrams, S I

1999-05-25

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T

Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients.

PubMed

Danese, S; Katz, J A; Saibeni, S; Papa, A; Gasbarrini, A; Vecchi, M; Fiocchi, C

2003-10-01

The CD40/CD40L system, a key regulator and amplifier of immune reactivity, is activated in inflammatory bowel disease (IBD) mucosa. To determine whether plasma levels of sCD40L are elevated in Crohn's disease (CD) and ulcerative colitis (UC) patients compared with normal controls, to investigate the cellular source of sCD40L, and to explore CD40L induction mechanisms. CD, UC, and normal control subjects were studied. The concentration of sCD40L in plasma and supernatants of freshly isolated platelets and autologous peripheral blood T cells (PBT) was measured by ELISA. Surface CD40L expression level was measured by flow cytometry in resting and thrombin activated platelets, and unstimulated and CD3/CD28 stimulated PBT before and after coculture with human intestinal microvascular endothelial cells (HIMEC). Compared with normal controls, plasma sCD40L levels were significantly higher in both CD and UC patients and proportional to the extent of mucosal inflammation. Platelets from IBD patients displayed a significantly higher surface CD40L expression than those from control subjects, and released greater amounts of sCD40L than autologous PBT. Contact with IL-1beta activated HIMEC induced significant upregulation of CD40L surface expression and release by platelets. Elevated levels of sCD40L in the circulation of IBD patients reflect enhanced surface expression and release of CD40L by platelets. This phenomenon translates to an increased platelet activation state apparently induced by passage through an inflamed mucosal microvascular bed, a conclusion supported by the positive correlation of plasma sCD40L levels with the extent of anatomical involvement by IBD. These results suggest that platelet-endothelial interactions critically contribute to activation of the CD40 pathway in IBD.

CD4 expression on EL4 cells as an epiphenomenon of retroviral transduction and selection.

PubMed

Logan, Grant J; Spinoulas, Afroditi; Alexander, Stephen I; Smythe, Jason A; Alexander, Ian E

2004-04-01

The EL4 murine tumour cell line, isolated from a chemically induced lymphoma over 50 years ago, has been extensively exploited in immunological research. The conclusions drawn from many of these studies have been based on the presumption that EL4 cells maintain a stable phenotype during experimental manipulation. To the contrary, we have observed 100-fold greater expression of cell surface CD4 (CD4(high)) on a subpopulation of EL4 cells following retroviral transduction and G418 selection when compared with unmodified populations. Although the mechanism responsible for this effect remains to be elucidated, the unexpected expression of CD4, a molecule that functions as both a coreceptor with the T-cell receptor and ligand for the pro-inflammatory cytokine IL-16, has the potential to influence experimental outcomes. Upregulation of CD4 should be excluded when EL4 cells are utilized in experiments requiring a consistent immuno-phenotype.

Mycobacterium avium subspecies induce differential expression of pro-inflammatory mediators in a murine macrophage model: evidence for enhanced pathogenicity of Mycobacterium avium subspecies paratuberculosis.

PubMed

Basler, Tina; Geffers, Robert; Weiss, Siegfried; Valentin-Weigand, Peter; Goethe, Ralph

2008-01-01

Mycobacterium avium subspecies (ssp.) paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic, non-treatable granulomatous enteritis of ruminants. MAP is the only mycobacterium affecting the intestinal tract, which is of interest since it is presently the most favoured pathogen linked to Crohn's disease (CD) in humans due to its frequent detection in CD tissues. MAP is genetically closely related to other M. avium ssp. such as M. avium ssp. avium (MAA) and M. avium ssp. hominissuis (MAH) which can cause mycobacteriosis in animals and immunocompromised humans. We have recently shown that murine macrophage cell lines represent suitable systems to analyse M. avium ssp. patho-mechanisms and could show that MAP, but not MAA, specifically inhibited the antigen-specific stimulatory capacity for CD4(+) T-cells. In the present study, we compared gene expression profiles of murine RAW264.7 macrophages in response to infections with MAP or MAA using murine high-density oligonucleotide Affymetrix microarrays. A comparison of MAP and MAA infection revealed 17 differentially expressed genes. They were expressed at a much lower level in MAP-infected macrophages than in MAA-infected macrophages. Among these were the genes for IL-1beta, IL-1alpha, CXCL2, PTGS2 (COX2), lipocalin (LCN2) and TNF, which are important pro-inflammatory factors. The microarray data were confirmed for selected genes by quantitative real-time reverse transcription PCR and, by protein array analyses and ELISA. Similar to MAA, infection with MAH also showed robust induction of IL-1beta, CXCL2, COX2, LCN2 and TNF. Taken together, our results from M. avium ssp.-infected murine macrophages provide evidence that MAP in contrast to MAA and MAH specifically suppresses the pro-inflammatory defence mechanisms of infected macrophages.

Murine Aortic Smooth Muscle Cells Acquire, Though Fail to Present Exogenous Protein Antigens on Major Histocompatibility Complex Class II Molecules

PubMed Central

Maddaluno, Marcella; MacRitchie, Neil; Grassia, Gianluca; Ialenti, Armando; Butcher, John P.; Garside, Paul; Brewer, James M.; Maffia, Pasquale

2014-01-01

In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323–339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4+ T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression. PMID:25136640

Noncanonical expression of a murine cytomegalovirus early protein CD8 T-cell epitope as an immediate early epitope based on transcription from an upstream gene.

PubMed

Fink, Annette; Büttner, Julia K; Thomas, Doris; Holtappels, Rafaela; Reddehase, Matthias J; Lemmermann, Niels A W

2014-02-14

Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I) glycoproteins, are often identified by "reverse immunology", a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs) based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype) mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E) phase protein, the m164 epitope is presented already during the Immediate Early (IE) phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

PubMed

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-10-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified Aromatic-Turmerone (AR)

PubMed Central

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-01-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone(ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs. PMID:23095866

Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle

PubMed Central

Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan

2016-01-01

Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281

Porcine, murine and human sialoadhesin (Sn/Siglec-1/CD169): portals for porcine reproductive and respiratory syndrome virus entry into target cells.

PubMed

Van Breedam, Wander; Verbeeck, Mieke; Christiaens, Isaura; Van Gorp, Hanne; Nauwynck, Hans J

2013-09-01

Porcine sialoadhesin (pSn; a sialic acid-binding lectin) and porcine CD163 (pCD163) are molecules that facilitate infectious entry of porcine reproductive and respiratory syndrome virus (PRRSV) into alveolar macrophages. In this study, it was shown that murine Sn (mSn) and human Sn (hSn), like pSn, can promote PRRSV infection of pCD163-expressing cells. Intact sialic acid-binding domains are crucial, since non-sialic acid-binding mutants of pSn, mSn and hSn did not promote infection. Endodomain-deletion mutants of pSn, mSn and hSn promoted PRRSV infection less efficiently, but also showed markedly reduced expression levels, making further research into the potential role of the Sn endodomain in PRRSV receptor activity necessary. These data further complement our knowledge on Sn as an important PRRSV receptor, and suggest - in combination with other published data - that species differences in the main PRRSV entry mediators Sn and CD163 do not account for the strict host species specificity displayed by the virus.

Increased expression of activation antigens on CD8+ T lymphocytes in Myalgic Encephalomyelitis/chronic fatigue syndrome: inverse associations with lowered CD19+ expression and CD4+/CD8+ ratio, but no associations with (auto)immune, leaky gut, oxidative and nitrosative stress biomarkers.

PubMed

Maes, Michael; Bosmans, Eugene; Kubera, Marta

2015-01-01

There is now evidence that specific subgroups of patients with Myalgic Encephalomyelitis / chronic fatigue syndrome (ME/CFS) suffer from a neuro-psychiatric-immune disorder. This study was carried out to delineate the expression of the activation markers CD38 and human leukocyte antigen (HLA) DR on CD4+ and CD8+ peripheral blood lymphocytes in ME/CFS. Proportions and absolute numbers of peripheral lymphocytes expressing CD3+, CD19+, CD4+, CD8+, CD38+ and HLA-DR+ were measured in ME/CFS (n=139), chronic fatigue (CF, n=65) and normal controls (n=40). The proportions of CD3+, CD8+, CD8+CD38+ and CD8+HLA-DR+ were significantly higher in ME/CFS patients than controls, while CD38+, CD8+CD38+, CD8+HLA-DR+ and CD38+HLA-DR+ were significantly higher in ME/CFS than CF. The percentage of CD19+ cells and the CD4+/CD8+ ratio were significantly lower in ME/CFS and CF than in controls. There were highly significant inverse correlations between the increased expression of CD38+, especially that of CD8+CD38+, and the lowered CD4+/CD8+ ratio and CD19+ expression. There were no significant associations between the flow cytometric results and severity or duration of illness and peripheral blood biomarkers of oxidative and nitrosative stress (O&NS, i.e. IgM responses to O&N modified epitopes), leaky gut (IgM or IgA responses to LPS of gut commensal bacteria), cytokines (interleukin-1, tumor necrosis factor-α), neopterin, lysozyme and autoimmune responses to serotonin. The results support that a) increased CD38 and HLA-DR expression on CD8+ T cells are biomarkers of ME/CFS; b) increased CD38 antigen expression may contribute to suppression of the CD4+/CD8+ ratio and CD19+ expression; c) there are different immune subgroups of ME/CFS patients, e.g. increased CD8+ activation marker expression versus inflammation or O&NS processes; and d) viral infections or reactivation may play a role in a some ME/CFS patients.

IL-25 promotes the function of CD4+CD25+ T regulatory cells and prolongs skin-graft survival in murine models.

PubMed

Tang, Jiayou; Zhou, Xiaohui; Liu, Jie; Meng, Qingshu; Han, Yang; Wang, Zhulin; Fan, Huimin; Liu, Zhongmin

2015-10-01

Interleukin (IL)-25, also known as IL-17E, belongs to the IL-17 family of cytokines. Unlike other IL-17 family members, IL-25 promotes Th2-type immune responses, stimulating IL-4, IL-5, and IL-13 production. Here, we employed murine models of skin graft to explore the role of IL-25 in suppression of graft rejection. We found that IL-25 expression is increased during allograft rejection, and allograft rejection was enhanced in IL-25 KO mice. IL-25 KO was associated with down-regulation of Foxp3 expression in CD4+ T cells. Further, while adoptive transfer of WT regulatory T cells (Tregs) protected against allograft rejection, adoptive transfer of IL-25 deficient Tregs failed to protect against allograft rejection. Exogenous IL-25 restored Foxp3 expression and Treg function in vitro. Moreover, IL-25 promoted phosphorylation of NFAT2. Thus, IL-25 may enhance Treg function by up-regulating NFAT2 phosphorylation. Our findings suggest that IL-25 can sustain Foxp3 expression, enhance the suppressive function of Tregs, and prolong skin-graft survival. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of CD10 predicts tumor progression and unfavorable prognosis in malignant melanoma.

PubMed

Oba, Junna; Nakahara, Takeshi; Hayashida, Sayaka; Kido, Makiko; Xie, Lining; Takahara, Masakazu; Uchi, Hiroshi; Miyazaki, Shogo; Abe, Takeru; Hagihara, Akihito; Moroi, Yoichi; Furue, Masutaka

2011-12-01

CD10 expression in malignant melanoma (MM) has been reported to increase according to tumor progression and metastasis; however, its association with patient outcome has not been clarified. We examined the immunohistochemical expression of CD10 in MM to determine whether or not it could serve as a marker for tumor progression and prognosis. A total of 64 formalin-fixed, paraffin-embedded samples of primary MM were immunostained for CD10. Similarly, 40 samples of melanocytic nevus and 20 of metastatic MM were analyzed for comparison. The following clinicopathologic variables were evaluated: age, gender, histologic type, tumor site, Breslow thickness, Clark level, the presence or absence of ulceration and tumor-infiltrating lymphocytes, and survival. Statistical analyses were performed to assess for associations. Several parameters were analyzed for survival using the Kaplan-Meier method and Cox proportional hazards model. Immunohistochemical analysis revealed that 34 of 64 cases (53%) of primary MM expressed CD10, compared with 15 of 20 cases (75%) of metastatic MM and only 4 of 40 cases (10%) of nevus. There was a significant positive relationship between CD10 expression and Breslow thickness, Clark level, and ulceration. Univariate analysis revealed 4 significant factors for shorter survival periods: CD10 expression, high Breslow thickness, high Clark level, and the presence of ulceration (P < .01 each). In multivariate analysis, CD10 expression was revealed to be a statistically significant and independent prognostic factor. The major limitation was the small sample size. CD10 expression may serve as a progression marker and can predict unfavorable prognosis in patients with MM. Copyright © 2010 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

PubMed Central

Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni

2017-01-01

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability. PMID:29155846

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

PubMed

Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni; Lupia, Enrico

2017-01-01

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Gut memories do not fade: epigenetic regulation of lasting gut homing receptor expression in CD4+ memory T cells.

PubMed

Szilagyi, B A; Triebus, J; Kressler, C; de Almeida, M; Tierling, S; Durek, P; Mardahl, M; Szilagyi, A; Floess, S; Huehn, J; Syrbe, U; Walter, J; Polansky, J K; Hamann, A

2017-11-01

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor α 4 β 7 was found in CD8 + T cells, questioning the concept. We now demonstrate that α 4 β 7 expression in murine CD4 + memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced α 4 β 7 . A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of α 4 β 7 expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by α 4 β 7 is imprinted in CD4 + memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.

Developmental Changes in Soluble CD40 Ligand

PubMed Central

Cholette, Jill M.; Blumberg, Neil; Phipps, Richard P.; McDermott, Michael P.; Gettings, Kelly F.; Lerner, Norma B.

2008-01-01

Objectives To determine if soluble CD40 ligand (sCD40L; formally CD154) levels vary with age and to identify age-dependent ranges in healthy pediatric and adult populations. Study design sCD40L was measured in 25 neonates, 74 children (3 months –15 years) and 20 adults using an enzyme-linked immunosorbent assay. For age group comparisons, Mann-Whitney tests were performed. Correlation coefficients assessed relationships between plasma and serum sCD40L. Results Plasma sCD40L levels were higher in neonates than in all other age groups, (p<0.001). All grouped pediatric plasma levels were significantly higher than in adults (p<0.0001). There were no significant differences in plasma sCD40L between pediatric age groups. Serum levels were significantly higher in neonates than in any other age group (p <0.0001). Pediatric and adult serum sCD40L levels were not significantly different. Conclusions Plasma sCD40L levels are highest at birth and remain higher than those in adults throughout childhood. Reasons for such developmental changes remain to be investigated. Age appropriate reference ranges should be used when sCD40L is being evaluated in pediatric disorders. PMID:18154898

Neighbor of Punc E 11: expression pattern of the new hepatic stem/progenitor cell marker during murine liver development.

PubMed

Schievenbusch, Stephanie; Sauer, Elisabeth; Curth, Harald-Morten; Schulte, Sigrid; Demir, Münevver; Toex, Ulrich; Goeser, Tobias; Nierhoff, Dirk

2012-09-20

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

1α,25-dihydroxyvitamin D3 acts via transforming growth factor-β to up-regulate expression of immunosuppressive CD73 on human CD4+ Foxp3- T cells.

PubMed

Mann, Elizabeth H; Chambers, Emma S; Chen, Yin-Huai; Richards, David F; Hawrylowicz, Catherine M

2015-11-01

Vitamin D deficiency is associated with increased incidence and severity of various immune-mediated diseases. Active vitamin D (1α,25-dihydroxyvitamin D3; 1,25(OH)2 D3) up-regulates CD4(+) T-cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti-inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)2 D3 on expression of the downstream ecto-5'-nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor-β (TGF-β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10(-8) to 10(-7) m, 1,25(OH)2 D3 significantly increased expression of CD73 on peripheral human CD4(+) T cells. Although 1,25(OH)2 D3 did not affect the mRNA expression of latent TGF-β1 , 1,25(OH)2 D3 did up-regulate expression of TGF-β-associated molecules [latency-associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin-1, thrombospondin-1 and αv integrin] which is likely to have contributed to the observed enhancement in TGF-β bioactivity. CD73 was highly co-expressed with LAP and GARP following 1,25(OH)2 D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4(+) Foxp3(-) T cells, rather than CD4(+) Foxp3(+) T cells. Notably, neutralization of TGF-β significantly impaired 1,25(OH)2 D3-mediated induction of CD73. Collectively, we show that 1,25(OH)2 D3 enhances expression of CD73 on CD4(+) Foxp3(-) T cells in a process that is at least partially TGF-β-dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune-mediated disease. © 2015 John Wiley & Sons Ltd.

Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial.

PubMed

Wayne, Alan S; Kreitman, Robert J; Findley, Harry W; Lew, Glen; Delbrook, Cynthia; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Fitzgerald, David J; Pastan, Ira

2010-03-15

Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. Lymphoblasts from children with B-lineage ALL were assessed for CD22 expression by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non-Hodgkin lymphoma. All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC(50), 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 microg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood.

Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells.

PubMed

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Samiee, Shahram; Ataee, Zahra; Tabei, Seyyed Ziyaoddin; Moazzeni, Seyed Mohammad

2009-01-01

RNA interference (RNAi) is an exciting mechanism for knocking down any target gene in transcriptional level. It is now clear that small interfering RNA (siRNA), a 19-21nt long dsRNA, can trigger a degradation process (RNAi) that specifically silences the expression of a cognate mRNA. Our findings in this study showed that down regulation of CD40 gene expression in dendritic cells (DCs) by RNAi culminated to immune modulation. Effective delivery of siRNA into DCs would be a reasonable method for the blocking of CD40 gene expression at the cell surface without any effect on other genes and cell cytotoxicity. The effects of siRNA against CD40 mRNA on the function and phenotype of DCs were investigated. The DCs were separated from the mice spleen and then cultured in vitro. By the means of Lipofectamine2000, siRNA was delivered to the cells and the efficacy of transfection was estimated by flow cytometry. By Annexine V and Propidium Iodide staining, we could evaluate the transfected cells viability. Also, the mRNA expression and protein synthesis were assessed by real-time PCR and flow cytometry, respectively. Knocking down the CD40 gene in the DCs caused an increase in IL-4 production, decrease in IL-12 production and allostimulation activity. All together, these effects would stimulate Th2 cytokines production from allogenic T-cells in vitro.

CD44 expression is related to poor prognosis of hypopharyngeal squamous cell carcinoma.

PubMed

Uwa, Nobuhiro; Kataoka, Tatsuki R; Torii, Ikuko; Sato, Ayuko; Nishigami, Takashi; Song, Misa; Daimon, Takashi; Saeki, Nobuo; Sagawa, Kousuke; Mouri, Takeshi; Terada, Tomonori; Sakagami, Masafumi; Tsujimura, Tohru

2011-03-01

CD44 expression in hypopharyngeal squamous cell carcinomas (SCCs) is closely associated with poor prognosis for patients. CD44 may serve as a prognostic marker for hypopharyngeal SCCs. CD44, an adhesion molecule binding to extracellular matrix, is believed to participate in the progression of malignancies. To clarify the role of CD44 in the progression of hypopharyngeal SCCs, we examined CD44 expression in relation to clinical parameters in hypopharyngeal SCCs. Biopsy specimens of hypopharyngeal SCCs were collected from 40 untreated patients, and their CD44 expression was examined immunohistochemically. Hypopharyngeal SCCs were classified into two groups: CD44-low SCCs comprising < 50% CD44-positive tumor cells and CD44-high SCCs comprising ≥ 50% CD44-positive tumor cells. The relation between CD44 expression and various parameters (clinical T and N stages, distant metastasis, and pathological T and N stages) was analyzed by Fisher's exact test. The relation between CD44 expression and the 5-year disease-free survival (DFS) rate was also analyzed by log rank test. The CD44 expression in hypopharyngeal SCCs was related to pathological N stage, but not to clinical T and N stages and pathological T stage, of the patients. Distant metastasis during the follow-up occurred more frequently in patients with CD44-high SCCs than those with CD44-low SCCs. The 5-year DFS was significantly lower in the former than in the latter.

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

PubMed

Akhmetzyanova, Ilseyar; Drabczyk, Malgorzata; Neff, C Preston; Gibbert, Kathrin; Dietze, Kirsten K; Werner, Tanja; Liu, Jia; Chen, Lieping; Lang, Karl S; Palmer, Brent E; Dittmer, Ulf; Zelinskyy, Gennadiy

2015-10-01

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.

Shikonin inhibits maturation of bone marrow-derived dendritic cells and suppresses allergic airway inflammation in a murine model of asthma

PubMed Central

Lee, Chen-Chen; Wang, Chien-Neng; Lai, Yu-Ting; Kang, Jaw-Jou; Liao, Jiunn-Wang; Chiang, Bor-Luen; Chen, Hui-Chen; Cheng, Yu-Wen

2010-01-01

BACKGROUND AND PURPOSE Shikonin exhibits a wide range of anti-inflammatory actions. Here, we assessed its effects on maturation of murine bone marrow-derived dendritic cells (BM-DCs) and on allergic reactions in a murine model of asthma. EXPERIMENTAL APPROACH Cultured murine BM-DCs were used to investigate the effects of shikonin on expression of cell surface markers and their stimulation of T-cell proliferation and cytokine production. The therapeutic potential of shikonin was evaluated in a model of allergic airway disease. KEY RESULTS Shikonin dose-dependently inhibited expression of major histocompatibility complex class II, CD80, CD86, CCR7 and OX40L on BM-DCs, induced by a mixture of ovalbumin (OVA; 100 µg·mL−1) and thymic stromal lymphopoietin (TSLP; 20 ng·mL−1). Shikonin-treated BM-DCs were poor stimulators of CD4+ T lymphocyte and induced lower levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor (TNF)-α release by responding T-cells. After intratracheal instillation of shikonin in OVA-immunized mice, OVA challenge induced lower IL-4, IL-5, IL-13, TNF-α and eotaxin release in bronchial alveolar lavage fluid, lower IL-4 and IL-5 production in lung cells and mediastinal lymph node cells and attenuated OVA-induced lung eosinophilia and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS Shikonin effectively suppressed OVA + TSLP-induced BM-DC maturation in vitro and inhibited allergic inflammation and airway hyperresponsiveness in a murine model of asthma, showing good potential as a treatment for allergic asthma. Also, our model provides a novel platform for screening drugs for allergic diseases. PMID:20735407

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

PubMed

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

PubMed Central

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

In vivo near-infrared fluorescence imaging of CD105 expression during tumor angiogenesis.

PubMed

Yang, Yunan; Zhang, Yin; Hong, Hao; Liu, Glenn; Leigh, Bryan R; Cai, Weibo

2011-11-01

Angiogenesis is an indispensable process during tumor development. The currently accepted standard method for quantifying tumor angiogenesis is to assess microvessel density (MVD) based on CD105 staining, which is an independent prognostic factor for survival in patients with most solid tumor types. The goal of this study is to evaluate tumor angiogenesis in a mouse model by near-infrared fluorescence (NIRF) imaging of CD105 expression. TRC105, a human/murine chimeric anti-CD105 monoclonal antibody, was conjugated to an NIRF dye (IRDye 800CW; Ex: 778 nm; Em: 806 nm). FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and 800CW-TRC105. In vivo/ex vivo NIRF imaging, blocking studies, and ex vivo histology were performed on 4T1 murine breast tumor-bearing mice to evaluate the ability of 800CW-TRC105 to target tumor angiogenesis. Another chimeric antibody, cetuximab, was used as an isotype-matched control. FACS analysis of human umbilical vein endothelial cells (HUVECs) revealed no difference in CD105 binding affinity between TRC105 and 800CW-TRC105, which was further validated by fluorescence microscopy. 800CW conjugation of TRC105 was achieved in excellent yield (> 85%), with an average of 0.4 800CW molecules per TRC105. Serial NIRF imaging after intravenous injection of 800CW-TRC105 revealed that the 4T1 tumor could be clearly visualized as early as 30 min post-injection. Quantitative region of interest (ROI) analysis showed that the tumor uptake peaked at about 16 h post-injection. Based on ex vivo NIRF imaging at 48 h post-injection, tumor uptake of 800CW-TRC105 was higher than most organs, thus providing excellent tumor contrast. Blocking experiments, control studies with 800CW-cetuximab and 800CW, as well as ex vivo histology all confirmed the in vivo target specificity of 800CW-TRC105. This is the first successful NIRF imaging study of CD105 expression in vivo. Fast, prominent, persistent, and CD105-specific

CD52, CD22, CD26, EG5 and IGF-1R expression in thymic malignancies.

PubMed

Remon, J; Abedallaa, N; Taranchon-Clermont, E; Bluthgen, V; Lindsay, C R; Besse, B; Thomas de Montpréville, V

2017-06-01

Thymic epithelial tumours are rare cancers for which new treatment options are required. Identification of putative predictive markers is important for developing clinical trials. We studied the expression of five putative predictive biomarkers, potentially actionable by approved experimental drugs. CD52, CD22, CD26, EG5, and IGF-1R expression were investigated by immunohistochemistry in formalin-fixed surgical samples of thymic epithelial tumour patients. All samples containing 10% positive epithelial tumour cells, independent of tumour cell intensity, were considered as positive. Correlation with histological subtype was performed. 106 surgical samples (89 thymomas, 12 thymic carcinoma, and 5 thymic neuroendocrine tumours) were evaluated. Overall, CD52, CD22, CD26, EG5 and IGF-1R expression was observed in 7%, 42%, 25%, 42% and 77% of samples, respectively. CD52 expression was more frequent in B2 and B3 thymoma. All TET subtypes stained for CD22, mainly AB thymoma (68%). CD26 expression also correlated with AB thymoma (68%), and A thymoma (50%) subtype, while IGFR1 was the most common marker expressed by thymic carcinoma samples (92%), followed by EG5 (60%). Only EG5 expression was significantly higher in thymic carcinomas than in thymomas (75% vs. 38%, p=0.026). Our data were consistent with a previous study of IGF-1R expression. Based on their expression, activity of agents targeting CD52, CD 22, CD26 and EG5 could be further explored in TET patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

PubMed

Eshima, Koji; Misawa, Kana; Ohashi, Chihiro; Iwabuchi, Kazuya

2018-05-01

Although CD4 + T cells are generally regarded as helper T cells, some activated CD4 + T cells have cytotoxic properties. Given that CD4 + cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4 + T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4 + T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4 + T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4 + T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4 + T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas - target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Incorporation of CD40 ligand enhances the immunogenicity of tumor‑associated calcium signal transducer 2 virus‑like particles against lung cancer.

PubMed

Xi, Wang; Ke, Dong; Min, Long; Lin, Wang; Jiahui, Zuo; Fang, Lin; Zhaowei, Gao; Zhe, Zhang; Xi, Chen; Huizhong, Zhang

2018-06-01

The cell surface glycoprotein Trop‑2 is overexpressed in various types of cancer, including in lung cancer, and has recently been used as an effective immunotherapeutic target. CD40 ligand (CD40L), a tumor necrosis factor superfamily member, is a promising immune adjuvant. Human immunodeficiency virus (HIV) gag‑based virus‑like particles (VLPs) are highly immunogenic, and foreign antigens can be incorporated onto their membrane envelope for cancer vaccine development. In the present study, a HIV gag‑based VLP strategy and Bac‑to‑Bac system were utilized to construct Trop‑2, CD40L and gag recombinant baculoviruses, which were then used to infect TN5 cells in order to form Trop‑2 VLPs or Trop‑2‑CD40L VLPs. These VLPs were characterized using transmission electron microscopy and western blot analysis methods. VLPs incorporating murine Trop‑2 only or incorporating Trop‑2 and CD40L were used to immunize C57BL/6 mice. Immunized mice demonstrated high humoral and cellular immunity responses, whereas the Trop‑2‑CD40L VLPs led to higher immune responses in comparison with Trop‑2 only VLPs. Immunization with Trop‑2‑CD40L VLPs also reduced tumor growth more effectively compared with Trop‑2 VLPs. Furthermore, Trop‑2‑CD40L VLP immunization increased the survival rate of Lewis tumor‑bearing mice more significantly when compared with Trop‑2 only VLPs. In conclusion, the present study provided a novel vaccine design by combination of a tumor antigen and an immune adjuvant based on a VLP strategy, which may be potentially applied as an alternative immunotherapeutic option in the treatment of lung cancer.

[Establishment of the retrovirus-mediated murine model with MLL-AF9 leukemia].

PubMed

Xu, Si-Miao; Yang, Yang; Zhou, Mi; Zhao, Xue-Jiao; Qin, Yu; Zhang, Pei-Ling; Yuan, Rui-Feng; Zhou, Jian-Feng; Fang, Yong

2013-10-01

This study was purposed to establish a retrovirus-mediated murine model with MLL-AF9 leukemia, so as to provide a basis for further investigation of the pathogenesis and therapeutic strategy of MLL associated leukemia. Murine (CD45.2) primary hematopoietic precursor positively selected for expression of the progenitor marker c-Kit by means of MACS were transduced with a retrovirus carrying MLL-AF9 fusion gene. After cultured in vitro, the transduced cells were injected intravenously through the tail vein into the lethally irradiated mice (CD45.1). PCR, flow cytometry and morphological observation were employed to evaluate the murine leukemia model system. The results showed that MLL-AF9 fusion gene was expressed in the infected cells, and the cells had a dramatically enhanced potential to generate myeloid colonies with primitive and immature morphology. Flow cytometric analysis revealed that the immortalized cells highly expressed myeloid lineage surface markers Gr-1 and Mac-1. Moreover, the expression levels of Hoxa9 and Meis1 mRNA were significantly higher in the MLL-AF9 cells than that in control. The mice transplanted with MLL-AF9 cells displayed typical signs of leukemia within 6-12 weeks. Extensive infiltration leukemic cells was observed in the Wright-Giemsa stained peripheral blood smear and bone marrow, and also in the histology of liver and spleen. Flow cytometric analysis of the bone marrow and spleen cells demonstrated that the CD45.2 populations expressed highly myeloid markers Gr-1 and Mac-1. The leukemic mice died within 12 weeks. It is concluded that the retrovirus-mediated murine model with MLL-AF9 leukemia is successfully established, which can be applied in the subsequent researches.

Citrus hallabong [(Citrus unshiu × C. sinensis) × C. reticulata)] exerts potent anti-inflammatory properties in murine splenocytes and TPA-induced murine ear oedema model.

PubMed

Herath, Kalahe Hewage Iresha Nadeeka Madushani; Bing, So Jin; Cho, Jinhee; Kim, Areum; Kim, Gi-Ok; Lee, Jong-Chul; Jee, Youngheun

2016-12-01

Hallabong [(Citrus unshiu × C. sinensis) X C. reticulata)] (Rutaceae) is a hybrid citrus cultivated in temperate regions of South Korea. Its fruit is well-known for pharmacological properties. This study examined the anti-inflammatory effect of 80% ethanol extract of Hallabong (HE) on concanavalin A (Con A)-stimulated splenocytes and mouse oedema model induced by 12-O-tetradecanoylphorbal acetate (TPA). Murine splenocytes treated with HE were stimulated with Con A (10 μg/mL, for 24 h) were evaluated for T-cell population and production of inflammatory cytokines IL-2, IL-4 and IFN-γ. Anti-inflammatory effect of topically applied HE (100 μg/20 μL) on TPA (4 μg/20 μL/ear)-induced ear oedema was investigated in mouse model. HE-treated Con A-stimulated murine splenocytes showed a marked decrease in CD44/CD62L + memory T-cell population, an important marker for anti-inflammatory activity, and a significant inhibition in the production of IL-2 and IFN-γ. HE treatment had reduced the mouse skin oedema (47%) and myeloperoxidase (MPO) activity significantly (40%) in TPA-challenged tissues. More importantly, immunohistochemical localization revealed the suppressed (p < 0.05) expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX2). HE decreased the infiltration of CD3 + T cells and F4/80 + macrophages to the site of inflammation and a topical application of HE significantly suppressed the expression of TNF-α (20.2%). A topical application of HE can exert a potential anti-inflammatory effect and HE can be explored further as a putative alternative therapeutic agent for inflammatory oedema.

Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

PubMed Central

Koguchi, Yoshinobu; Gardell, Jennifer L.; Thauland, Timothy J.; Parker, David C.

2011-01-01

CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery. PMID:21677130

A novel CD4-CD8alpha+CD205+CD11b- murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis.

PubMed

Ruiz, Sophie; Beauvillain, Céline; Mévélec, Marie-Noëlle; Roingeard, Philippe; Breton, Pascal; Bout, Daniel; Dimier-Poisson, Isabelle

2005-11-01

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8alpha(+) DC line (H-2(k)) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4(-)CD8alpha(+)CD205(+)CD11b(-) DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen-pulsed SRDCs, which synthesize large amounts of interleukin-12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2-type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4(-)CD8alpha(+)CD205(+)CD11b(-) DC line can be expected to be a very useful tool for immunobiology studies of DC.

Soluble CD40 Ligand Stimulates the Pro-Angiogenic Function of Peripheral Blood Angiogenic Outgrowth Cells via Increased Release of Matrix Metalloproteinase-9

PubMed Central

Bou Khzam, Lara; Boulahya, Rahma; Abou-Saleh, Haissam; Hachem, Ahmed; Zaid, Younes; Merhi, Yahye

2013-01-01

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway. PMID:24358353

Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9.

PubMed

Bou Khzam, Lara; Boulahya, Rahma; Abou-Saleh, Haissam; Hachem, Ahmed; Zaid, Younes; Merhi, Yahye

2013-01-01

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway.

Evaluation of Microvascularity by CD34 Expression in Esophagus and Oral Squamous Cell Carcinoma.

PubMed

Shahsavari, Fatemeh; Farhadi, Sareh; Sadri, Donia; Sedehi, Marzieh

2015-06-01

The present study was scheduled to evaluate microvascularity by CD34 expression in esophagus and oral squamous cell carcinoma. This study was scheduled using 40 paraffin blocked samples including 20 of oral SCC and 20 of esophagus ones and Immunohistochemical staining was conducted using CD34 monoclonal antibody. Exact fisher test was used to evaluate frequency of expression between two studied groups. There was significant correlation between age and tumor size with CD34 expression in oral SCC samples (p < 0.05) and no significant correlation between sex and tumor differentiation level (grading) (p > 0.05). Also, there was no significant correlation between age, sex, tumor size and tumor differentiation level (grading) with CD34 expression in esophagus SCC samples (p > 0.05). There was no significant difference of CD34 expression frequency in oral and esophagus SCC (p = 0/583). Finally, CD34 expression was reported 'high' for major cases of esophagus and oral SCCs. It seems, other angiogenetic or nonangiogenetic factors except CD34 may play more important role and explain the different clinical behavior of SCC at recent different locations. Other factors would be considered along with CD34 expression to interpret different clinical behavior of SCC at recent different locations.

Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression

PubMed Central

Moyo, Nathifa A; Marchi, Emanuele; Steinbach, Falko

2013-01-01

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system. PMID:23461413

CD40-CD40 Ligand Pathway is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis.

PubMed

Michels, Monique; Danieslki, Lucinéia Gainski; Vieira, Andriele; Florentino, Drielly; Dall'Igna, Dhébora; Galant, Letícia; Sonai, Beatriz; Vuolo, Francieli; Mina, Franciele; Pescador, Bruna; Dominguini, Diogo; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Petronilho, Fabrícia

2015-03-26

Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40-CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40-CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40-CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40-CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis.

Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway

PubMed Central

Wang, Xiao-Lin; Zhou, Yuan-Li; Sun, Wei; Li, Li

2016-01-01

CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques. PMID:27120457

Human CD1c+ dendritic cells drive the differentiation of CD103+ CD8+ mucosal effector T cells via the cytokine TGF-β

PubMed Central

Yu, Chun I; Becker, Christian; Wang, Yuanyuan; Marches, Florentina; Helft, Julie; Leboeuf, Marylene; Anguiano, Esperanza; Pourpe, Stephane; Goller, Kristina; Pascual, Virginia; Banchereau, Jacques; Merad, Miriam; Palucka, Karolina

2013-01-01

Summary In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, using lung tissues from humans and humanized mice, the role of human CD1c+ and CD141+ DCs in determining the type of CD8+ T cell immunity generated to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8+ T cells in vitro. However, lung-tissue-resident CD1c+ DCs but not CD141+ DCs were able to drive CD103 expression on CD8+ T cells and promoted CD8+ T cell accumulation in lung epithelia in vitro and in vivo. CD1c+ DCs induction of CD103 expression was dependent on membrane-bound cytokine TGF-β1. Thus, CD1c+ and CD141+ DCs generate CD8+ T cells with different properties, and CD1c+ DCs specialize in the regulation of mucosal CD8+ T cells. PMID:23562160

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

PubMed

Nagamata, Satoshi; Nagasaka, Miwako; Kawabata, Akiko; Kishimoto, Kenji; Hasegawa, Daiichiro; Kosaka, Yoshiyuki; Mori, Takeshi; Morioka, Ichiro; Nishimura, Noriyuki; Iijima, Kazumoto; Yamada, Hideto; Kawamoto, Shinichiro; Yakushijin, Kimikazu; Matsuoka, Hiroshi; Mori, Yasuko

2018-05-01

CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4 + and CD8 + cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4 + T cells, p = .02 in CD8 + T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4 + T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT. Copyright © 2018 Elsevier B.V. All rights reserved.

Combination therapy with an OX40L fusion protein and a vaccine targeting the transcription factor twist inhibits metastasis in a murine model of breast cancer.

PubMed

Malamas, Anthony S; Hammond, Scott A; Schlom, Jeffrey; Hodge, James W

2017-10-31

OX40 is a costimulatory receptor that potentiates proliferation, survival, memory formation, and effector function of CD4 + and CD8 + T-cells, while overcoming the suppressive activity of regulatory T-cells (Tregs). Here, we explored the combination of an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) to inhibit tumor metastasis in the 4T1 murine breast cancer model. Contrary to the single agent treatments, the combination therapy significantly decreased the number of metastatic colonies per lung and prolonged survival. Depletion studies demonstrated that these effects were mediated by both CD4 + and CD8 + T-cells. The combination therapy a) increased the total number of T-cells in the CD4 + Foxp3 - population and the CD4 + central and effector memory subsets within the lung, spleen, and draining lymph node, b) enhanced infiltration of CD4 + T-cells into metastatic areas of the lung, and (c) increased the number of functional CD8 + T-cells that produced IFNγ and TNFα. The combination therapy also promoted the development of KLRG1 - CD127 + memory precursor CD8 + T-cells, while reducing those with a KLRG1 + terminally differentiated phenotype. Moreover, the combination of OX40L-FP and vaccine induced greater CD4 + and CD8 + Twist-specific responses. In addition, Tregs isolated from mice receiving the combination were also less immunosuppressive in ex-vivo proliferation assays than those from the OX40L-FP and MVA-Twist-TRICOM monotherapy groups. Such results provide the rationale to combine co-stimulatory agonists with cancer vaccines for the treatment of tumor metastasis.

Combination therapy with an OX40L fusion protein and a vaccine targeting the transcription factor twist inhibits metastasis in a murine model of breast cancer

PubMed Central

Malamas, Anthony S.; Hammond, Scott A.; Schlom, Jeffrey; Hodge, James W.

2017-01-01

OX40 is a costimulatory receptor that potentiates proliferation, survival, memory formation, and effector function of CD4+ and CD8+ T-cells, while overcoming the suppressive activity of regulatory T-cells (Tregs). Here, we explored the combination of an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) to inhibit tumor metastasis in the 4T1 murine breast cancer model. Contrary to the single agent treatments, the combination therapy significantly decreased the number of metastatic colonies per lung and prolonged survival. Depletion studies demonstrated that these effects were mediated by both CD4+ and CD8+ T-cells. The combination therapy a) increased the total number of T-cells in the CD4+Foxp3- population and the CD4+ central and effector memory subsets within the lung, spleen, and draining lymph node, b) enhanced infiltration of CD4+ T-cells into metastatic areas of the lung, and (c) increased the number of functional CD8+ T-cells that produced IFNγ and TNFα. The combination therapy also promoted the development of KLRG1-CD127+ memory precursor CD8+ T-cells, while reducing those with a KLRG1+ terminally differentiated phenotype. Moreover, the combination of OX40L-FP and vaccine induced greater CD4+ and CD8+ Twist-specific responses. In addition, Tregs isolated from mice receiving the combination were also less immunosuppressive in ex-vivo proliferation assays than those from the OX40L-FP and MVA-Twist-TRICOM monotherapy groups. Such results provide the rationale to combine co-stimulatory agonists with cancer vaccines for the treatment of tumor metastasis. PMID:29207606

CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

PubMed

Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

2017-02-01

Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

PubMed Central

MIRANDA-HERNÁNDEZ, D.F.; FRANCO-MOLINA, M.A.; MENDOZA-GAMBOA, E.; ZAPATA-BENAVIDES, P.; SIERRA-RIVERA, C.A.; CORONADO-CERDA, E.E.; ROSAS-TARACO, A.G.; TAMÉZ-GUERRA, R.S.; RODRÍGUEZ-PADILLA, C.

2013-01-01

The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-β cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression. PMID:24179494

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

PubMed

Ramonell, Kimberly M; Zhang, Wenxiao; Hadley, Annette; Chen, Ching-Wen; Fay, Katherine T; Lyons, John D; Klingensmith, Nathan J; McConnell, Kevin W; Coopersmith, Craig M; Ford, Mandy L

2017-01-01

Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

Murine Polyomavirus Virus-Like Particles Carrying Full-Length Human PSA Protect BALB/c Mice from Outgrowth of a PSA Expressing Tumor

PubMed Central

Eriksson, Mathilda; Andreasson, Kalle; Weidmann, Joachim; Lundberg, Kajsa; Tegerstedt, Karin

2011-01-01

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies. PMID:21858228

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, bothmore » sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.« less

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

PubMed

Bower, Joseph F; Green, Thomas D; Ross, Ted M

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

CD40 AND THE IMUNE RESPONSE TO PARASITIC INFECTIONS

PubMed Central

Subauste, Carlos S.

2009-01-01

The interaction between CD40 and CD154 regulates many aspects of cellular and humoral immunity. The CD40 — CD154 pathway is important for resistance against a variety of parasites. Studies done with these pathogens have provided important insight into the various mechanisms by which this pathway enhances host protection, mechanisms by which pathogens subvert CD40 signaling, conditions in which the CD40 — CD154 pathway promotes disease and on modulation of this pathway for immunotherapy. PMID:19616968

CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism.

PubMed

Wilhelm, Annika; Aldridge, Victoria; Haldar, Debashis; Naylor, Amy J; Weston, Christopher J; Hedegaard, Ditte; Garg, Abhilok; Fear, Janine; Reynolds, Gary M; Croft, Adam P; Henderson, Neil C; Buckley, Christopher D; Newsome, Philip N

2016-07-01

CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chattopadhyay, K.; Ramagopal, U; Nathenson, S

2009-01-01

Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with themore » murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.« less

CD40–CD40 Ligand Pathway Is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis

PubMed Central

Michels, Monique; Danieslki, Lucinéia Gainski; Vieira, Andriele; Florentino, Drielly; Dall’Igna, Dhébora; Galant, Letícia; Sonai, Beatriz; Vuolo, Francieli; Mina, Franciele; Pescador, Bruna; Dominguini, Diogo; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Petronilho, Fabrícia

2015-01-01

Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40–CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40–CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40–CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40–CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis. PMID:25822797

Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells.

PubMed

Ma, Hak-Ling; Napierata, Lee; Stedman, Nancy; Benoit, Stephen; Collins, Mary; Nickerson-Nutter, Cheryl; Young, Deborah A

2010-02-01

Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor alpha (TNFalpha) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNFalpha blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNFalpha blockade-induced exacerbation of skin inflammation in murine psoriasis-like skin disease. Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RB(high)CD25- (naive CD4) T cells from donor mice. These mice were treated with either anti-interleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNFalpha. Cytokine gene expression from these differentiated cells was also determined. Neutralization of TNFalpha exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1beta, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNFalpha also demonstrated a divergent role during priming and reactivation of naive T cells. These results reveal a novel immunoregulatory role of TNFalpha on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.

Lactoferrin Expression in Human and Murine Ocular Tissue.

PubMed

Rageh, Abrar A; Ferrington, Deborah A; Roehrich, Heidi; Yuan, Ching; Terluk, Marcia R; Nelson, Elizabeth F; Montezuma, Sandra R

2016-07-01

Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

ImmunoPET Imaging of Murine CD4+ T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging.

PubMed

Freise, Amanda C; Zettlitz, Kirstin A; Salazar, Felix B; Lu, Xiang; Tavaré, Richard; Wu, Anna M

2017-08-01

Molecular imaging of CD4 + T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4 + T cell viability, proliferation, CD4 expression, and function. The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89 Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed. For immunoPET imaging, the lowest protein dose of 2 μg of 89 Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4 + T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4 + T cells. Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4 + T cells in the context of

Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes.

PubMed

Castan, J; Klauenberg, U; Kalmár, P; Fleischer, B; Bröker, B M

1998-06-01

CTLA-4 (CD152) is a T cell surface receptor with sequence homology to the co-stimulatory molecule CD28. The molecule, which is essential for the inhibitory regulation of the immune response, becomes transiently expressed on mature T cells after stimulation in vitro. In situ, CTLA-4+ T cells are enriched in the light zones of the germinal centers in human peripheral lymphoid organs. In this study we have studied expression of CTLA-4 in human thymus in situ. CTLA-4 was expressed on about one third of CD4+/CD8-/CD1- medullary thymocytes. CTLA-4 was acquired by a subset of immature (CD1+) thymocytes and lost from the mature (CD1-) subpopulation within 48 h of cell culture, suggesting that the expression on medullary thymocytes is transient. The demonstration of CTLA-4 on a substantial subpopulation of mature CD4+ thymocytes adds a new dimension to the understanding of this important molecule. When contemplating application of anti-CTLA-4 for therapy its potential influence on T cell maturation has to be taken into account.

Virus-specific CD8+ T cells infiltrate melanoma lesions and retain function independent of PD-1 expression

PubMed Central

Erkes, Dan A.; Smith, Corinne J.; Wilski, Nicole A.; Caldeira-Dantas, Sofia; Mohgbeli, Toktam; Snyder, Christopher M.

2017-01-01

It is well known that CD8+ tumor infiltrating lymphocytes (TIL) are correlated with positive prognoses in cancer patients and used to determine efficacy of immune therapies. While it is generally assumed that CD8+ TIL will be tumor associated antigen (TAA)-specific, it is unknown whether CD8+ T cells with specificity for common pathogens also infiltrate tumors. If so, the presence of these T cells could alter the interpretation of prognostic and diagnostic TIL assays. We compared TAA-specific and virus-specific CD8+ T cells in the same tumors using murine cytomegalovirus (MCMV), a herpesvirus that causes a persistent/latent infection, and Vaccinia virus (VacV), a poxvirus that is cleared by the host. Virus-specific CD8+ TIL migrated into cutaneous melanoma lesions during acute infection with either virus, as well as after a cleared VacV infection, and during a persistent/latent MCMV infection. Virus-specific TILs developed independent of viral antigen in the tumor and interestingly, expressed low or intermediate levels of full-length PD-1 in the tumor environment. Importantly, PD-1 expression could be markedly induced by antigen, but did not correlate with dysfunction for virus-specific TIL, in sharp contrast to TAA-specific TIL in the same tumors. These data suggest that CD8+ TIL can reflect an individual's immune status, rather than exclusively representing TAA-specific T cells, and that PD-1 expression on CD8+ TIL is not always associated with repeated antigen encounter or dysfunction. Thus, functional virus-specific CD8+ TIL could skew the results of prognostic or diagnostic TIL assays. PMID:28202614

Vaccination against CD99 inhibits atherogenesis in low-density lipoprotein receptor-deficient mice.

PubMed

van Wanrooij, Eva J A; de Vos, Paula; Bixel, M Gabriele; Vestweber, Dietmar; van Berkel, Theo J C; Kuiper, Johan

2008-06-01

Murine CD99 was recently found to be expressed on leukocytes and endothelial cells, where it is concentrated at inter-endothelial contacts. Blockade of CD99 by specific antibodies inhibits leukocyte extravasation to inflamed sites in vivo. The aim of the present study is to show the role of CD99 in atherosclerosis using a CD99 vaccination protocol to block the function of CD99 during atherosclerosis. We constructed a DNA vaccine against CD99 by cloning the extracellular domain of murine CD99 into pcDNA3. Vaccination was performed by oral administration of attenuated Salmonella typhimurium transformed with pcDNA3-CD99. This vaccination results in a CD99-specific, CD8-mediated cytotoxic response and subsequent reduction of CD99-expressing cells. We showed that CD99 is expressed on vascular endothelium overlying atherosclerotic plaques and found that CD99 expression is upregulated during western-type diet feeding. CD99 vaccination induced the formation of CD8-positive T cells that were cytotoxic against cells transfected with pcDNA3-CD99. Activation of CD8(+) T cells was demonstrated by a 30% increase in CD8(+)CD69(+) double-positive T cells in spleen and mediastinal lymph nodes. Furthermore, lymphocytes isolated from CD99-vaccinated mice specifically lysed CD99-expressing cells. More importantly, vaccination against CD99 attenuated atherosclerotic lesion formation in the aortic valve leaflets by 38% and in the carotid artery by 69% compared with mice that were vaccinated with a control vector. Furthermore, a lower number of cells were found in atherosclerotic lesions, implying that fewer leukocytes were recruited to these sites. These observations were accompanied by a decrease in CD99 expression on leukocytes. We conclude that vaccination against CD99 decreases atherogenesis by the selective removal of CD99-expressing cells, which could reduce leukocyte recruitment into atherosclerotic lesions and attenuate atherogenesis.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models

PubMed Central

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-01-01

Background Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. Methods CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. Results CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4+ and CD8+ T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Conclusions Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. PMID:26917236

CD44s and CD44v6 Expression in Head and Neck Epithelia

PubMed Central

Mack, Brigitte; Gires, Olivier

2008-01-01

Background CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia. Methods Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC. Results In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6. Conclusion CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. PMID:18852874

Involvement of nuclear factor {kappa}B in platelet CD40 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hachem, Ahmed; Yacoub, Daniel; Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1

Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Givenmore » that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against

Phenotypic, molecular, and functional characterization of human peripheral blood CD34+/THY1+ cells.

PubMed

Humeau, L; Bardin, F; Maroc, C; Alario, T; Galindo, R; Mannoni, P; Chabannon, C

1996-02-01

A subset of mobilized CD34+ cells present in patient aphereses expresses Thy1 (CDw90). This population contains most long-term culture initiating cells, as assayed with a murine stromal cell line. It also contains a significant proportion of colony-forming unit granulocyte macrophage, but very few burst-forming unit erythroid. The limited differentiation towards the erythroid lineage is further confirmed by the absence of GATA-1 mRNA in the CD34+/Thy1+ subset, and by the low level of c-kit expression. The CD34+/Thy1+ subset appears phenotypically and functionally heterogeneous, a finding consistent with its high representation, compared to phenotypes such as CD34+/CD38-. Therefore, while at least some of CD34+/Thy1+ cells may be infectable by retroviral vectors, as shown by the presence of a transcript for the receptor for murine amphotropic retroviruses, the use of this selection strategy to specifically target human stem cells appears questionable.

1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu

2009-05-01

1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure,more » murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.« less

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

PubMed

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-06-09

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

Involvement of nuclear factor κB in platelet CD40 signaling.

PubMed

Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-08-17

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Possible role of CD22, CD79b and CD20 expression in distinguishing small lymphocytic lymphoma from chronic lymphocytic leukemia.

PubMed

Jovanovic, Danijela; Djurdjevic, Predrag; Andjelkovic, Nebojsa; Zivic, Ljubica

2014-01-01

Flow cytometry has an important role in diagnosis and classification of B-cell lymphoproliferative disorders (BCLPDs). However, in distinguishing chronic lymphocytic leukemia (CLL) from small lymphocytic lymphoma (SLL) only clinical criteria are available so far. Aim of the study was to determine differences in the expression of common B cell markers (CD22, CD79b and CD20) on the malignant lymphocytes in the peripheral blood samples of CLL and SLL patients. Peripheral blood samples of 56 CLL and 11 SLL patients were analyzed by 5-color flow cytometry on the CD45/CD19/CD5 gate for CD22, CD79b and CD20. In the samples collected from the CLL patients, CD22 expression was detected in only 20% of patients in the low pattern, while in SLL patients the expression was medium and present in 90.9% of patients (p < 0.0001). For CD79b expression, statistical significance is reached both in the expression pattern, which was low/medium for CLL and high for SLL, and expression level (p = 0.006). The expression of CD20 was counted as the CD20/CD19 ratio. The average ratio was 0.512 in the CLL patients vs. 0.931 in the SLL patients (p = 0.0001). The pattern of expression and expression level of CD22, CD79b and CD20 in peripheral blood could be used for distinguishing SLL from CLL patients.

Decreased serum levels of sCD40L and IL-31 correlate in treated patients with Relapsing-Remitting Multiple Sclerosis.

PubMed

de J Guerrero-García, José; Rojas-Mayorquín, Argelia E; Valle, Yeminia; Padilla-Gutiérrez, Jorge R; Castañeda-Moreno, Víctor A; Mireles-Ramírez, Mario A; Muñoz-Valle, José F; Ortuño-Sahagún, Daniel

2018-01-01

The CD40/CD40L system is a binding key for co-stimulation of immune cells. Soluble form of CD40L has been widely studied as marker of inflammatory and autoimmune diseases. Here we analyze serum concentrations of sCD40L, as well as 14 cytokines, in patients with Multiple Sclerosis (MS) treated with Glatiramer acetate or Interferon beta. In the healthy control group, we found in serum a highly positive correlation between sCD40L and Interleukin (IL)-31, an anti-inflammatory Th2 cytokine. Additionally, an important reduction in IL-31 and sCD40L serum levels, as well as a significant reduction in CD40 mRNA expression and complete depletion of CD40L mRNA, detected from peripheral blood cells, was found in treated patients with MS. Therefore, sCD40L and IL-31 must be taken into account as possible prognostic markers when analyzing the disease progress of MS in order to provide more personalized treatment. Copyright © 2017 Elsevier GmbH. All rights reserved.

Interaction of Macrophage Antigen 1 and CD40 Ligand Leads to IL-12 Production and Resistance in CD40-Deficient Mice Infected with Leishmania major.

PubMed

Okwor, Ifeoma; Jia, Ping; Uzonna, Jude E

2015-10-01

Although some studies indicate that the interaction of CD40 and CD40L is critical for IL-12 production and resistance to cutaneous leishmaniasis, others suggest that this pathway may be dispensable. In this article, we compared the outcome of Leishmania major infection in both CD40- and CD40L-deficient mice after treatment with rIL-12. We show that although CD40 and CD40L knockout (KO) mice are highly susceptible to L. major, treatment with rIL-12 during the first 2 wk of infection causes resolution of cutaneous lesions and control of parasite replication. Interestingly, although treated CD40 KO mice remained healed, developed long-term immunity, and were resistant to secondary L. major challenge, treated CD40L KO reactivated their lesion after cessation of rIL-12 treatment. Disease reactivation in CD40L KO mice was associated with impaired IL-12 and IFN-γ production and a concomitant increase in IL-4 production by cells from lymph nodes draining the infection site. We show that IL-12 production by dendritic cells and macrophages via CD40L-macrophage Ag 1 (Mac-1) interaction is responsible for the sustained resistance in CD40 KO mice after cessation of rIL-12 treatment. Blockade of CD40L-Mac-1 interaction with anti-Mac-1 mAb led to spontaneous disease reactivation in healed CD40 KO mice, which was associated with impaired IFN-γ response and loss of infection-induced immunity after secondary L. major challenge. Collectively, our data reveal a novel role of CD40L-Mac-1 interaction in IL-12 production, development, and maintenance of optimal Th1 immunity in mice infected with L. major. Copyright © 2015 by The American Association of Immunologists, Inc.

Bone marrow-derived CD13+ cells sustain tumor progression

PubMed Central

Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2014-01-01

Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b+CD13+ myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b+CD13+ myeloid cells could become a non-malignant target for the development of novel anticancer regimens. PMID:25339996

CD40 Ligand Is Increased in Mast Cells in Psoriasis and Actinic Keratosis but Less So in Epithelial Skin Carcinomas.

PubMed

Haimakainen, Salla; Kaukinen, Antti P; Suttle, Mireille-Maria; Pelkonen, Jukka; Harvima, Ilkka T

2017-03-16

The expression of CD40 ligand (CD40L) in mast cells was investigated in biopsies from lesional and non-lesional skin samples of patients with psoriasis, actinic keratosis (AK), basal cell carcinoma, and squamous cell carcinoma using a sequential double-staining technique. The percentage of CD40L + mast cells was higher in the lesional than in the non-lesional skin (p < .003). Interestingly, this percentage was lower in both carcinomas than in psoriasis and actinic keratosis (p < .025). Cells immunopositive for CD40 receptor were increased in all lesion types but especially so in carcinomas. The results suggest a dysregulated anti-tumoral immune response by mast cell CD40L in skin carcinomas.

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules*

PubMed Central

El Fakhry, Youssef; Alturaihi, Haydar; Yacoub, Daniel; Liu, Lihui; Guo, Wenyan; Leveillé, Claire; Jung, Daniel; Khzam, Lara Bou; Merhi, Yahye; Wilkins, John A.; Li, Hongmin; Mourad, Walid

2012-01-01

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors. PMID:22461623

CD127 and CD25 expression defines CD4+ T cell subsets that are differentially depleted during HIV infection.

PubMed

Dunham, Richard M; Cervasi, Barbara; Brenchley, Jason M; Albrecht, Helmut; Weintrob, Amy; Sumpter, Beth; Engram, Jessica; Gordon, Shari; Klatt, Nichole R; Frank, Ian; Sodora, Donald L; Douek, Daniel C; Paiardini, Mirko; Silvestri, Guido

2008-04-15

Decreased CD4(+) T cell counts are the best marker of disease progression during HIV infection. However, CD4(+) T cells are heterogeneous in phenotype and function, and it is unknown how preferential depletion of specific CD4(+) T cell subsets influences disease severity. CD4(+) T cells can be classified into three subsets by the expression of receptors for two T cell-tropic cytokines, IL-2 (CD25) and IL-7 (CD127). The CD127(+)CD25(low/-) subset includes IL-2-producing naive and central memory T cells; the CD127(-)CD25(-) subset includes mainly effector T cells expressing perforin and IFN-gamma; and the CD127(low)CD25(high) subset includes FoxP3-expressing regulatory T cells. Herein we investigated how the proportions of these T cell subsets are changed during HIV infection. When compared with healthy controls, HIV-infected patients show a relative increase in CD4(+)CD127(-)CD25(-) T cells that is related to an absolute decline of CD4(+)CD127(+)CD25(low/-) T cells. Interestingly, this expansion of CD4(+)CD127(-) T cells was not observed in naturally SIV-infected sooty mangabeys. The relative expansion of CD4(+)CD127(-)CD25(-) T cells correlated directly with the levels of total CD4(+) T cell depletion and immune activation. CD4(+)CD127(-)CD25(-) T cells were not selectively resistant to HIV infection as levels of cell-associated virus were similar in all non-naive CD4(+) T cell subsets. These data indicate that, during HIV infection, specific changes in the fraction of CD4(+) T cells expressing CD25 and/or CD127 are associated with disease progression. Further studies will determine whether monitoring the three subsets of CD4(+) T cells defined based on the expression of CD25 and CD127 should be used in the clinical management of HIV-infected individuals.

Effects of a Shuangling Fuzheng anticancer preparation on the proliferation of SGC-7901 cells and immune function in a cyclophosphamide-treated murine model.

PubMed

Chen, Hua-Sheng; Chen, Jue; Cui, De-Li; Zheng, Yuan-Yuan; Xu, Ai-Hua; Chen, Gang; Jia, Ling-Chang

2007-12-28

To study the inhibitory effects of a Shuangling Fuzheng anticancer preparation (SFAP) on the human gastric cancer cell line SGC-7901 in vitro as well as its immune-modulated effects in a cyclophosphamide-treated murine model. MTT experiments and immunocytochemistry ABC experiments were performed for detecting the proliferation of SGC-7901 cells in vitro and protein expression of c-myc. The staphylococcal protein A (SPA) rosette test was utilized for measuring the ratio of T-lymphocyte subsets from peripheral blood in a cyclophosphamide-treated murine model. Enzyme-linked immunosorbant assay (ELISA) was performed for measuring the levels of serum sIL-2R in treated mice, while immunoturbidimetry was used for measuring the levels of immunoglobulins (Ig). SFAP (40-640 mg/L, 48 h) inhibited the proliferation of SGC-7901 cells, and a positive correlation was noted between inhibitory effects and dosage. At a dosage of 160-320 mg/L in cultured cells, the expression of c-myc was decreased. SFAP (50-200 mg/kg) increased the percentage of CD3+ and CD4+ T-lymphocytes, the ratio of CD4/CD8, and the contents of Ig such as IgM, IgG or IgA, but decreased the levels of serum sIL-2R in peripheral blood from cyclophosphamide-treated mice. SFAP can inhibit the proliferation of SGC-7901 cells via the c-myc gene. In addition, SFAP can modulat the cellular and humoral immunity in cyclophosphamide-induced immunosuppressed mice.

Expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder.

PubMed

Ai, Xing; Zhang, Xu; Wu, Zhun; Ma, Xin; Ju, Zhenghua; Wang, Baojun; Shi, Taoping

2007-02-01

The expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder (TCCB) and its clinical significance were investigated. Immunohistochemistry was used to detect KAI1/CD82 and MRP-1/CD9 protein expression in 52 TCCB specimens. Correlation between the expression of KAI1/CD82 and MRP-1/CD9 to clinicopathologic factors was statistically analyzed. The results showed that the positive rate of KAI1/CD82 and MRP-1/CD9 in TCCB was 50% and 61.5%, respectively. The MRP-1/CD9 and KAI1/CD82 expression was significantly associated with grade of TCCB (P<0.05), but no correlation was found between MRP-1/CD9 or KAI1/CD82 expression and clinical stage of TCCB (P>0.05). The expression level of MRP-1/CD9 and KAI1/CD82 in recurrent TCCB samples was lower than that in non-recurrent samples (P<0.05). Meanwhile, the correlation between the KAI1/CD82 expression and MRP-1/CD9 expression was statistically significant (r=0.316, P<0.05). It was concluded that KAI1/CD82 and MRP-1/CD9 expression may be important prognostic indicators and potentially useful for assessing the biological behavior of TCCB.

Human CD40 ligand deficiency dysregulates the macrophage transcriptome causing functional defects that are improved by exogenous IFN-γ.

PubMed

Cabral-Marques, Otavio; Ramos, Rodrigo Nalio; Schimke, Lena F; Khan, Taj Ali; Amaral, Eduardo Pinheiro; Barbosa Bomfim, Caio César; Junior, Osvaldo Reis; França, Tabata Takahashi; Arslanian, Christina; Carola Correia Lima, Joanna Darck; Weber, Cristina Worm; Ferreira, Janaíra Fernandes; Tavares, Fabiola Scancetti; Sun, Jing; D'Imperio Lima, Maria Regina; Seelaender, Marília; Garcia Calich, Vera Lucia; Marzagão Barbuto, José Alexandre; Costa-Carvalho, Beatriz Tavares; Riemekasten, Gabriela; Seminario, Gisela; Bezrodnik, Liliana; Notarangelo, Luigi; Torgerson, Troy R; Ochs, Hans D; Condino-Neto, Antonio

2017-03-01

CD40 ligand (CD40L) deficiency predisposes to opportunistic infections, including those caused by fungi and intracellular bacteria. Studies of CD40L-deficient patients reveal the critical role of CD40L-CD40 interaction for the function of T, B, and dendritic cells. However, the consequences of CD40L deficiency on macrophage function remain to be investigated. We sought to determine the effect of CD40L absence on monocyte-derived macrophage responses. After observing the improvement of refractory disseminated mycobacterial infection in a CD40L-deficient patient by recombinant human IFN-γ (rhIFN-γ) adjuvant therapy, we investigated macrophage functions from CD40L-deficient patients. We analyzed the killing activity, oxidative burst, cytokine production, and in vitro effects of rhIFN-γ and soluble CD40 ligand (sCD40L) treatment on macrophages. In addition, the effect of CD40L absence on the macrophage transcriptome before and after rhIFN-γ treatment was studied. Macrophages from CD40L-deficient patients exhibited defective fungicidal activity and reduced oxidative burst, both of which improved in the presence of rhIFN-γ but not sCD40L. In contrast, rhIFN-γ and sCD40L ameliorate impaired production of inflammatory cytokines. Furthermore, rhIFN-γ reversed defective control of Mycobacterium tuberculosis proliferation by patients' macrophages. The absence of CD40L dysregulated the macrophage transcriptome, which was improved by rhIFN-γ. Additionally, rhIFN-γ increased expression levels of pattern recognition receptors, such as Toll-like receptors 1 and 2, dectin 1, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin in macrophages from both control subjects and patients. Absence of CD40L impairs macrophage development and function. In addition, the improvement of macrophage immune responses by IFN-γ suggests this cytokine as a potential therapeutic option for patients with CD40L deficiency. Copyright © 2016 American Academy of

Novel Markers to Delineate Murine M1 and M2 Macrophages

PubMed Central

Jablonski, Kyle A.; Amici, Stephanie A.; Webb, Lindsay M.; Ruiz-Rosado, Juan de Dios; Popovich, Phillip G.; Partida-Sanchez, Santiago; Guerau-de-Arellano, Mireia

2015-01-01

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages. PMID:26699615

Glutathione S-transferase pi isoform (GSTP1) expression in murine retina increases with developmental maturity.

PubMed

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2009). We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to ultraviolet (UV) light, and GSTP1 over-expression protects them against UV light damage (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2010). In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Eyes from BALB/c mice at postnatal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lx of white fluorescent light for 24 h, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. GSTP1 levels in the murine retina increased in ascending order from postnatal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at postnatal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina.

Glutathione S-Transferase Pi Isoform (GSTP1) Expression in Murine Retina Increases with Developmental Maturity

PubMed Central

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Background and Aims Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls [1]. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage [2]. In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Methods Eyes from BALB/c mice at post-natal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lux of white fluorescent light for 24 hours, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. Results GSTP1 levels in the murine retina increased in ascending order from post-natal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at post-natal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. Conclusions GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina. PMID:24664677

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.

PubMed

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-08-01

Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

PubMed Central

2012-01-01

Introduction The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. Methods The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. Results Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. Conclusions The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-Jun N terminal kinase activation independent from the interferon pathway.

PubMed

Rau, Sibylle J; Hildt, Eberhard; Himmelsbach, Kiyoshi; Thimme, Robert; Wakita, Takaji; Blum, Hubert E; Fischer, Richard

2013-01-01

CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection. Copyright © 2012 American Association for the Study of Liver Diseases.

CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming

PubMed Central

Martín-Fontecha, Alfonso; Baumjohann, Dirk; Guarda, Greta; Reboldi, Andrea; Hons, Miroslav; Lanzavecchia, Antonio; Sallusto, Federica

2008-01-01

There is growing evidence that the maturation state of dendritic cells (DCs) is a critical parameter determining the balance between tolerance and immunity. We report that mouse CD4+ effector memory T (TEM) cells, but not naive or central memory T cells, constitutively expressed CD40L at levels sufficient to induce DC maturation in vitro and in vivo in the absence of antigenic stimulation. CD4+ TEM cells were excluded from resting lymph nodes but migrated in a CD62P-dependent fashion into reactive lymph nodes that were induced to express CD62P, in a transient or sustained fashion, on high endothelial venules. Trafficking of CD4+ TEM cells into chronic reactive lymph nodes maintained resident DCs in a mature state and promoted naive T cell responses and experimental autoimmune encephalomyelitis (EAE) to antigens administered in the absence of adjuvants. Antibodies to CD62P, which blocked CD4+ TEM cell migration into reactive lymph nodes, inhibited DC maturation, T cell priming, and induction of EAE. These results show that TEM cells can behave as endogenous adjuvants and suggest a mechanistic link between lymphocyte traffic in lymph nodes and induction of autoimmunity. PMID:18838544

Expression of CD56 in patients with adenomyosis and its correlation with dysmenorrhea.

PubMed

Wang, Fei; Shi, Xixi; Qin, Xiaoyan; Wen, ZeQing; Zhao, Xingbo; Li, Changzhong

2015-11-01

To investigate the expression of CD56 in endometrial samples from patients with adenomyosis and its relationship with menstrual cycle phase and severity of dysmenorrhea. 40 patients with histologically proved adenomyosis (proliferative n=20; secretory n=20) and dysmenorrhea were examined in this study, control groups includes 20 patients with adenomyosis without dysmenorrhea (main complaint: menorrhagia) and 20 patients without adenomyosis who had undergone hysterectomy for non-endometrial pathology (no dysmenorrhea medical history). Immunohistochemical staining against CD56 was performed for the eutopic and ectopic endometrium from patients with adenomyosis and the control samples. The expression of CD56 was determined by calculating the H-score and the severity of dysmenorrhea was determined using the visual analogue scale. The menstrual cycle status and the disease severity were compared to the levels of staining. CD56 was expressed mainly in the endometrial glandular epithelium in patients with adenomyosis and normal endometrium. The epithelial staining intensity of CD56 in ectopic lesions of adenomyosis with dysmenorrhea was obviously higher than in the corresponding eutopic endometrium and control groups (P<0.01). There were no statistical differences in the expression between normal endometrium, eutopic endometrium of adenomyosis with dysmenorrhea and adenomyostic samples without dysmenorrhea. For eutopic endometrium in adenomyosis with dysmenorrhea, expression was higher in the secretory phases than in the proliferative phase (P<0.05). The increased CD56 immunoreactivity correlated with the severity of dysmenorrhea (spearman rho=0.84, P<0.01). These findings suggest that the expression of CD56 in adenomyosis is positively associated with the severity of dysmenorrhea. Endometrial glandular epithelium is likely to secrete more CD56 and stimulating nerve growth in the stroma, which could then play a role in the pathogenesis of adenomoysis-related dysmenorrhea

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells

PubMed Central

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-01-01

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/– cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/– cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/– and CD34–MPL– cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL– cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/– SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34– SRCs generate CD34+CD38–CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL– SRCs reside at the apex of the human HSC hierarchy. PMID:27938494

CD127 and CD25 Expression Defines CD4+ T Cell Subsets That Are Differentially Depleted during HIV Infection1

PubMed Central

Dunham, Richard M.; Cervasi, Barbara; Brenchley, Jason M.; Albrecht, Helmut; Weintrob, Amy; Sumpter, Beth; Engram, Jessica; Gordon, Shari; Klatt, Nichole R.; Frank, Ian; Sodora, Donald L.; Douek, Daniel C.; Paiardini, Mirko; Silvestri, Guido

2009-01-01

Decreased CD4+ T cell counts are the best marker of disease progression during HIV infection. However, CD4+ T cells are heterogeneous in phenotype and function, and it is unknown how preferential depletion of specific CD4+ T cell subsets influences disease severity. CD4+ T cells can be classified into three subsets by the expression of receptors for two T cell-tropic cytokines, IL-2 (CD25) and IL-7 (CD127). The CD127+CD25low/− subset includes IL-2-producing naive and central memory T cells; the CD127−CD25− subset includes mainly effector T cells expressing perforin and IFN-γ; and the CD127lowCD25high subset includes FoxP3-expressing regulatory T cells. Herein we investigated how the proportions of these T cell subsets are changed during HIV infection. When compared with healthy controls, HIV-infected patients show a relative increase in CD4+CD127−CD25− T cells that is related to an absolute decline of CD4+CD127+CD25low/− T cells. Interestingly, this expansion of CD4+CD127− T cells was not observed in naturally SIV-infected sooty mangabeys. The relative expansion of CD4+CD127−CD25− T cells correlated directly with the levels of total CD4+ T cell depletion and immune activation. CD4+CD127−CD25− T cells were not selectively resistant to HIV infection as levels of cell-associated virus were similar in all non-naive CD4+ T cell subsets. These data indicate that, during HIV infection, specific changes in the fraction of CD4+ T cells expressing CD25 and/or CD127 are associated with disease progression. Further studies will determine whether monitoring the three subsets of CD4+ T cells defined based on the expression of CD25 and CD127 should be used in the clinical management of HIV-infected individuals. PMID:18390743

Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality

PubMed Central

Schneidawind, Dominik; Baker, Jeanette; Pierini, Antonio; Buechele, Corina; Luong, Richard H.; Meyer, Everett H.

2015-01-01

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4+ iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4+ iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4+ iNKT cells from third-party mice were as protective as CD4+ iNKT cells from donor mice although third-party CD4+ iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4+ iNKT cells resulted in a robust expansion of donor CD4+CD25+FoxP3+ regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation. PMID:25795920

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

PubMed Central

Shmelkov, Sergey V.; Butler, Jason M.; Hooper, Andrea T.; Hormigo, Adilia; Kushner, Jared; Milde, Till; St. Clair, Ryan; Baljevic, Muhamed; White, Ian; Jin, David K.; Chadburn, Amy; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; D’Angelica, Michael; Kemeny, Nancy; Lyden, David; Rafii, Shahin

2008-01-01

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10–/–CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133– population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133– metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133– cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24–), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic

Murine cell glycolipids customization by modular expression of glycosyltransferases.

PubMed

Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

2013-01-01

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

CD133 expression in osteosarcoma and derivation of CD133⁺ cells.

PubMed

Li, Ji; Zhong, Xiao-Yan; Li, Zong-Yu; Cai, Jin-Fang; Zou, Lin; Li, Jian-Min; Yang, Tao; Liu, Wei

2013-02-01

Cluster of differentiation 133 (CD133) is recognized as a stem cell marker for normal and cancerous tissues. Using cell culture and real‑time fluorescent polymerase chain reaction, CD133 expression was analyzed in osteosarcoma tissue and Saos‑2 cell lines. In addition, cancer stem cell‑related gene expression in the Saos‑2 cell line was determined to explore the mechanisms underlying tumorigenesis and high drug resistance in osteosarcoma. CD133+ cells were found to be widely distributed in various types of osteosarcoma tissue. Following cell culture, cells entered the G2/M and S cell cycle stages from G0/G1. Levels of CD133+ cells decreased to normal levels rapidly over the course of cell culture. Colony forming efficiency was higher in the CD133+ compared with the CD133‑ subpopulation of Saos‑2 cells. Expression levels of stem cell‑related genes, including multidrug resistance protein 1 (MDR1) and sex determining region Y‑box 2 (Sox2) in the CD133+ subpopulation of cells were found to be significantly higher compared with the CD133‑ subpopulation. These observations indicate that CD133+ Saos‑2 cells exhibit stem cell characteristics, including low abundance, quiescence and a high potential to undergo differentiation, as well as expression of key stem cell regulatory and drug resistance genes, which may cause osteosarcoma and high drug resistance.

Evaluation of the role of CD207 on Langerhans cells in a murine model of atopic dermatitis by in situ imaging using Cr:forsterite laser-based multimodality nonlinear microscopy

NASA Astrophysics Data System (ADS)

Lee, Jyh-Hong; Tsai, Ming-Rung; Sun, Chi-Kuang; Chiang, Bor-Luen

2012-11-01

Atopic dermatitis (AD) is an allergic inflammatory disease of skin. It remains unclear that CD207 of Langerhans cells (LCs) plays a central role in the development of allergic sensitization. There is little data on LCs within the microenviroment in vivo. We used a murine model of epicutaneous (EC) ovalbumin (OVA) sensitization inducing an inflammatory skin resembling AD to explore the role of CD207 in the pathogenesis of AD. Cr:forsterite laser-based multimodality nonlinear microscopy was applied for in situ imaging. Peritoneal injections of Alexa Fluor 647-rat anti-mouse CD207 into mice were performed to specifically trace the LCs. Peritoneal injections of OVA-Alexa Fluor 647 conjugate into mice were performed to specifically trace the OVA. We found that combining Alexa Fluor fluorescent probes with multimodality nonlinear microscopy permitted the unequivocal in situ imaging of CD207-expressing LCs. The relevant time-course, expressional, and functional studies reveal that CD207 of LCs plays an essential role during the induction of EC sensitization. We establish and validate that Cr:forsterite laser-based multimodality nonlinear microscopy is applicable for the specific detection of labeled mAb-bound LCs and labeled antigen. We suggest that CD207-expressing LCs initiate the allergic response through the CD207 mediated epicutaneous sensitization associated with the development of AD.

Flagellin is a Th1 polarizing factor for human CD4+ T cells and induces protection in a murine neonatal vaccination model of rotavirus infection.

PubMed

Labastida-Conde, Rosario Guadalupe; Ramírez-Pliego, Oscar; Peleteiro-Olmedo, Mercedes; Lopez-Guerrero, Delia Vanessa; Badillo-Godinez, Oscar Daniel; Gutiérrez-Xicoténcatl, María de Lourdes; Rosas-Salgado, Gabriela; González-Fernández, África; Esquivel-Guadarrama, Fernando R; Santana, M Angélica

2018-07-05

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4 + T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4 + and CD8 + T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4 + T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4 + T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effective therapy for a murine model of human anaplastic large-cell lymphoma with the anti-CD30 monoclonal antibody, HeFi-1, does not require activating Fc receptors

PubMed Central

Zhang, Meili; Yao, Zhengsheng; Zhang, Zhuo; Garmestani, Kayhan; Goldman, Carolyn K.; Ravetch, Jeffrey V.; Janik, John; Brechbiel, Martin W.; Waldmann, Thomas A.

2006-01-01

CD30 is a member of the tumor necrosis factor receptor family. Overexpression of CD30 on some neoplasms versus its limited expression on normal tissues makes this receptor a promising target for antibody-based therapy. Anaplastic large-cell lymphoma (ALCL) represents a heterogeneous group of aggressive non-Hodgkin lymphomas characterized by the strong expression of CD30. We investigated the therapeutic efficacy of HeFi-1, a mouse IgG1 monoclonal antibody, which recognizes the ligand-binding site on CD30, and humanized anti-Tac antibody (daclizumab), which recognizes CD25, in a murine model of human ALCL. The ALCL model was established by intravenous injection of karpas299 cells into nonobese diabetic/severe combined immuno-deficient (SCID/NOD) wild-type or SCID/NOD Fc receptor common γ chain–deficient (FcRγ–/–) mice. HeFi-1, given at a dose of 100 μg weekly for 4 weeks, significantly prolonged survival of the ALCL-bearing SCID/NOD wild-type and SCID/NOD FcRγ–/– mice (P < .01) as compared with the control groups. In vitro studies showed that HeFi-1 inhibited the proliferation of karpas299 cells, whereas daclizumab did not inhibit cell proliferation. We demonstrated that the expression of FcRγ on polymorphonuclear leukocytes and monocytes was not required for HeFi-1–mediated tumor growth inhibition in vivo, although it was required for daclizumab. PMID:16551968

CD1d-Expressing Breast Cancer Cells Modulate NKT Cell-Mediated Antitumor Immunity in a Murine Model of Breast Cancer Metastasis

PubMed Central

Hix, Laura M.; Shi, Yihui H.; Brutkiewicz, Randy R.; Stein, Paul L.; Wang, Chyung-Ru; Zhang, Ming

2011-01-01

Background Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression. Methodology/Principal Findings In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors. Conclusions/Significance The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer. PMID:21695190

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

PubMed

Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L; Siminovitch, Katherine A; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K; Worthington, Jane; Gupta, Namrata; Clemons, Paul A; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M

2013-05-01

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in

Metformin Inhibits Advanced Glycation End Products-Induced Inflammatory Response in Murine Macrophages Partly through AMPK Activation and RAGE/NFκB Pathway Suppression

PubMed Central

Zhou, Zhong'e; Tang, Yong; Chen, Chengjun; Lu, Yi; Liu, Liang

2016-01-01

Advanced glycation end products (AGEs) are major inflammatory mediators in diabetes, affecting atherosclerosis progression via macrophages. Metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated. The present study of murine bone marrow derived macrophages showed that (1) AGEs enhanced proinflammatory cytokines (interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)) mRNA expression, RAGE expression, and NFκB activation; (2) metformin pretreatment inhibited AGEs effects and AGEs-induced cluster designation 86 (CD86) (M1 marker) expression, while promoting CD206 (M2 marker) surface expression and anti-inflammatory cytokine (IL-10) mRNA expression; and (3) the AMPK inhibitor, Compound C, attenuated metformin effects. In conclusion, metformin inhibits AGEs-induced inflammatory response in murine macrophages partly through AMPK activation and RAGE/NFκB pathway suppression. PMID:27761470

Comparative Analysis of Hepatic CD14 Expression between Two Different Endotoxin Shock Model Mice: Relation between Hepatic Injury and CD14 Expression

PubMed Central

Hozumi, Hiroyasu; Tada, Rui; Murakami, Taisuke; Adachi, Yoshiyuki; Ohno, Naohito

2013-01-01

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo. PMID:23308276

Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists

PubMed Central

Beatty, Gregory L.; Li, Yan; Long, Kristen B.

2017-01-01

INTRODUCTION CD40 is a promising therapeutic target for cancer immunotherapy. In patients with advanced solid malignancies, CD40 agonists have demonstrated some anti-tumor activity and a manageable toxicity profile. A 2nd generation of CD40 agonists has now been designed with optimized Fc receptor (FcR) binding based on preclinical evidence suggesting a critical role for FcR engagement in defining the potency of CD40 agonists in vivo. AREAS COVERED We provide a comprehensive review using PubMed and Google Patent databases on the current clinical status of CD40 agonists, strategies for applying CD40 agonists in cancer therapy, and the preclinical data that supports and is guiding the future development of CD40 agonists. EXPERT COMMENTARY There is a wealth of preclinical data that provide rationale on several distinct approaches for using CD40 agonists in cancer immunotherapy. This data illustrates the need to strategically combine CD40 agonists with other clinically active treatment regimens in order to realize the full potential of activating CD40 in vivo. Thus, critical to the success of this class of immune-oncology drugs, which have the potential to restore both innate and adaptive immunosurveillance, will be the identification of biomarkers for monitoring and predicting responses as well as informing mechanisms of treatment resistance. PMID:27927088

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

PubMed

Winchester, Lee J; Veeranki, Sudhakar; Givvimani, Srikanth; Tyagi, Suresh C

2015-07-01

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Soluble CD40 ligand in prediction of acute severe pancreatitis

PubMed Central

Frossard, Jean Louis; Morel, Philippe; Kwak, Brenda; Pastor, Catherine; Berney, Thierry; Buhler, Léo; Von Laufen, Alain; Demulder, Sandrine; Mach, Francois

2006-01-01

AIM: To assess the early predictability of the soluble CD40L (sCD40L) in pancreatitis severity. METHODS: Between February 2000 and February 2003, 279 consecutive patients with acute pancreatitis were prospectively enrolled in our study. In this report, 40 patients with mild and 40 patients with severe pancreatitis were randomly studied. sCD40L concentrations were measured 48 hours after admission. RESULTS: sCD40L levels were significantly higher 48 hours after admission in severe pancreatitis than in mild pancreatitis. Using a cutoff of 1 000 pg/L, the sensitivity and specificity of sCD40L to detect a severe course of the disease were 78% and 62% respectively compared to 72% and 81% for CRP. Logistic regression analysis found that CRP was the only statistically significant marker able to detect a severe course of the disease. CONCLUSION: These findings indicate that CRP remains a valuable marker to determine the severity and prognosis of acute pancreatitis whereas sCD40L levels should be assessed in further studies. PMID:16570356

IL‐12 and IL‐15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells

PubMed Central

Hydes, Theresa; Noll, Angela; Salinas‐Riester, Gabriela; Abuhilal, Mohammed; Armstrong, Thomas; Hamady, Zaed; Primrose, John; Takhar, Arjun; Walter, Lutz

2017-01-01

Abstract Introduction Murine hepatic NK cells exhibit adaptive features, with liver‐specific adhesion molecules CXCR6 and CD49a acting as surface markers. Methods We investigated human liver‐resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. Results Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver‐resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver‐resident double‐positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single‐positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL‐12 and IL‐15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver‐resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. Conclusion IL‐12 and IL‐15 may be key for generating NK cells with a tissue‐homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue‐homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease. PMID:28952190

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

PubMed

Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

1994-04-01

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO

Expression of LLT1 and its receptor CD161 in lung cancer is associated with better clinical outcome.

PubMed

Braud, Véronique M; Biton, Jérôme; Becht, Etienne; Knockaert, Samantha; Mansuet-Lupo, Audrey; Cosson, Estelle; Damotte, Diane; Alifano, Marco; Validire, Pierre; Anjuère, Fabienne; Cremer, Isabelle; Girard, Nicolas; Gossot, Dominique; Seguin-Givelet, Agathe; Dieu-Nosjean, Marie-Caroline; Germain, Claire

2018-01-01

Co-stimulatory and inhibitory receptors expressed by immune cells in the tumor microenvironment modulate the immune response and cancer progression. Their expression and regulation are still not fully characterized and a better understanding of these mechanisms is needed to improve current immunotherapies. Our previous work has identified a novel ligand/receptor pair, LLT1/CD161, that modulates immune responses. Here, we extensively characterize its expression in non-small cell lung cancer (NSCLC). We show that LLT1 expression is restricted to germinal center (GC) B cells within tertiary lymphoid structures (TLS), representing a new hallmark of the presence of active TLS in the tumor microenvironment. CD161-expressing immune cells are found at the vicinity of these structures, with a global enrichment of NSCLC tumors in CD161 + CD4 + and CD8 + T cells as compared to normal distant lung and peripheral blood. CD161 + CD4 + T cells are more activated and produce Th1-cytokines at a higher frequency than their matched CD161-negative counterparts. Interestingly, CD161 + CD4 + T cells highly express OX40 co-stimulatory receptor, less frequently 4-1BB, and display an activated but not completely exhausted PD-1-positive Tim-3-negative phenotype. Finally, a meta-analysis revealed a positive association of CLEC2D (coding for LLT1) and KLRB1 (coding for CD161) gene expression with favorable outcome in NSCLC, independently of the size of T and B cell infiltrates. These data are consistent with a positive impact of LLT1/CD161 on NSCLC patient survival, and make CD161-expressing CD4 + T cells ideal candidates for efficient anti-tumor recall responses.

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

PubMed

Kalantari, Elham; Asgari, Mojgan; Nikpanah, Seyedehmoozhan; Salarieh, Naghme; Asadi Lari, Mohammad Hossein; Madjd, Zahra

2017-10-01

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers

PubMed Central

Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A.; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

2016-01-01

B-cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL+ MHC class-IHi CD86Hi B cells of unknown origin. Here we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. The 4BL cells induce expression of 4-1BBL and IFNγR1 on B1a cells resulting in subsequent up regulation of membrane TNFα (mTNFα) and CD86. As a result, B1a cells induce expression of granzyme B in CD8+T cells by targeting TNFR2 via mTNFα while providing co-stimulation with CD86. Thus, for the first time, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8+T cells. PMID:26983789

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells.

PubMed

Watanabe, Masashi; Fujihara, Chiharu; Radtke, Andrea J; Chiang, Y Jeffrey; Bhatia, Sumeena; Germain, Ronald N; Hodes, Richard J

2017-09-04

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

CD30 expression in follicular lymphoma.

PubMed

Gardner, L J; Polski, J M; Evans, H L; Perkins, S L; Dunphy, C H

2001-08-01

CD30(+) anaplastic large cell lymphomas were originally described as being of T-cell, null cell, and B-cell origin. CD30, however, is not a specific marker of anaplastic large cell lymphoma and has been found to be expressed in reactive as well as neoplastic populations as a probable activation marker. In addition, CD30(+) cells have also been described in both diffuse large B-cell and follicular lymphomas (FLs), resembling the pattern seen in reactive tonsils and lymph nodes. We report an index case of FL with CD30 expression, which on initial touch preparations and flow cytometric immunophenotyping revealed a prominent population of CD30(+) cells with marked cellular pleomorphism (anaplasia) in a background of typical FL. Immunohistochemistry of the paraffin section for CD30 in our index case confirmed unequivocal CD30(+) pleomorphic cells in the malignant nodules in occasional clusters. This case prompted a study of additional cases of FL for pattern of immunoreactivity with CD30 on paraffin sections. Twenty-two additional cases of FL (grades 1-3) were retrieved for CD30 immunoperoxidase staining as in the index case. This study demonstrated 32% of the additional cases of FL had definitive CD30(+), large, pleomorphic malignant cells by paraffin immunohistochemistry. In 2 cases (9%), the pattern of immunoreactivity with CD30 showed clustering and variable staining of large cells, as our index case. This study underscores the morphologic and immunophenotypic spectrum of FL that includes CD30 staining and cellular pleomorphism.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

PubMed

Mariappan, Devi; Winkler, Johannes; Chen, Shuhua; Schulz, Herbert; Hescheler, Jürgen; Sachinidis, Agapios

2009-02-01

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells. CD31(+) cells exhibit endothelial-like behavior by being able to incorporate DiI-labeled acetylated low-density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi-quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31(+) cells, large-scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31(+) cell population. Based on the transcriptomic signatures of the CD31(+) cells, we conclude that this ES cell-derived population contains endothelial-like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31(+) cells enables an in vitro functional genomic model to identify genes required for angiogenesis.

Murine Sarcoma Virus Gene Expression: Transformants Which Express Viral Envelope Glycoprotein In The Absence Of The Major Internal Protein And Infectious Particles

PubMed Central

Bilello, John A.; Strand, Mette; August, J. T.

1974-01-01

Expression of the major internal protein and the envelope glycoprotein of murine C-type viruses in focus-derived lines of normal rat kidney cells infected with Kirsten murine sarcoma virus was measured by radioimmunoassay. Of the clones selected, which do not produce virus particles or the major viral structural protein, approximately half express the viral envelope glycoprotein at concentrations found in productively infected cells. Expression of the envelope glycoprotein did not appear to alter significantly the properties of the transformed cells in culture. PMID:4370209

Host CD40 Is Essential for DCG Treatment Against Metastatic Lung Cancer.

PubMed

Yamashita, Kimihiro; Hasegawa, Hiroshi; Fujita, Mitsugu; Nishi, Masayasu; Tanaka, Tomoko; Arimoto, Akira; Suzuki, Satoshi; Kamigaki, Takashi; Kakeji, Yoshihiro

2016-07-01

For the application of invariant natural killer T (iNKT) cells in cancer therapy, the CD40-CD40L interaction is indispensable in administering alpha-galactosylceramide (αGalCer). We hypothesized that CD40 plays an important role in dendritic cells (DC) pulsed with αGalCer (DCGs) in the treatment of lung metastases. Wild-type (WT) and CD40(-/-) mice were treated with DCGs isolated from WT or CD40(-/-) mice in a B16F10 lung metastases model and NK and NKT cell activity in lungs and the spleen were examined. DCG treatment improved WT mice survival but CD40(-/-) hosts received no survival benefit. Conversely, attenuation of a therapeutic effect in mice treated with CD40(-/-) DCGs was not observed. The functional activities of NK and NKT cells in DCG-treated CD40(-/-) mice were partially suppressed. Host CD40 is essential for DCG treatment to have a therapeutic effect on B16F10 lung metastases. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Intratumoral Infection with Murine Cytomegalovirus Synergizes with PD-L1 Blockade to Clear Melanoma Lesions and Induce Long-term Immunity

PubMed Central

Erkes, Dan A; Xu, Guangwu; Daskalakis, Constantine; Zurbach, Katherine A; Wilski, Nicole A; Moghbeli, Toktam; Hill, Ann B; Snyder, Christopher M

2016-01-01

Cytomegalovirus is an attractive cancer vaccine platform because it induces strong, functional CD8+ T-cell responses that accumulate over time and migrate into most tissues. To explore this, we used murine cytomegalovirus expressing a modified gp100 melanoma antigen. Therapeutic vaccination by the intraperitoneal and intradermal routes induced tumor infiltrating gp100-specific CD8+ T-cells, but provided minimal benefit for subcutaneous lesions. In contrast, intratumoral infection of established tumor nodules greatly inhibited tumor growth and improved overall survival in a CD8+ T-cell-dependent manner, even in mice previously infected with murine cytomegalovirus. Although murine cytomegalovirus could infect and kill B16F0s in vitro, infection was restricted to tumor-associated macrophages in vivo. Surprisingly, the presence of a tumor antigen in the virus only slightly increased the efficacy of intratumoral infection and tumor-specific CD8+ T-cells in the tumor remained dysfunctional. Importantly, combining intratumoral murine cytomegalovirus infection with anti-PD-L1 therapy was synergistic, resulting in tumor clearance from over half of the mice and subsequent protection against tumor challenge. Thus, while a murine cytomegalovirus-based vaccine was poorly effective against established subcutaneous tumors, direct infection of tumor nodules unexpectedly delayed tumor growth and synergized with immune checkpoint blockade to promote tumor clearance and long-term protection. PMID:27434584

Chemotherapeutic Effect of CD147 Antibody-labeled Micelles Encapsulating Doxorubicin Conjugate Targeting CD147-Expressing Carcinoma Cells.

PubMed

Asakura, Tadashi; Yokoyama, Masayuki; Shiraishi, Koichi; Aoki, Katsuhiko; Ohkawa, Kiyoshi

2018-03-01

CD147 (basigin/emmprin) is expressed on the surface of carcinoma cells. For studying the efficacy of CD147-targeting medicine on CD147-expressing cells, we studied the effect of anti-CD147-labeled polymeric micelles (CD147ab micelles) that encapsulated a conjugate of doxorubicin with glutathione (GSH-DXR), with specific accumulation and cytotoxicity against CD147-expressing A431 human epidermoid carcinoma cells, Ishikawa human endometrial adenocarcinoma cells, and PC3 human prostate carcinoma cells. By treatment of each cell type with CD147ab micelles for 1 h, a specific accumulation of CD147ab micelles in CD147-expressing cells was observed. In addition, the cytotoxicity of GSH-DXR-encapsulated micelles against each cell type was measured by treatment of the micelles for 1 h. The cytotoxic effect of CD147ab micelles carrying GSH-DXR was 3- to 10-fold higher for these cells than that of micelles without GSH-DXR. These results suggest that GSH-DXR-encapsulated CD147ab micelles could serve as an effective drug delivery system to CD147-expressing carcinoma cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit.

PubMed

Tanaka, Junji; Sugita, Junichi; Kato, Naoko; Toubai, Tomomi; Ibata, Makoto; Shono, Yusuke; Ota, Shuichi; Kondo, Takeshi; Kobayashi, Takahiko; Kobayashi, Masanobu; Asaka, Masahiro; Imamura, Masahiro

2007-10-01

Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

PubMed

Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

1992-12-01

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

Identification, characterization and isolation of a common progenitor for osteoclasts, macrophages and dendritic cells from murine bone marrow and periphery

PubMed Central

Jacome-Galarza, Christian E.; Lee, Sun-Kyeong; Lorenzo, Joseph A.; LeonardoAguila, Hector

2012-01-01

Osteoclasts are specialized bone resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype: B220−CD3−CD11b−/low CD115+ and either CD117hi, CD117intermediate or CD117low. We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages and antigen-presenting dendritic cells in vitro with efficiencies of over 90 percent, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined. PMID:23165930

CD28 T-cell costimulatory molecule expression in pemphigus vulgaris.

PubMed

Alecu, M; Ursaciuc, C; Surcel, M; Coman, G; Ciotaru, D; Dobre, M

2009-03-01

CD28 superfamily of immune costimulatory molecules could play an important role in autotolerance control. CD28 costimulation seems to be necessary for regulatory T cell (Treg) activation and successive suppressive activities involved in autoimmunity protection. This study investigates CD28 expression, especially inducible costimulator fraction, on T lymphocytes in pemphigus vulgaris (PV) patients. CD28 expression on T lymphocytes was assessed in 16 PV patients during acute attack. All patients and 10 healthy control subjects were tested for lymphocyte populations, T-cell subpopulations (T-CD4+, T-CD8+), Treg and CD28 expression on T-cell subpopulations. T, B and natural killer cells average values in PV patients were close to the control group values. Compared with control group, PV values showed lower Treg (2.2% compared with 4.7%), slightly decreased CD4+ CD28+ T cells (91% compared with 95%), higher CD4+ CD28- T cells (9% compared with 5%), decreased CD8+ CD28+ T cells (57% and 73%, respectively) and significantly enhanced CD8+ CD28- T cells (43% compared with 27%). These data suggest that Treg-mediated suppressor T-cell effects could be diminished in PV, together with an abnormal or ineffective subsequent helper T-cell suppression. CD28 high expression on helper T cells and low expression on suppressor T cells are arguments for a potential CD28 role in PV autoimmune response mechanism.

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties.

PubMed

Paduano, Francesco; Marrelli, Massimo; Palmieri, Francesca; Tatullo, Marco

2016-10-01

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146 Low and CD146 High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146 Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146 High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146 Low than in CD146 High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells.

PubMed

Hydes, Theresa; Noll, Angela; Salinas-Riester, Gabriela; Abuhilal, Mohammed; Armstrong, Thomas; Hamady, Zaed; Primrose, John; Takhar, Arjun; Walter, Lutz; Khakoo, Salim I

2018-03-01

Murine hepatic NK cells exhibit adaptive features, with liver-specific adhesion molecules CXCR6 and CD49a acting as surface markers. We investigated human liver-resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver-resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver-resident double-positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single-positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL-12 and IL-15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver-resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. IL-12 and IL-15 may be key for generating NK cells with a tissue-homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue-homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes.

PubMed

Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Vastolo, Viviana; Petrosino, Giuseppe; Visconte, Feliciano; Raia, Maddalena; Scalia, Giulia; Loffredo, Stefania; Varricchi, Gilda; Galdiero, Maria Rosaria; Granata, Francescopaolo; Del Vecchio, Luigi; Portella, Giuseppe; Marone, Gianni

2017-05-01

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14 + monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14 + CD16 - monocytes, TSLPR + monocytes (TSLPR + mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR + mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6 , ALOX15B , FCGR2B , LAIR1 ). Strikingly, TSLPR + mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14 + CD1c + cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14 + CD16 - monocytes and prompt further ontogenetic and functional analysis of CD14 + CD1c + and LPS-activated CD14 + CD1c + TSLPR + mono. Copyright © 2017 by The American Association of Immunologists, Inc.

A reevaluation of CD22 expression in human lung cancer.

PubMed

Pop, Laurentiu M; Barman, Stephen; Shao, Chunli; Poe, Jonathan C; Venturi, Guglielmo M; Shelton, John M; Pop, Iliodora V; Gerber, David E; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I; Richardson, James A; Minna, John D; Tedder, Thomas F; Vitetta, Ellen S

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

[Expression and clinical significance of CD147 in parathyroid carcinoma].

PubMed

Du, X M; Wang, L L; Chang, H; Meng, W; Zhang, J Y; Shen, B

2016-06-08

To study the expression and clinical significance of CD147 in the patients of parathyroid carcinoma. Fourteen cases of parathyroid carcinoma encountered during the period from 2012 to 2015 were enrolled. Thirty three cases of parathyroid adenoma encountered during the same period were enrolled. The expression of CD147 in parathyroid carcinoma and parathyroid adenoma was studied by means of immunohistochemistry (EnVision method). CD147 positive color was brown and yellow, and positive position was located mainly in the cytomembrane, and a small amount of cytoplasm was appeared. Among 14 cases of parathyroid carcinoma, 11 cases of CD147 positive score was 3+ , 3 cases of CD147 positive score was 2+ ; Among 33 cases of parathyroid adenoma , 8 cases of CD147 positive score was 2+ , 15 cases of it was 1+ , 10 cases of it was negative. CD147 was highly expressed in parathyroid carcinoma tissues, and the expression of CD147 was significantly different from the expression of parathyroid adenoma(P<0.05). CD147 immunohistochemical staining can help to diagnose parathyroid carcinoma.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges

PubMed Central

Gardner, Matthew R.; Kattenhorn, Lisa M.; Kondur, Hema R.; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J.; Haworth, Kevin G.; Decker, Julie M.; Alpert, Michael D.; Bailey, Charles C.; Neale, Ernest S.; Fellinger, Christoph H.; Joshi, Vinita R.; Fuchs, Sebastian P.; Martinez-Navio, Jose M.; Quinlan, Brian D.; Yao, Annie Y.; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C.; Burton, Dennis R.; Kwong, Peter D.; Piatak, Michael; Lifson, Jeffrey D.; Gao, Guangping; Desrosiers, Ronald C.; Evans, David T.; Hahn, Beatrice H.; Ploss, Alexander; Cannon, Paula M.; Seaman, Michael S.; Farzan, Michael

2015-01-01

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

Syk Mediates BCR- and CD40-Signaling Intergration during B Cell Activation

PubMed Central

Ying, Haiyan; Li, Zhenping; Yang, Lifen; Zhang, Jian

2010-01-01

CD40 is essential for optimal B cell activation. It has been shown that CD40 stimulation can augment BCR-induced B cell responses, but the molecular mechanism(s) by which CD40 regulates BCR signaling is poorly understood. In this report, we attempted to characterize the signaling synergy between BCR- and CD40-mediated pathways during B cell activation. We found that spleen tyrosine kinase (Syk) is involved in CD40 signaling, and is synergistically activated in B cells in response to BCR/CD40 costimulation. CD40 stimulation alone also activates B cell linker (BLNK), Bruton tyrosine kinase (Btk), and Vav-2 downstream of Syk, and significantly enhances BCR-induced formation of complex consisting of, Vav-2, Btk, BLNK, and phospholipase C-gamma2 (PLC-γ2) leading to activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase, Akt, and NF-κB required for optimal B cell activation. Therefore, our data suggest that CD40 can strengthen BCR-signaling pathway and quantitatively modify BCR signaling during B cell activation. PMID:21074890

[The expression and significance of CD(276) and CD(133) in colorectal cancer and precancerous lesions].

PubMed

Lu, G F; Huang, L N; Ren, J L; Hu, G M; Zheng, Z H; Wu, J X; Zhu, Y P; Tang, F A

2018-06-01

In order to study the significance of CD(276) and CD(133) in the development and progression of colorectal cancer (CRC), the expression of CD(276) and CD(133) was detected by immunohistochemistry in CRC and precancerous lesions. The results showed that the intensity of CD(276) and CD(133) in CRC samples was higher than that in adenoma group and non-adenoma group. CD(276) and CD(133) single and double positive expression were significantly correlated with CRC lymph node metastasis, distant metastasis and survival. CD(276) and CD(133) are significantly correlated to the development and progression of CRC and associated with poor prognosis.

Characterization of CD22 Expression in Acute Lymphoblastic Leukemia

PubMed Central

Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Richards, Kelly; Delbrook, Cindy; Kreitman, Robert J.; Pastan, Ira; Wayne, Alan S.

2015-01-01

Background CD22 is a B-lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL). Procedure Properties of CD22 expression relevant to therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B-precursor (pre-B) ALL. Results CD22 expression was demonstrated in all subjects (n=163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349 – 19,653, n=160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349-3,624, n=20 versus 3,853 sites/cell, range 451-19,653, n=140; p=<0.0001) and 6 of 21 cases had sub-populations of blasts lacking CD22 expression (22% – 82% CD22+). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti-CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti-CD22 directed agents. Conclusions These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy-refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22-negative blast population. PMID:25728039

Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells.

PubMed

Cho, Jae-Ho; Kim, Hee-Ok; Webster, Kylie; Palendira, Mainthan; Hahm, Bumsuk; Kim, Kyu-Sik; King, Cecile; Tangye, Stuart G; Sprent, Jonathan

2011-07-07

Immune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8(+) T cells; these receptors restrain CD8(+) responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8(+) (but not CD4(+)) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-β) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.

Comparison of Adipose-Derived and Bone Marrow Mesenchymal Stromal Cells in a Murine Model of Crohn's Disease.

PubMed

Xie, Minghao; Qin, Huabo; Luo, Qianxin; He, Xiaosheng; He, Xiaowen; Lan, Ping; Lian, Lei

2017-01-01

Mesenchymal stromal cells (MSCs) have been used in the treatment of Crohn's disease (CD) because of the immunomodulatory ability. The aim of this study was to investigate the therapeutic effect of adipose-derived MSCs (AD-MSCs) and to compare the therapeutic effect of AD-MSCs with that of bone marrow MSCs (BM-MSCs) in a murine model of CD. Murine colitis model of CD was created by trinitrobenzene sulfonic acid (TNBS). Twelve hours after treatment with TNBS, the mouse model was injected with MSCs intraperitoneally. Real-time polymerase chain reaction and immunohistochemistry staining were used to measure the expression levels of inflammatory cytokines in colonic tissues to investigate the therapeutic effect of AD-MSCs. The ten-day survival was recorded after infusion of MSCs. Intraperitoneal injection of MSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and improved the survival of the TNBS-induced mouse model of CD. AD-MSCs could effectively increase the expression of interleukin-10 and reduce the secretion of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-12, and vascular endothelial growth factor. The mucosal injury was repaired by AD-MSCs. These effects were comparable between AD-MSCs and BM-MSCs. The therapeutic effect appears similar between AD-MSCs and BM-MSCs in treating CD. AD-MSCs may be a potential alternative of cell-based therapy for CD.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

PubMed

Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

2016-04-15

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Expression and prognostic value of soluble CD97 and its ligand CD55 in intrahepatic cholangiocarcinoma.

PubMed

Meng, Ze-Wu; Liu, Min-Chao; Hong, Hai-Jie; Du, Qiang; Chen, Yan-Ling

2017-03-01

The incidence rate of intrahepatic cholangiocarcinoma is rising, and treatment options are limited. Therefore, new biological markers of intrahepatic cholangiocarcinoma are needed. Immunohistochemistry and enzyme-linked immunosorbent assay were applied to analyze the expressions of CD97, CD55, and soluble CD97 in 71 patients with intrahepatic cholangiocarcinoma and 10 patients with hepatolithiasis. CD97 and CD55 were not expressed in hepatolithiatic tissues, but positive expression was observed in 76.1% (54/71) and 70.4% (50/71) of intrahepatic cholangiocarcinoma patients. The univariate analyses indicated that the positive expressions of CD97 and CD55 were related to short intrahepatic cholangiocarcinoma survival of patients (both p = 0.001). Furthermore, CD97 and CD55 expressions and biliary soluble CD97 levels were significantly associated with histological grade (p = 0.004, 0.002, and 0.012, respectively), lymph node metastases (p = 0.020, 0.038, and 0.001, respectively), and venous invasion (p = 0.003, 0.002, and 0.001, respectively). The multivariate analyses indicated that lymph node metastases (hazard ratio: 2.407, p = 0.003), positive CD55 expression (hazard ratio: 4.096, p = 0.003), and biliary soluble CD97 levels (hazard ratio: 2.434, p = 0.002) were independent risk factors for the intrahepatic cholangiocarcinoma survival. The receiver operating characteristic (ROC) curve analysis indicated that when the cutoff values of biliary soluble CD97 were 1.15 U/mL, the diagnostic value for predicting lymph node metastasis had a sensitivity of 87.5% and a specificity of 51.3%. For intrahepatic cholangiocarcinoma patient death within 60 months at a cutoff value of 0.940 U/mL, the diagnostic value sensitivity was 89.3% and the specificity was 93.3%. Biliary soluble CD97 may be a new biological marker for early diagnosis, prediction of lymph node metastasis and poor prognosis, and discovery of a therapeutic target.

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants.

PubMed

Hartkamp, Linda M; Fine, Jay S; van Es, Inge E; Tang, Man Wai; Smith, Michael; Woods, John; Narula, Satwant; DeMartino, Julie; Tak, Paul P; Reedquist, Kris A

2015-08-01

Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

PubMed

Mitachi, Takafumi; Mezaki, Minori; Yamashita, Kunihiko; Itagaki, Hiroshi

2018-01-01

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model

PubMed Central

Lee, Young Bok; Kim, Su Jin; Park, Sae Mi; Lee, Kyung Ho; Han, Hyung Jin; Yu, Dong Soo; Woo, So Youn; Yun, Seong Taek; Hamm, Se-Yeong; Kim, Hong Jig

2016-01-01

Background Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. Objective This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. Methods The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Results Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4+ Foxp3+ regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Conclusion Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis. PMID:27081266

Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model.

PubMed

Lee, Young Bok; Kim, Su Jin; Park, Sae Mi; Lee, Kyung Ho; Han, Hyung Jin; Yu, Dong Soo; Woo, So Youn; Yun, Seong Taek; Hamm, Se-Yeong; Kim, Hong Jig; Kim, Jin-Wou

2016-04-01

Although the therapeutic mechanism of balneotherapy for atopic dermatitis has not been clarified, many atopic patients who visit thermomineral springs have shown clinical improvements. This study was aimed to evaluate the immunomodulatory effect of thermomineral water balneotherapy on the atopic dermatitis murine model. The oxazolone-induced atopic dermatitis murine model was used to evaluate the therapeutic effect of balneotherapy with Deokgu thermomineral water compared with distilled water. Histologic evaluation and confocal microscopic imaging were performed to analyze the lesional expression of cluster-of-differentiation (CD)4 and forkhead box p3 (Foxp3). Lesional mRNA expression of interleukin (IL) 33, thymic stromal lymphopoietin (TSLP), and Foxp3 was evaluated by real-time reverse transcription polymerase chain reaction. Compared with the distilled water bath group, confocal microscopic evaluation of CD4 and Foxp3 merged images showed increased expression of regulatory T cells in the thermomineral balneotherapy group. The lesional mRNA level of IL-33 showed a reduced trend in the thermomineral balneotherapy group, whereas the level of mRNA of Foxp3 was increased. TSLP showed a decreased trend in both distilled water and thermomineral water bath groups. There was a trend of reduced expression in lesional IL-33 mRNA but increased cell count of CD4(+) Foxp3(+) regulatory T cells in thermomineral balneotherapy compared with distilled water bath. Therefore, thermomineral balneotherapy can be an effective and safe adjuvant therapeutic option for atopic dermatitis.

Characterization of CD22 expression in acute lymphoblastic leukemia.

PubMed

Shah, Nirali N; Stevenson, Maryalice Stetler; Yuan, Constance M; Richards, Kelly; Delbrook, Cindy; Kreitman, Robert J; Pastan, Ira; Wayne, Alan S

2015-06-01

CD22 is a B-lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL). Properties of CD22 expression relevant to therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B-precursor (pre-B) ALL. CD22 expression was demonstrated in all subjects (n = 163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349-19,653, n = 160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349-3,624, n = 20 versus 3,853 sites/cell, range 451-19,653, n = 140; P = <0.0001) and 6 of 21 cases had sub-populations of blasts lacking CD22 expression (22%-82% CD22 +). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti-CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti-CD22 directed agents. These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy-refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22-negative blast population. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Anti-CD45 radioimmunotherapy using 211At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orozco, Johnnie J.; Back, Tom; Kenoyer, Aimee L.

2013-05-15

Anti-CD45 Radioimmunotherapy using an Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model ABSTRACT Despite aggressive chemotherapy combined with hematopoietic cell transplant (HCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using antibodies (Ab) labeled primarily with beta-emitting radionuclides has been explored to reduce relapse.

Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133- cells, and the CD133+ sector is enlarged by hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blazek, Ed R.; Foutch, Jennifer L.; Maki, Guitta

2007-01-01

Purpose: Primary medulloblastoma and glioblastoma multiforme tumor cells that express the surface marker CD133 are believed to be enriched for brain tumor stem cells because of their unique ability to initiate or reconstitute tumors in immunodeficient mice. This study sought to characterize the radiobiological properties and marker expression changes of CD133+ vs. CD133- cells of an established medulloblastoma cell line. Methods and Materials: Daoy and D283 Med cell lines were stained with fluorescently labeled anti-CD133 antibody and sorted into CD133+ and CD133- populations. The effect of oxygen (2% vs. 20%) on CD133 expression was measured. Both populations were analyzed formore » marker stability, cell cycle distribution, and radiosensitivity. Results: CD133+ Daoy cells restored nearly native CD133+ and CD133- populations within 18 days, whereas CD133- cells remained overwhelmingly CD133-. Culturing Daoy cells in 2% oxygen rather than the standard 20% oxygen increased their CD133 expression 1.6-fold. CD133+ Daoy cells were radioresistant via the {beta}-parameter of the linear-quadratic model relative to CD133- Daoy cells, although their {alpha}-parameters and cell cycle distributions were identical. Conclusions: Restoration of the original CD133+ and CD133- populations from CD133+ Daoy cells in serum is further evidence that CD133+ cells are functionally distinct from CD133- cells. The radioresistance of CD133+ compared with CD133- Daoy cells is consistent with better repair of sublethal damage. Enlargement of the CD133+ sector is a new feature of the hypoxic response.« less