Production of cloned embryos from caprine mammary epithelial cells expressing recombinant human β-defensin-3.

PubMed

Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-03-01

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells

PubMed Central

Lankford, Susan; Petty, Christopher; LaVoy, Alora; Reckling, Stacie; Tompkins, Wayne; Dean, Gregg A.

2008-01-01

Regulatory T cells (Treg) are increased and directly infected by feline immunodeficiency virus (FIV) and likely play a role in other feline autoimmune, neoplastic, and infectious diseases. Phenotypically, Treg are best characterized by surface expression of CD4 and CD25 and intranuclear expression of the forkhead transcription factor Foxp3. Our objective was to clone and sequence feline FOXP3 for the purpose of developing assays to enhance studies of feline Treg. We determined the feline FOXP3 is 1293 nucleotides in length and codes for a protein that shares high homology to other species. A splice variant devoid of exon 2 was also identified. A real-time PCR assay was developed and used to show Foxp3 mRNA expression occurs primarily in CD4+CD25+ T cells. Two cross-reacting antibodies were identified by immunocytochemical staining of HEK293 cells transfected with feline FOXP3. The antibody labeling confirmed the nuclear localization of the protein. A flow cytometric assay was also validated and used to correlate the phenotypic and functional characteristics of feline Treg induced by treatment of lymph node lymphocytes with flagellin or LPS in combination with mitogen or IL2. Together, these studies provide useful tools to further investigate Foxp3 and Tregs in cats. PMID:18180044

B cells as accessory cells in a Con A response of a T cell clone.

PubMed

Takeuchi, M; Kakiuchi, T; Taira, S; Nariuchi, H

1987-12-01

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo.

PubMed

Pestel, J; Dissous, C; Dessaint, J P; Louis, J; Engers, H; Capron, A

1985-06-01

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1990-01-01

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

PubMed Central

1993-01-01

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

PubMed Central

Hsu, Cary; Jones, Stephanie A.; Cohen, Cyrille J.; Zheng, Zhili; Kerstann, Keith; Zhou, Juhua; Robbins, Paul F.; Peng, Peter D.; Shen, Xinglei; Gomes, Theotonius J.; Dunbar, Cynthia E.; Munroe, David J.; Stewart, Claudia; Cornetta, Kenneth; Wangsa, Danny; Ried, Thomas; Rosenberg, Steven A.

2007-01-01

Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation. PMID:17353346

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

1997-12-02

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

1999-02-09

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

Accumulation of human T lymphotropic virus (HTLV)-I-specific T cell clones in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

PubMed

Höger, T A; Jacobson, S; Kawanishi, T; Kato, T; Nishioka, K; Yamamoto, K

1997-08-15

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraperesis (HAM/TSP) is a slowly progressive neurologic disorder following infection with HTLV-I. It is characterized by spasticity and hyper-reflexia of the lower extremities, urinary bladder disturbance, lower extremity muscle weakness, and sensory disturbances. HTLV-I, as an inducer of a strong humoral and cytotoxic response, is a well-known pathogenic factor for the progression of HAM/TSP. Peptides derived from proviral tax and env genes provide epitopes recognized by T cells. We herein report an accumulation of distinct clonotypes of alpha/beta TCR+ peripheral blood T lymphocytes from HAM/TSP patients in comparison with that observed in both asymptomatic carriers and healthy controls, using the reverse-transcriptase PCR/single-strand conformation polymorphism method. We also found that some of the accumulated T cell clones in the peripheral blood and cerebrospinal fluid are HTLV-I Tax(11-19) peptide specific. Such clones were found to expand strongly after being cultured with an HTLV-I Tax(11-19) peptide. Moreover, the cultured samples exhibited a strong MHC class I-restricted cytotoxic activity against HTLV-I Tax(11-19) peptide-expressing targets, and therefore most likely also include the disease-associated T cell clones observed in the patients. This is the first report of a direct assessment of Ag-specific T cell responses in fresh PBL and cerebrospinal fluid.

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

NASA Technical Reports Server (NTRS)

Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

1986-01-01

Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

Urushiol (poison ivy)-triggered suppressor T cell clone generated from peripheral blood.

PubMed Central

Kalish, R S; Morimoto, C

1988-01-01

Allergic contact dermatitis to Toxicodendron radicans (poison ivy) is mediated by the hapten urushiol. An urushiol-specific, interleukin 2 (IL-2)-dependent T cell clone (RLB9-7) was generated from the peripheral blood of a patient with a history of allergic contact dermatitis to T. radicans. This clone proliferated specifically to both leaf extract and pure urushiol. Although the clone had the phenotype CD3+CD4+CD8+, proliferation to antigen was blocked by anti-CD8 and anti-HLA-A, B, C, but not by anti-CD4, suggesting that CD4 was not functionally associated with the T cell receptor. Furthermore, studies with antigen-presenting cells from MHC-typed donors indicated that the clone was MHC class 1 restricted. RLB9-7 was WT31 positive, indicating it bears the alpha beta T cell receptor. The clone lacked significant natural killer cell activity and produced only low levels of IL-2 or gamma-interferon upon antigen stimulation. Addition of RLB9-7 to autologous peripheral blood mononuclear cells in the presence of urushiol inhibited the pokeweed mitogen-driven IgG synthesis. This suppression was resistant to irradiation (2,000 rad) and was not seen when RLB9-7 was added to allogeneic cells, even in the presence of irradiated autologous antigen-presenting cells, suggesting that suppression was MHC restricted and not mediated by nonspecific soluble factors. However, RLB9-7 cells in the presence of urushiol inhibited the synthesis of tetanus toxoid-specific IgG by autologous lymphocytes, indicating that the suppression, although triggered specifically by urushiol, was nonspecific. PMID:2458387

Reproductive performance and expression of imprinted genes in somatic cell cloned boars.

PubMed

Kawarasaki, Tatsuo; Enya, Satoko; Otake, Masayoshi; Shibata, Masatoshi; Mikawa, Satoshi

2017-11-01

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin-like growth factor -2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot-and-mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic. © 2017 Japanese Society of Animal Science.

Functional Analysis of Dendritic Cells Generated from T-iPSCs from CD4+ T Cell Clones of Sjögren's Syndrome.

PubMed

Iizuka-Koga, Mana; Asashima, Hiromitsu; Ando, Miki; Lai, Chen-Yi; Mochizuki, Shinji; Nakanishi, Mahito; Nishimura, Toshinobu; Tsuboi, Hiroto; Hirota, Tomoya; Takahashi, Hiroyuki; Matsumoto, Isao; Otsu, Makoto; Sumida, Takayuki

2017-05-09

Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells (DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Conformation-dependent recognition of a protein by T-lymphocytes: apomyoglobin-specific T-cell clone recognizes conformational changes between apomyoglobin and myoglobin

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

1988-01-01

A T-cell clone specific to apomyoglobin was generated. It was prepared from a T-cell culture obtained by in vitro driving of lymph node cells with apomyoglobin from SJL mice that have been primed in vivo with apomyoglobin. In proliferative assays, the T-cell clone responded to apomyoglobin but did not recognize native myoglobin or any of the synthetic peptides corresponding to the six T sites of myoglobin. The demonstration that a T-cell clone can be isolated, whose specificity is directed entirely to apomyoglobin and not to its counterpart myoglobin, with an identical amino acid composition, indicates the importance of the three-dimensional structure in the presentation of the protein to T cells.

Allogeneic substitution for nominal antigen-specific T-cell clone reactivity in schistosomiasis.

PubMed Central

Linette, G P; Lammie, P J; Phillips, S M

1986-01-01

The present studies have established the nature of a T-cell clone which demonstrates dual reactivity directed against Schistosoma mansoni antigen presented by syngeneic antigen presenting cells and against allogeneic cells. Clone G4, when stimulated by either antigen (SEA) or allogeneic cells (PL/J), exhibits similar functional and phenotypic characteristics. A subclone of G4, G4A.1, which has been maintained in continuous mixed lymphocyte culture for 12 months (in the absence of SEA), retains comparable reactivity with respect to proliferation and ability to produce lymphokines, transfer delayed-type hypersensitivity, and produce in vitro granulomas in response to SEA. Normal antigenic stimulation is highly contingent upon I-Ab compatibility while antibody blocking experiments map allo-reactivity to I-Eu. The failure of B10.PL spleen cells to stimulate G4, however, suggests that alloreactivity may be directed against the recently described Mls X locus. Both allogeneic and nominal antigen induced T-cell activation are blocked by antibody directed against L3T4A, confirming Class II MHC restriction for both types of stimulation. These studies suggest that stimulation of T cells by either alloantigen or nominal antigen elicits qualitatively similar functional profiles, and further suggest the feasibility of producing large numbers of nominal antigen reactive cloned T cells in the absence of nominal antigen under mixed lymphocyte culture conditions. PMID:2420707

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

PubMed

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection.

PubMed

Akazawa, Daisuke; Date, Tomoko; Morikawa, Kenichi; Murayama, Asako; Miyamoto, Michiko; Kaga, Minako; Barth, Heidi; Baumert, Thomas F; Dubuisson, Jean; Wakita, Takaji

2007-05-01

Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.

Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells.

PubMed

Ioannides, C G; Freedman, R S; Platsoucas, C D; Rashed, S; Kim, Y P

1991-03-01

CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA-1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer.

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

PubMed Central

Miltenburg, A M; Van Laar, J M; De Kuiper, P; Daha, M R; Breedveld, F C

1990-01-01

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. PMID:2148285

Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyer's patches.

PubMed

Tsuji, Noriko M; Mizumachi, Koko; Kurisaki, Jun-Ichi

2003-04-01

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

NASA Astrophysics Data System (ADS)

Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

1992-07-01

The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

Cloning of the transgenic pigs expressing human decay accelerating factor and N-acetylglucosaminyltransferase III.

PubMed

Fujimura, Tatsuya; Kurome, Mayuko; Murakami, Hiroshi; Takahagi, Yoichi; Matsunami, Katsuyoshi; Shimanuki, Shinichi; Suzuki, Kohei; Miyagawa, Shuji; Shirakura, Ryota; Shigehisa, Tamotsu; Nagashima, Hiroshi

2004-01-01

The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

PubMed

de Koster, H S; Vermeulen, C J; Hiemstra, H S; Amons, R; Drijfhout, J W; Koning, F

1999-04-01

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression.

PubMed

Xu, Huanbin; Wang, Xiaolei; Pahar, Bapi; Moroney-Rasmussen, Terri; Alvarez, Xavier; Lackner, Andrew A; Veazey, Ronald S

2010-12-15

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

PubMed

Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

2007-02-01

To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

T-helper cell receptors from long-term survivors after telomerase cancer vaccination for use in adoptive cell therapy.

PubMed

Kyte, Jon Amund; Gaudernack, Gustav; Faane, Anne; Lislerud, Kari; Inderberg, Else Marit; Brunsvig, Paal; Aamdal, Steinar; Kvalheim, Gunnar; Wälchli, Sébastien; Pule, Martin

2016-01-01

We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4 + Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted T-cell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNγ, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4 + and CD8 + T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.

Identification of candidate vaccine antigens of bovine hemoparasites Theileria parva and Babesia bovis by use of helper T cell clones.

PubMed

Brown, W C; Zhao, S; Logan, K S; Grab, D J; Rice-Ficht, A C

1995-03-01

reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.

Desmoglein 3-specific T regulatory 1 cells consist of two subpopulations with differential expression of the transcription factor Foxp3

PubMed Central

Veldman, Christian; Pahl, Andreas; Hertl, Michael

2009-01-01

Pemphigus vulgaris (PV) is an autoimmune bullous skin disorder associated with autoantibodies against desmoglein (Dsg) 3. An imbalance of type 1 regulatory T (Tr1) cells and T helper type 2 (Th2) cells specific for Dsg3 may be critical for the loss of tolerance against Dsg3 in PV. Within the population of Dsg3-responsive, interleukin (IL)-10-secreting Tr1 cell clones, two major subpopulations were identified and sorted by fluorescence-activated cell sorting (FACS) based on their size and granularity. Upon in vitro culture, the larger subpopulation differentiated back into the two former subpopulations of the Tr1 cell clones, while the smaller subpopulation died within 2 weeks. The smaller subpopulation of the Tr1 cell clones was characterized by the expression of Foxp3, the secretion of IL-10, transforming growth factor (TGF)-β and IL-5 upon stimulation with Dsg3, a proliferative response to IL-2 but not to Dsg3 or mitogenic stimuli, and an inhibitory effect on the proliferative response of Dsg3-responsive Th clones in a Dsg3-specific manner. In contrast, the larger subpopulation showed a Th-like phenotype, lacking Foxp3, cytotoxic T-lymphocyte antigen 4 (CTLA4) and glucocorticoid-induced tumour necrosis factor receptor (GITR) expression and IL-2 secretion, and did not mount a proliferative response to Dsg3 and mitogenic stimuli. The two Tr1 subpopulations showed expression of identical T-cell receptor (TCR) Vβ chains which varied among the PV patients studied. Upon inhibition of Foxp3, the smaller Tr1 subpopulation developed a proliferate response to Dsg3 and mitogenic stimuli, no longer suppressed Dsg3-specific Th cells, lost expression of GITR and CTLA4 and secreted IL-2. Thus, our observations suggest a distinct relationship between Dsg3-specific Tr1 and Th-like cells which may be critical for the continuous generation and survival of Dsg3-specific Tr1 cells. PMID:18800988

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

[TSA improve transgenic porcine cloned embryo development and transgene expression].

PubMed

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

PubMed Central

Ebrahimi, Mina; Kazemi, Tohid; Ganjalikhani-hakemi, Mazdak; Majidi, Jafar; khanahmad, Hossein; Rahimmanesh, Ilnaz; Homayouni, Vida; Kohpayeh, Shirin

2015-01-01

Background Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1. PMID:28959306

Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

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

Londei, M.; Savill, C.M.; Verhoef, A.

Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovialmore » tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis.« less

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

PubMed

Wang, Fen; Ye, Bin

2016-10-01

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

Cloning and expression of the gene for bacteriophage T7 RNA polymerase

DOEpatents

Studier, F.W.; Davanloo, P.; Rosenberg, A.H.

1984-03-30

This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties.

Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity

PubMed Central

1989-01-01

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN- gamma after stimulation with dengue 3 antigens, and also produced IFN- gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

PubMed

Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

2007-05-15

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Characteristics of Prevotella intermedia-specific CD4+ T cell clones from peripheral blood of a chronic adult periodontitis patient

PubMed Central

Wassenaar, A; Reinhardus, C; Abraham-Inpijn, L; Snijders, A; Kievits, F

1998-01-01

Periodontitis is a chronic destructive inflammatory disease associated with periodontopathic bacteria. In addition, autoantigens such as collagen and heat shock proteins (hsp) have been suggested to play a role. Established periodontal lesions are characterized by dense infiltrations of immune cells such as cytokine-producing CD4+ and CD8+ T cells. CD4+ T cells specific for Prevotella intermedia can be isolated from lesional gingiva, suggesting an active role for CD4+ T cells in the response to this bacterium. We therefore investigated the characteristics of a panel of 13 P. intermedia-specific CD4+ T cells generated from the peripheral blood of a patient with chronic adult periodontitis. All 13 P. intermedia-specific CD4+ T cells recognized the antigens in the context of HLA-DR. The T cell clones were mainly classified as Th0, producing comparable amounts of interferon-gamma (IFN-γ) and IL-4, and Th2, producing high amounts of IL-4 and almost no IFN-γ. None of the P. intermedia-specific T cell clones recognized antigens of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis and of the autoantigens collagen and hsp. The reactivity profile of the T cell clones to size-fractionated cell envelope antigens of P. intermedia indicated that P. intermedia-specific CD4+ T cell clones recognize probably five different antigen specificities in the context of the MHC class II molecules, DR7 or DR15. These results suggest that a broad panel of cell-associated protein antigens play a role in the induction of P. intermedia-specific CD4+ T cell response. PMID:9697992

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT.

PubMed

Sununliganon, Laddawun; Singhatanadgit, Weerachai

2012-01-01

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

PubMed Central

Neophytou, P I; Roep, B O; Arden, S D; Muir, E M; Duinkerken, G; Kallan, A; de Vries, R R; Hutton, J C

1996-01-01

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases. PMID:8700877

Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase.

PubMed

van Gennip, H G; van Rijn, P A; Widjojoatmodjo, M N; Moormann, R J

1999-03-01

A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.

Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase.

PubMed

Yi, Jicai; Liu, Lanna; Cao, Youpei; Li, Jiazuo; Mei, Mantong

2013-12-01

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

PubMed Central

Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

2015-01-01

Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

PubMed

Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

PubMed

Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

2016-02-01

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells.

PubMed

Ghosh-Choudhury, N; Butcher, M; Ghosh, H P

1990-03-01

A DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.

PubMed

Matsunari, Hitomi; Onodera, Masafumi; Tada, Norihiro; Mochizuki, Hideki; Karasawa, Satoshi; Haruyama, Erika; Nakayama, Naoki; Saito, Hitoshi; Ueno, Satoshi; Kurome, Mayuko; Miyawaki, Atsushi; Nagashima, Hiroshi

2008-09-01

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Human immunodeficiency virus infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and interleukin 4 secretion.

PubMed Central

Laurence, J; Friedman, S M; Chartash, E K; Crow, M K; Posnett, D N

1989-01-01

HIV selectively inhibited the proliferative response of clonal CD4+ T lymphocytes to alloantigen while other alloantigen-dependent responses were unperturbed. Specifically, impaired blastogenesis could be dissociated from alloantigen-specific induction of the B cell activation molecule CD23, IL-4 release, and inositol lipid hydrolysis. In addition, membrane expression of pertinent T cell receptor molecules, including CD2, CD3, and T cell antigen receptor (Ti), remained intact. Using two MHC class II-specific human CD4+ helper T cell clones, the proliferative defect was shown to be an early consequence of HIV infection, occurring within 4 d of viral inoculation and preceding increases in mature virion production. It was generalizable to three distinct methods of T cell activation, all independent of antigen-presenting cells: anti-CD3 mediated cross-linking of the CD3/Ti complex; anti-CD2 and phorbol 12-myristic 13-acetate (PMA); and anti-CD28 plus PMA. These abnormalities were not mitigated by addition of exogenous IL-2, even though expression of the IL-2 receptor (CD25) was unaltered. These studies define a selective blockade in T cell function early after HIV exposure that could serve as a model for certain in vivo manifestations of AIDS. PMID:2470786

Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies

PubMed Central

Peifang, S.; Pira, G. L.; Fenoglio, D.; Harris, S.; Costa, M. G.; Venturino, V.; Dessì, V.; Layton, G.; Laman, J.; Huisman, J. G.; Manca, F.

1994-01-01

Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4+ T cell lines and clones. VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization. It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones. Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells. Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety. This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct. PMID:7915974

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

PubMed Central

Yoshimura, M; Cooper, D M

1992-01-01

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+. Images PMID:1379717

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

PubMed

Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

2018-01-26

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

PubMed

Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

PubMed Central

O'Connor, C D; Timmis, K N

1987-01-01

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational. Images PMID:2443481

Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

PubMed Central

Farshadpour, Fatemeh; Makvandi, Manoochehr; Taherkhani, Reza

2015-01-01

Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. PMID

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

PubMed Central

Mamalaki, Clio; Elliott, James; Norton, Trisha; Yannoutsos, Nicholas; Townsend, Alain R.; Chandler, Phillip; Simpson, Elizabeth

1993-01-01

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired. PMID:8281031

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence.

PubMed

Mazzatti, Dawn J; Pawelec, Graham; Longdin, Robin; Powell, Jonathan R; Forsey, Rosalyn J

2007-06-05

The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays. Discriminant analysis identified several protein or peptide peaks in the region of 14.5-16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis. Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.

Properties of HTLV-I transformed CD8+ T-cells in response to HIV-1 infection.

PubMed

Gulzar, N; Shroff, A; Buberoglu, B; Klonowska, D; Kim, J E; Copeland, K F T

2010-10-25

HIV-1 infection studies of primary CD8(+) T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8(+) T-cells in the context of HIV-1 infection. In this study, a set of CD8(+) T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4(+) T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8(+) T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8(+) T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8(+) T-cell dysfunction. Copyright © 2010 Elsevier Inc. All rights reserved.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

1988-01-01

Non-immune SJL (H-2s) spleen cells were fused with (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B- cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones were able to present both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Zouhair Atassi, M. Z.

1989-01-01

Non-immune SJL (H-2s) spleen cells were fused with non-secreting, non-antigen presenting (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B-cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones presented both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

The proliferative response of cloned Peyer's patch switch T cells to syngeneic and allogeneic stimuli.

PubMed

Kawanishi, H; Ozato, K; Strober, W

1985-06-01

We previously defined a concanavalin A (Con A)-induced cloned T cell population in Peyer's patches (PP) that causes sIgM-bearing B cells to switch to sIgA-bearing B cells. In the present study we show that such IgA-specific switch T cells proliferate when exposed to syngeneic stimulator cells, i.e., the switch T cells are autoreactive. Detailed study of this phenomenon disclosed that both B cells and macrophages were capable of causing switch T cell proliferation, and in both cases, stimulation was enhanced by preactivation of the stimulator cells with lipopolysaccharide (LPS). In addition, fresh T cells can act as stimulators, but only if preactivated with Con A. Finally, it was clearly shown in blocking studies with the use of various antibodies directed at class II MHC specificities that class II MHC antigens were the stimulatory determinants. These studies suggest that IgA-specific switch T cells arise in PP as a result of autologous cell-cell interactions with activated (antigen-stimulated) B cells, macrophages, or T cells.

Lmo2 expression defines tumor cell identity during T-cell leukemogenesis.

PubMed

García-Ramírez, Idoia; Bhatia, Sanil; Rodríguez-Hernández, Guillermo; González-Herrero, Inés; Walter, Carolin; González de Tena-Dávila, Sara; Parvin, Salma; Haas, Oskar; Woessmann, Wilhelm; Stanulla, Martin; Schrappe, Martin; Dugas, Martin; Natkunam, Yasodha; Orfao, Alberto; Domínguez, Verónica; Pintado, Belén; Blanco, Oscar; Alonso-López, Diego; De Las Rivas, Javier; Martín-Lorenzo, Alberto; Jiménez, Rafael; García Criado, Francisco Javier; García Cenador, María Begoña; Lossos, Izidore S; Vicente-Dueñas, Carolina; Borkhardt, Arndt; Hauer, Julia; Sánchez-García, Isidro

2018-06-07

The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

CLONING AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS FROM THE RAW264.7 CELL LINE

PubMed Central

Cuetara, Bethany L. V.; Crotti, Tania N.; O'Donoghue, Anthony J.

2006-01-01

SUMMARY Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell culture, which are poorly suited to molecular and transgene studies due to the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP) positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP positive multinuclear cells. Clones capable of forming large TRAP positive multinuclear cells also expressed β3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation. PMID:16948499

IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules.

PubMed

Knolle, P A; Uhrig, A; Hegenbarth, S; Löser, E; Schmitt, E; Gerken, G; Lohse, A W

1998-12-01

Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-gamma) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2 (PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver.

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

PubMed

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed Central

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-01-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

PubMed

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-04-07

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

PubMed Central

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-01-01

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. PMID:20150895

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

PubMed Central

1989-01-01

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy. PMID:2793940

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

PubMed

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

PubMed Central

Catakovic, Kemal; Gassner, Franz Josef; Ratswohl, Christoph; Zaborsky, Nadja; Rebhandl, Stefan; Schubert, Maria; Steiner, Markus; Gutjahr, Julia Christine; Pleyer, Lisa; Egle, Alexander; Hartmann, Tanja Nicole; Greil, Richard; Geisberger, Roland

2018-01-01

ABSTRACT While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment. PMID:29296521

Persistence of Multiple Tumor-Specific T-Cell Clones Is Associated with Complete Tumor Regression in a Melanoma Patient Receiving Adoptive Cell Transfer Therapy

PubMed Central

Zhou, Juhua; Dudley, Mark E.; Rosenberg, Steven A.; Robbins, Paul F.

2007-01-01

Summary The authors recently reported that adoptive immunotherapy with autologous tumor-reactive tumor infiltrating lymphocytes (TILs) immediately following a conditioning nonmyeloablative chemotherapy regimen resulted in an enhanced clinical response rate in patients with metastatic melanoma. These observations led to the current studies, which are focused on a detailed analysis of the T-cell antigen reactivity as well as the in vivo persistence of T cells in melanoma patient 2098, who experienced a complete regression of all metastatic lesions in lungs and soft tissues following therapy. Screening of an autologous tumor cell cDNA library using transferred TILs resulted in the identification of novel mutated growth arrest-specific gene 7 (GAS7) and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts. Direct sequence analysis of the expressed T-cell receptor beta chain variable regions showed that the transferred TILs contained multiple T-cell clonotypes, at least six of which persisted in peripheral blood for a month or more following transfer. The persistent T cells recognized both the mutated GAS7 and GAPDH. These persistent tumor-reactive T-cell clones were detected in tumor cell samples obtained from the patient following adoptive cell transfer and appeared to be represented at higher levels in the tumor sample obtained 1 month following transfer than in the peripheral blood obtained at the same time. Overall, these results indicate that multiple tumor-reactive T cells can persist in the peripheral blood and at the tumor site for prolonged times following adoptive transfer and thus may be responsible for the complete tumor regression in this patient. PMID:15614045

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

PD-1 Blockade Promotes Epitope Spreading in Anticancer CD8+ T Cell Responses by Preventing Fratricidal Death of Subdominant Clones To Relieve Immunodomination.

PubMed

Memarnejadian, Arash; Meilleur, Courtney E; Shaler, Christopher R; Khazaie, Khashayarsha; Bennink, Jack R; Schell, Todd D; Haeryfar, S M Mansour

2017-11-01

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8 + T cell (T CD8 ) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse T CD8 exhaustion/anergy. However, whether they alter the epitope breadth of T CD8 responses remains unclear. This is an important question because subdominant T CD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of T CD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant T CD8 , which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant T CD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I-restricted cytotoxicity. The selective increase in subdominant T CD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti-PD-1 invigorates subdominant T CD8 responses by relieving their lysis-dependent suppression by immunodominant T CD8 To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination. Copyright © 2017 by The American Association of Immunologists, Inc.

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.

Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

PubMed

Hoogendoorn, M; Wolbers, J Olde; Smit, W M; Schaafsma, M R; Barge, R M Y; Willemze, R; Falkenburg, J H F

2004-07-01

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

PubMed

Takeda, Kazuyoshi; Kitaura, Kazutaka; Suzuki, Ryuji; Owada, Yuki; Muto, Satoshi; Okabe, Naoyuki; Hasegawa, Takeo; Osugi, Jun; Hoshino, Mika; Tsunoda, Takuya; Okumura, Ko; Suzuki, Hiroyuki

2018-06-01

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca 2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.

Vertebrate Cells Express Protozoan Antigen after Hybridization

NASA Astrophysics Data System (ADS)

Crane, Mark St. J.; Dvorak, James A.

1980-04-01

Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

Antitumor effects of a tumor cell vaccine expressing a membrane-bound form of the IL-12 p35 subunit.

PubMed

Lim, Ho Yong; Ju, Hee Young; Chung, Hee-Yong; Kim, Young Sang

2010-08-15

We investigated whether expression of the IL-12 p35 subunit in membrane-bound form in tumor cells enhanced their immunogenicity. Since p35 is only secreted when associated with the IL-12 p40 subunit, we generated tumor cells expressing membrane-bound forms of p35 and p40 as chimeras with the transmembrane/cytoplasmic region of TNFα (mbIL-12p35 and mbIL-12p40). The relevant vectors were transfected into MethA fibrosarcoma cells, and mbIL-12p35 or mbIL-12p40-expressing tumor clones were isolated and their ability to induce antitumor immunity studied. Cells of the mbIL-12p35 tumor clone induced CD69 expression and IFNγ production in purified CD8(+) T cells in vitro, and their in vivo tumorigenicity was reduced. Cells of the mbIL-12p40 tumor clone failed to show either of these activities. Mice that had rejected cells of the mbIL-12p35 tumor clone possessed systemic antitumor immunity to wild type tumor cells. The growth rate of mbIL-12p35 tumor cells was greater in CD8(+) T cell-depleted mice than in CD4(+) T-cell- and NK cell-depleted mice or normal mice, suggesting that CD8(+) T cells were mainly responsible for the antitumor immunity. These results indicate that expression of mbIL-12p35 on tumor cells enhances their immunogenicity by increasing their ability to activate CD8(+) T cells, possibly by direct priming.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

PubMed Central

2012-01-01

Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS. PMID:22480370

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

PubMed

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: molecular cloning and functional characterization.

PubMed

Huang, Xian-De; Wei, Guo-jian; Zhang, Hua; He, Mao-Xian

2015-01-01

Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of a G protein coupled receptor induced in activated T cells.

PubMed

Kaplan, M H; Smith, D I; Sundick, R S

1993-07-15

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

PubMed

Cerosaletti, Karen; Barahmand-Pour-Whitman, Fariba; Yang, Junbao; DeBerg, Hannah A; Dufort, Matthew J; Murray, Sara A; Israelsson, Elisabeth; Speake, Cate; Gersuk, Vivian H; Eddy, James A; Reijonen, Helena; Greenbaum, Carla J; Kwok, William W; Wambre, Erik; Prlic, Martin; Gottardo, Raphael; Nepom, Gerald T; Linsley, Peter S

2017-07-01

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4 + memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4 + T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4 + memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood. Copyright © 2017 by The American Association of Immunologists, Inc.

Effect of Cytomegalovirus (CMV) and Ageing on T-Bet and Eomes Expression on T-Cell Subsets.

PubMed

Hassouneh, Fakhri; Lopez-Sejas, Nelson; Campos, Carmen; Sanchez-Correa, Beatriz; Tarazona, Raquel; Pera, Alejandra; Solana, Rafael

2017-06-29

The differential impact of ageing and cytomegalovirus (CMV) latent infection on human T-cell subsets remains to some extent controversial. The purpose of this study was to analyse the expression of the transcription factors T-bet and Eomes and CD57 on CD4+, CD4 hi CD8 lo and CD8+ T-cell subsets in healthy individuals, stratified by age and CMV serostatus. The percentage of CD4+ T-cells expressing T-bet or Eomes was very low, in particular in CD4+ T-cells from young CMV-seronegative individuals, and were higher in CMV-seropositive older individuals, in both CD57- and CD57+ CD4+ T-cells. The study of the minor peripheral blood double-positive CD4 hi CD8 lo T-cells showed that the percentage of these T-cells expressing both Eomes and T-bet was higher compared to CD4+ T-cells. The percentage of CD4 hi CD8 lo T-cells expressing T-bet was also associated with CMV seropositivity and the coexpression of Eomes, T-bet and CD57 on CD4 hi CD8 lo T-cells was only observed in CMV-seropositive donors, supporting the hypothesis that these cells are mature effector memory cells. The percentage of T-cells expressing Eomes and T-bet was higher in CD8+ T-cells than in CD4+ T-cells. The percentages of CD8+ T-cells expressing Eomes and T-bet increased with age in CMV-seronegative and -seropositive individuals and the percentages of CD57- CD8+ and CD57+ CD8+ T-cells coexpressing both transcription factors were similar in the different groups studied. These results support that CMV chronic infection and/or ageing are associated to the expansion of highly differentiated CD4+, CD4 hi CD8 lo and CD8+ T-cells that differentially express T-bet and Eomes suggesting that the expression of these transcription factors is essential for the generation and development of an effector-memory and effector T lymphocytes involved in conferring protection against chronic CMV infection.

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

PubMed

Graser, R T; DiLorenzo, T P; Wang, F; Christianson, G J; Chapman, H D; Roopenian, D C; Nathenson, S G; Serreze, D V

2000-04-01

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-05-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

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

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesusmore » monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.« less

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

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

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

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.

Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells.

PubMed

Takeshige, Keiko; Sekido, Takashi; Kitahara, Jun-ichirou; Ohkubo, Yousuke; Hiwatashi, Dai; Ishii, Hiroaki; Nishio, Shin-ichi; Takeda, Teiji; Komatsu, Mitsuhisa; Suzuki, Satoru

2014-01-01

μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells.

PubMed

Zaborsky, Nadja; Gassner, Franz Josef; Asslaber, Daniela; Reinthaler, Petra; Denk, Ursula; Flenady, Sabine; Hofbauer, Josefina Piñón; Danner, Barbara; Rebhandl, Stefan; Harrer, Andrea; Geisberger, Roland; Greil, Richard; Egle, Alexander

2016-08-02

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

PubMed

van den Heuvel, Heleen; Heutinck, Kirstin M; van der Meer-Prins, Ellen M W; Franke-van Dijk, Marry E I; van Miert, Paula P M C; Zhang, Xiaoqian; Ten Berge, Ineke J M; Claas, Frans H J

2018-01-01

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8 + T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8 + T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

Characterization of CTLA-4 Structure and Expression on Human T Cells

DTIC Science & Technology

1993-10-01

prevents induction of anergy in T-cell plastic B cells. J. Immunol. 143:2714. clones. Nature 356:607. 7. Selvakumar , A., B. K. Mohanraj, R . L. Eddy, T... r ~n~~1 Form Approved ,, "൘ rmi OCUMENTATION PAGE orm Ap.r•ved Onorraoe is estimated Ic Ae.C.. I e 1C.;W fp$ei . ý’~t.cr.cenq Ile Urn* fo 1’ e...associated antigen," CTLA-4 (13). The genes for the geninterestid to the r 5 iacids o both human and mouse CI’IA-4 share a similar exon and J3

Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells.

PubMed

Wu, S Vincent; Rozengurt, Nora; Yang, Moon; Young, Steven H; Sinnett-Smith, James; Rozengurt, Enrique

2002-02-19

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

Changes in the expression of potassium channels during mouse T cell development

PubMed Central

1986-01-01

In this report we have combined the whole-cell electrophysiological recording technique with flow microfluorometry to isolate phenotypically defined thymocytes and T lymphocytes. Results obtained showed that J11d-/Lyt-2-/L3T4- cells express none or very few delayed rectifier K+ channels, whereas most other Lyt-2-/L3T4- cells, as well as typical cortical thymocytes (Lyt-2+/L3T4+), do express K+ channels. Mature (Lyt-2+/L3T4- or Lyt-2-/L3T4+) thymocytes, which are heterogeneous for J11d expression, were also found to be heterogeneous for K+ channel expression. Consistent with this finding was the observation that the cortisone-resistant subpopulation of thymocytes, which express low levels of J11d, were enriched for cells expressing low levels of K+ channels. Mature phenotype peripheral T lymphocytes expressed very low levels of K+ channels, but upon activation with Con A were found to express high levels of K+ channels. The results suggest that K+ channel expression in T cells is developmentally regulated. Increased expression of the channel is induced in response to mitogenic signals throughout the T cell lineage. Expression of the channel, therefore, serves as a useful marker in defining steps in the T cell differentiation pathway. PMID:2431091

Functional analysis of T cells expressing Ia antigens. I. Demonstration of helper T-cell heterogeneity.

PubMed

Swierkosz, J E; Marrack, P; Kappler, J W

1979-12-01

We have examined the expression of I-region antigens on functional subpopulations of murine T cells. A.TH anti-A.TL (anti-Ik, Sk, Gk) alloantiserum was raised by immunization of recipients with concanavalin A (Con A) stimulated thymic and peripheral T-cell blasts. In contrast to similar antisera made by conventional methods, the anti-Ia blast serum was highly cytotoxic for purified T lymphocytes. Moreover, it reacted in a specific fashion with T cells having particular functions. Treatment of keyhole limpet hemocyanin (KLH)-primed B10.A (H-2 alpha) T cells with this antiserum plus complement resulted in the elimination of helper activity for B-cell responses to trinitrophenyl-KLH. Inhibition was shown to be a result of the selective killing of one type of helper T cell whose activity could be replaced by a factor(s) found in the supernate of Con A-activated spleen cells. A second type of helper cell required for responses to protein-bound antigens appeared to be Ia-. By absorption and analysis on H-2 recombinants, at least two specificities were detectable on helper T cells; one mapping in the I-A subregion and a second in a region(s) to the right of I-J. In addition, the helper T cell(s) involved in the generation of alloreactive cytotoxic lymphocytes was shown to be Ia+, whereas cytotoxic effector cells and their precursors were Ia- with this antiserum. These results provide strong evidence for the selective expression of I-region determinants on T-cell subsets and suggest that T-cell-associated Ia antigens may play an important role in T-lymphocyte function.

Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells

PubMed Central

Weiss, Jonathan M.; Bilate, Angelina M.; Gobert, Michael; Ding, Yi; Curotto de Lafaille, Maria A.; Parkhurst, Christopher N.; Xiong, Huizhong; Dolpady, Jayashree; Frey, Alan B.; Ruocco, Maria Grazia; Yang, Yi; Floess, Stefan; Huehn, Jochen; Oh, Soyoung; Li, Ming O.; Niec, Rachel E.; Rudensky, Alexander Y.; Dustin, Michael L.; Littman, Dan R.

2012-01-01

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-β. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1low. We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease. PMID:22966001

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

PubMed Central

Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

1986-01-01

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells. Images PMID:3785169

Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

PubMed

Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

2013-05-01

Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

IRF-4 and c-Rel expression in antiviral-resistant adult T-cell leukemia/lymphoma

PubMed Central

Ramos, Juan Carlos; Ruiz, Phillip; Ratner, Lee; Reis, Isildinha M.; Brites, Carlos; Pedroso, Celia; Byrne, Gerald E.; Toomey, Ngoc L.; Andela, Valentine; Harhaj, Edward W.; Lossos, Izidore S.

2007-01-01

Adult T-cell leukemia/lymphoma (ATLL) is a generally fatal malignancy. Most ATLL patients fare poorly with conventional chemotherapy; however, antiviral therapy with zidovudine (AZT) and interferon alpha (IFN-α) has produced long-term clinical remissions. We studied primary ATLL tumors and identified molecular features linked to sensitivity and resistance to antiviral therapy. Enhanced expression of the proto-oncogene c-Rel was noted in 9 of 27 tumors. Resistant tumors exhibited c-Rel (6 of 10; 60%) more often than did sensitive variants (1 of 9; 11%). This finding was independent of the disease form. Elevated expression of the putative c-Rel target, interferon regulatory factor-4 (IRF-4), was observed in 10 (91%) of 11 nonresponders and in all tested patients with c-Rel+ tumors and occurred in the absence of the HTLV-1 oncoprotein Tax. In contrast, tumors in complete responders did not express c-Rel or IRF-4. Gene rearrangement studies demonstrated the persistence of circulating T-cell clones in long-term survivors maintained on antiviral therapy. The expression of nuclear c-Rel and IRF-4 occurs in the absence of Tax in primary ATLL and is associated with antiviral resistance. These molecular features may help guide treatment. AZT and IFN-α is a suppressive rather than a curative regimen, and patients in clinical remission should remain on maintenance therapy indefinitely. PMID:17138822

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

PubMed Central

Laurence, J; Kulkosky, J; Friedman, S M; Posnett, D N; Ts'o, P O

1987-01-01

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS. Images PMID:2960696

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in

The globally disseminated M1T1 clone of Group A Streptococcus evades autophagy for intracellular replication

PubMed Central

Barnett, Timothy C.; Liebl, David; Seymour, Lisa M.; Gillen, Christine M.; Lim, Jin Yan; LaRock, Christopher N.; Davies, Mark R.; Schulz, Benjamin L.; Nizet, Victor; Teasdale, Rohan D.; Walker, Mark J.

2014-01-01

SUMMARY Autophagy is reported to be an important innate immune defence against the intracellular bacterial pathogen Group A Streptococcus (GAS). However, the GAS strains examined to-date belong to serotypes infrequently associated with human disease. We find that the globally disseminated serotype M1T1 clone of GAS can evade autophagy and replicate efficiently in the cytosol of infected cells. Cytosolic M1T1 GAS (strain 5448), but not M6 GAS (strain JRS4), avoids ubiquitylation and recognition by the host autophagy marker LC3 and ubiquitin-LC3 adaptor proteins NDP52, p62 and NBR1. Expression of SpeB, a streptococcal cysteine protease, is critical for this process, as an isogenic M1T1 ΔspeB mutant is targeted to autophagy and attenuated for intracellular replication. SpeB degrades p62, NDP52 and NBR1 in vitro and within the host cell cytosol. These results uncover a proteolytic mechanism utilized by GAS to escape the host autophagy pathway which may underpin the success of the M1T1 clone. PMID:24331465

CD30 Expression by B and T Cells: A Frequent Finding in Angioimmunoblastic T-Cell Lymphoma and Peripheral T-Cell Lymphoma-Not Otherwise Specified.

PubMed

Onaindia, Arantza; Martínez, Nerea; Montes-Moreno, Santiago; Almaraz, Carmen; Rodríguez-Pinilla, Socorro M; Cereceda, Laura; Revert, Jose B; Ortega, César; Tardio, Antoni; González, Lucía; García, Sonia; Camacho, Francisca I; González-Vela, Carmen; Piris, Miguel A

2016-03-01

CD30 expression in peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) is currently of great interest because therapy targeting CD30 is of clinical benefit, but the clinical and therapeutic relevance of CD30 expression in these neoplasms still remains uncertain. The aim of this study was to better quantify CD30 expression in AITL and PTCL-not otherwise specified (NOS). The secondary objective was to determine whether CD30 cells exhibit a B-cell or a T-cell phenotype. Gene expression profiling was studied in a series of 37 PTCL cases demonstrating a continuous spectrum of TNFRSF8 expression. This prompted us to study CD30 immunohistochemical (IHC) expression and mRNA levels by reverse transcription polymerase chain reaction (RT-PCR) in a different series of 51 cases (43 AITLs and 8 PTCL-NOSs) in routine samples. Double stainings with PAX5/CD30, CD3/CD30, and LEF1/CD30 were performed to study the phenotype of CD30 cells. Most (90%) of the cases showed some level of CD30 expression by IHC (1% to 95%); these levels were high (>50% of tumoral cells) in 14% of cases. CD30 expression was not detected in 10% of the cases. Quantitative RT-PCR results largely confirmed these findings, demonstrating a moderately strong correlation between global CD30 IHC and mRNA levels (r=0.65, P=1.75e-7). Forty-four of the positive cases (98%) contained CD30-positive B cells (PAX5), whereas atypical CD30-positive T cells were detected in 42 cases (93%). In conclusion, our data show that most AITL and PTCL-NOS cases express CD30, exhibiting very variable levels of CD30 expression that may be measured by IHC or RT-PCR techniques.

T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

PubMed

Sanecka, Anna; Yoshida, Nagisa; Kolawole, Elizabeth Motunrayo; Patel, Harshil; Evavold, Brian D; Frickel, Eva-Maria

2018-01-01

T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii- infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103 + ) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

Differential expression of GPR15 on T cells during ulcerative colitis

PubMed Central

Adamczyk, Alexandra; Gageik, Daniel; Frede, Annika; Pastille, Eva; Hansen, Wiebke; Rueffer, Andreas; Buer, Jan; Büning, Jürgen; Langhorst, Jost

2017-01-01

G protein–coupled receptor 15 (GPR15) was recently highlighted as a colon-homing receptor for murine and human CD4+ T cells. The aim of this study was to explore the functional phenotype of human GPR15+CD4+ T cells, focusing on Tregs and effector T cells (Teffs), and to determine whether GPR15 is the driver for the migration of T cells to the colon during ulcerative colitis (UC). In the peripheral blood, GPR15 was expressed on Tregs and Teffs; both GPR15+ T cell subsets produced less IFN-γ and IL-4 but more IL-17 after stimulation and showed a higher migration activity compared with GPR15–CD4+ T cells. In UC patients, GPR15 expression was increased on Tregs in the peripheral blood but not on Teffs. Interestingly, the expression of GPR15 was significantly enhanced on colonic T cells of UC patients in noninflamed biopsies but not in inflamed biopsies. The differential expression of GPR15 in UC patients was accompanied by a significant reduction of bacterial immunoregulatory metabolites in the feces. In conclusion, GPR15 expression on CD4+ T cells is altered in UC patients, which may have implications for the development of therapeutic approaches to target T cell trafficking to the colon. PMID:28422750

Molecular cloning and expression in mammalian cells of ricin B chain

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

Chang, M.

1987-01-01

In these studies, the cDNA encoding the B chain of ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with {sup 35}S-methionine and {sup 35}S-cysteine and demonstrating secretion of a protein with a Mr of 30-32,000 which was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B chain antibody. The amount of recombinant B chain secreted by the COS-M6 cells was determined by radioimmunoassay to be 1-10 ng/ml of media. Virtually all the recombinant B chain formedmore » active ricin when mixed with native A chain; it could also bind as effectively as native B chain to the galactose-containing glycoprotein, asialofetuin. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function.« less

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

Human Naive T Cells Express Functional CXCL8 and Promote Tumorigenesis.

PubMed

Crespo, Joel; Wu, Ke; Li, Wei; Kryczek, Ilona; Maj, Tomasz; Vatan, Linda; Wei, Shuang; Opipari, Anthony W; Zou, Weiping

2018-05-25

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4 + T cells in umbilical cord and peripheral blood. We found that naive CD4 + T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8 + naive T cells were preferentially enriched CD31 + T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses. Copyright © 2018 by The American Association of Immunologists, Inc.

Clonal predominance of CD8+ T cells in patients with unexplained neutropenia

PubMed Central

Wlodarski, Marcin Wojciech; Nearman, Zachary; Jiang, Ying; Lichtin, Alan; Maciejewski, Jaroslaw Pawel

2008-01-01

Objective T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. Materials and Methods Using rational algorithms for T-cell receptor analysis and in vivo tracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-g expression in CD8+cells using Taqman polymerase chain reaction. Results Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-γ in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones. Conclusions These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the “extreme” end of the clonal continuum. PMID:18279717

CXCR6 is expressed on T cells in both T helper type 1 (Th1) inflammation and allergen-induced Th2 lung inflammation but is only a weak mediator of chemotaxis

PubMed Central

Latta, Markus; Mohan, Karkada; Issekutz, Thomas B

2007-01-01

Numerous chemokine receptors are increased in number on T cells in inflamed tissues. Our objective was to examine CXCR6 expression on lymphocytes during immune and inflammatory reactions and its potential for mediating T-cell recruitment. The cDNA for rat CXCR6 was cloned and monoclonal antibodies (mAbs) to CXCR6 were developed. CXCR6 was present on 4–6% of CD4 and CD8 T cells in blood, normal lymph nodes (LNs) and the spleen, primarily on memory T cells. In vitro antigen re-stimulation of LN T cells from animals with autoimmune arthritis and experimental autoimmune encephalomyelitis (EAE) increased the proportion of CXCR6+ T cells to 35–50% and anti-T-cell receptor (TCR) activation to 60–80%. In vivo, after antigen challenge of LNs there was only a small increase in CXCR6+ T cells on the lymphoblasts in the LNs, and a much higher percentage of T cells were CXCR6+ in virus-induced peritoneal exudates (∼47%) and in allergen-induced lung inflammation (33%). Chemotaxis of CXCR6-expressing inflammatory T cells to CXCL16 was poor, but that to CXCL10 was robust. We conclude that few T cells in normal and antigen-challenged LNs are CXCR6+, whereas a high proportion of in vitro activated T cells and T cells from inflammatory sites are CXCR6+, but these cells migrate poorly to CXCL16. This suggests that CXCR6 may contribute to T-cell positioning and activation, rather than recruitment. CXCR6 is also expressed on T cells not only in T helper type 1 (Th1) inflammation (arthritis and EAE) but also, as shown here, in Th2 inflammation, where it is increased after allergen challenge. PMID:17437534

CXCR6 is expressed on T cells in both T helper type 1 (Th1) inflammation and allergen-induced Th2 lung inflammation but is only a weak mediator of chemotaxis.

PubMed

Latta, Markus; Mohan, Karkada; Issekutz, Thomas B

2007-08-01

Numerous chemokine receptors are increased in number on T cells in inflamed tissues. Our objective was to examine CXCR6 expression on lymphocytes during immune and inflammatory reactions and its potential for mediating T-cell recruitment. The cDNA for rat CXCR6 was cloned and monoclonal antibodies (mAbs) to CXCR6 were developed. CXCR6 was present on 4-6% of CD4 and CD8 T cells in blood, normal lymph nodes (LNs) and the spleen, primarily on memory T cells. In vitro antigen re-stimulation of LN T cells from animals with autoimmune arthritis and experimental autoimmune encephalomyelitis (EAE) increased the proportion of CXCR6(+) T cells to 35-50% and anti-T-cell receptor (TCR) activation to 60-80%. In vivo, after antigen challenge of LNs there was only a small increase in CXCR6(+) T cells on the lymphoblasts in the LNs, and a much higher percentage of T cells were CXCR6(+) in virus-induced peritoneal exudates (approximately 47%) and in allergen-induced lung inflammation (33%). Chemotaxis of CXCR6-expressing inflammatory T cells to CXCL16 was poor, but that to CXCL10 was robust. We conclude that few T cells in normal and antigen-challenged LNs are CXCR6(+), whereas a high proportion of in vitro activated T cells and T cells from inflammatory sites are CXCR6(+), but these cells migrate poorly to CXCL16. This suggests that CXCR6 may contribute to T-cell positioning and activation, rather than recruitment. CXCR6 is also expressed on T cells not only in T helper type 1 (Th1) inflammation (arthritis and EAE) but also, as shown here, in Th2 inflammation, where it is increased after allergen challenge.

Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System

PubMed Central

Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence JN; Rosenberg, Steven A

2016-01-01

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAKS2580F or ERBB2H473Y or the HLA-DQB*0601-restricted neoantigen ERBB2IPE805G were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ+ T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27−CD45RA−) and less-differentiated (CD27+CD45RA+) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006

Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

PubMed

Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

2018-04-01

Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on

TLX homeodomain oncogenes mediate T cell maturation arrest in T-ALL via interaction with ETS1 and suppression of TCRα gene expression.

PubMed

Dadi, Saïda; Le Noir, Sandrine; Payet-Bornet, Dominique; Lhermitte, Ludovic; Zacarias-Cabeza, Joaquin; Bergeron, Julie; Villarèse, Patrick; Vachez, Elodie; Dik, Willem A; Millien, Corinne; Radford, Isabelle; Verhoeyen, Els; Cosset, François-Loïc; Petit, Arnaud; Ifrah, Norbert; Dombret, Hervé; Hermine, Olivier; Spicuglia, Salvatore; Langerak, Anton W; Macintyre, Elizabeth A; Nadel, Bertrand; Ferrier, Pierre; Asnafi, Vahid

2012-04-17

Acute lymphoblastic leukemias (ALLs) are characterized by multistep oncogenic processes leading to cell-differentiation arrest and proliferation. Specific abrogation of maturation blockage constitutes a promising therapeutic option in cancer, which requires precise understanding of the underlying molecular mechanisms. We show that the cortical thymic maturation arrest in T-lineage ALLs that overexpress TLX1 or TLX3 is due to binding of TLX1/TLX3 to ETS1, leading to repression of T cell receptor (TCR) α enhanceosome activity and blocked TCR-Jα rearrangement. TLX1/TLX3 abrogation or enforced TCRαβ expression leads to TCRα rearrangement and apoptosis. Importantly, the autoextinction of clones carrying TCRα-driven TLX1 expression supports TLX "addiction" in TLX-positive leukemias and provides further rationale for targeted therapy based on disruption of TLX1/TLX3. Copyright © 2012 Elsevier Inc. All rights reserved.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

PubMed Central

Bengtsson, Å; Lundberg, M; Avila-Cariño, J; Jacobsson, G; Holmgren, A; Scheynius, A

2001-01-01

The thiol antioxidant N-acetyl-l-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 mm NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases. PMID:11298119

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

PubMed

Huang, Xiang-Ying; Yu, Shuang-Qing; Cheng, Zhan; Ye, Jing-Rong; Xu, Ke; Feng, Xia; Zeng, Yi

2013-04-01

To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

PubMed

Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

2013-01-30

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

PubMed Central

Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

2013-01-01

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

PubMed

Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

T-cell lymphomas associated gene expression signature: Bioinformatics analysis based on gene expression Omnibus.

PubMed

Zhou, Lei-Lei; Xu, Xiao-Yue; Ni, Jie; Zhao, Xia; Zhou, Jian-Wei; Feng, Ji-Feng

2018-06-01

Due to the low incidence and the heterogeneity of subtypes, the biological process of T-cell lymphomas is largely unknown. Although many genes have been detected in T-cell lymphomas, the role of these genes in biological process of T-cell lymphomas was not further analyzed. Two qualified datasets were downloaded from Gene Expression Omnibus database. The biological functions of differentially expressed genes were evaluated by gene ontology enrichment and KEGG pathway analysis. The network for intersection genes was constructed by the cytoscape v3.0 software. Kaplan-Meier survival curves and log-rank test were employed to assess the association between differentially expressed genes and clinical characters. The intersection mRNAs were proved to be associated with fundamental processes of T-cell lymphoma cells. These intersection mRNAs were involved in the activation of some cancer-related pathways, including PI3K/AKT, Ras, JAK-STAT, and NF-kappa B signaling pathway. PDGFRA, CXCL12, and CCL19 were the most significant central genes in the signal-net analysis. The results of survival analysis are not entirely credible. Our findings uncovered aberrantly expressed genes and a complex RNA signal network in T-cell lymphomas and indicated cancer-related pathways involved in disease initiation and progression, providing a new insight for biotargeted therapy in T-cell lymphomas. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

PubMed

Bell, Catherine C; Faulkner, Lee; Martinsson, Klara; Farrell, John; Alfirevic, Ana; Tugwood, Jonathan; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

2013-05-20

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

PubMed

Que, Zujun; Zou, Fangyuan; Zhang, Anle; Zheng, Yuanhong; Bi, Ling; Zhong, Jianjiang; Tian, Jianhui; Liu, Jianwen

2014-11-01

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

PubMed

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-08-11

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

PubMed Central

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-01-01

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation. PMID:19666573

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

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

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

Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

2005-12-30

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells

PubMed Central

Tone, Masahide; Tone, Yukiko; Adams, Elizabeth; Yates, Stephen F.; Frewin, Mark R.; Cobbold, Stephen P.; Waldmann, Herman

2003-01-01

Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+ regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+ T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences. PMID:14608036

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes

PubMed Central

Jones, R. Brad; Mueller, Stefanie; O’Connor, Rachel; Rimpel, Katherine; Sloan, Derek D.; Karel, Dan; Wong, Hing C.; Jeng, Emily K.; Thomas, Allison S.; Whitney, James B.; Lim, So-Yon; Kovacs, Colin; Benko, Erika; Karandish, Sara; Huang, Szu-Han; Buzon, Maria J.; Lichterfeld, Mathias; Irrinki, Alivelu; Murry, Jeffrey P.; Tsai, Angela; Yu, Helen; Geleziunas, Romas; Trocha, Alicja; Ostrowski, Mario A.; Irvine, Darrell J.; Walker, Bruce D.

2016-01-01

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies. PMID:27082643

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

PubMed

Jones, R Brad; Mueller, Stefanie; O'Connor, Rachel; Rimpel, Katherine; Sloan, Derek D; Karel, Dan; Wong, Hing C; Jeng, Emily K; Thomas, Allison S; Whitney, James B; Lim, So-Yon; Kovacs, Colin; Benko, Erika; Karandish, Sara; Huang, Szu-Han; Buzon, Maria J; Lichterfeld, Mathias; Irrinki, Alivelu; Murry, Jeffrey P; Tsai, Angela; Yu, Helen; Geleziunas, Romas; Trocha, Alicja; Ostrowski, Mario A; Irvine, Darrell J; Walker, Bruce D

2016-04-01

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

PubMed

Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

2012-01-01

TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review

PubMed Central

Matnani, Rahul G.; Stewart, Rachel L.; Pulliam, Joseph; Jennings, Chester D.; Kesler, Melissa

2013-01-01

A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones. PMID:24066244

Phenotypic effects of somatic cell cloning in the mouse.

PubMed

Ogura, A; Inoue, K; Ogonuki, N; Lee, J; Kohda, T; Ishino, F

2002-01-01

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

PubMed

Ono, Yukiko; Kono, Tomohiro

2006-08-01

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Elements Recognized by Abacavir-Induced T Cells.

PubMed

Yerly, Daniel; Pompeu, Yuri Andreiw; Schutte, Ryan J; Eriksson, Klara K; Strhyn, Anette; Bracey, Austin W; Buus, Soren; Ostrov, David A

2017-07-07

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976-984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230-238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues.

Structural Elements Recognized by Abacavir-Induced T Cells

PubMed Central

Yerly, Daniel; Pompeu, Yuri Andreiw; Schutte, Ryan J.; Eriksson, Klara. K.; Strhyn, Anette; Bracey, Austin. W.; Buus, Soren; Ostrov, David A.

2017-01-01

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976–984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230–238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues. PMID:28686208

Requirements for flare reactions of joint inflammation induced in mice by cloned MT4+, Lyt-2- T cells.

PubMed

Klasen, I S; Ladestein, R M; van den Berg, W B; Benner, R

1989-03-01

Joint inflammation was induced in C57B1/6 mice by injection of cloned MT4+, Lyt-2- T cells specific for the antigen methylated bovine serum albumin (mBSA), together with mBSA. In this model, after waning of the inflammation, flare reactions can be induced by a rechallenge with the specific antigen. Herein we show that such flare reactions can still be induced several weeks after waning of the joint inflammation, as was demonstrated both in normal C57B1/6 mice and in athymic C57B1 nude mice. The results in the latter group indicate that T cells of the recipient mice are not necessary for the elicitation of flare reactions. On histologic examination, the inflammatory infiltrates in the knee joints of the nude mice appeared to be mainly granulocytic. The cloned T cells persisted and remained functionally reactive in the knee joint for at least 2 weeks in the absence of the antigen, and thus, in the absence of inflammation. In view of the similarities between induced joint inflammation in mice and rheumatoid arthritis in humans, these data may be relevant to our understanding of the processes involved in the latter disease.

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

PubMed

Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

2016-11-01

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. © 2016 The Authors.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

PubMed

Sternberg, Hal; Jiang, Jianjie; Sim, Pamela; Kidd, Jennifer; Janus, Jeffrey; Rinon, Ariel; Edgar, Ron; Shitrit, Alina; Larocca, David; Chapman, Karen B; Binette, Francois; West, Michael D

2014-01-01

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Taste information derived from T1R-expressing taste cells in mice.

PubMed

Yoshida, Ryusuke; Ninomiya, Yuzo

2016-03-01

The taste system of animals is used to detect valuable nutrients and harmful compounds in foods. In humans and mice, sweet, bitter, salty, sour and umami tastes are considered the five basic taste qualities. Sweet and umami tastes are mediated by G-protein-coupled receptors, belonging to the T1R (taste receptor type 1) family. This family consists of three members (T1R1, T1R2 and T1R3). They function as sweet or umami taste receptors by forming heterodimeric complexes, T1R1+T1R3 (umami) or T1R2+T1R3 (sweet). Receptors for each of the basic tastes are thought to be expressed exclusively in taste bud cells. Sweet (T1R2+T1R3-expressing) taste cells were thought to be segregated from umami (T1R1+T1R3-expressing) taste cells in taste buds. However, recent studies have revealed that a significant portion of taste cells in mice expressed all T1R subunits and responded to both sweet and umami compounds. This suggests that sweet and umami taste cells may not be segregated. Mice are able to discriminate between sweet and umami tastes, and both tastes contribute to behavioural preferences for sweet or umami compounds. There is growing evidence that T1R3 is also involved in behavioural avoidance of calcium tastes in mice, which implies that there may be a further population of T1R-expressing taste cells that mediate aversion to calcium taste. Therefore the simple view of detection and segregation of sweet and umami tastes by T1R-expressing taste cells, in mice, is now open to re-examination. © 2016 Authors; published by Portland Press Limited.

Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells.

PubMed

Sharma, Sonia; Grandvaux, Nathalie; Mamane, Yael; Genin, Pierre; Azimi, Nazli; Waldmann, Thomas; Hiscott, John

2002-09-15

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-kappaB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4(+) T lymphocytes.

Expression of immune checkpoints in T cells of esophageal cancer patients.

PubMed

Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

2016-09-27

Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer.

Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

PubMed Central

Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

2014-01-01

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

Scrutinizing the Expression and Blockade of Inhibitory Molecules Expressed on T Cells from Acute Myeloid Leukemia Patients.

PubMed

Abdolmaleki, Mohsen; Mojtabavi, Nazanin; Zavvar, Mahdi; Vaezi, Mohammad; Noorbakhsh, Farshid; Nicknam, Mohammad Hossein

2018-06-01

T cell exhaustion is an immunosuppressive mechanism which occurs in chronic viral infections, solid tumors and hematologic malignancies. Exhausted T cell has increased the expression of inhibitory receptors, and functional impairment. In this study, we investigated the expression from some of those inhibitory receptors being Programmed death 1 (PD-1), T cell immunoglobulin and mucin domain containing molecules 3 (TIM-3) and CD244 on T cells from Iranian acute myeloid leukemia (AML) patients. Peripheral blood samples were collected from Iranian newly diagnosed AML patients and flow cytometric analysis was accomplished for cell surface expression of PD-1, TIM-3, and CD244 on T lymphocytes. Functionality and proliferation assay were done in the presence of anti-PD-1 and anti-CD244 blocking antibodies. Immunophenotyping of T cells showed a significant increase of PD-1 and CD244 expression on CD4+ and CD8+ T cells of AML patients. Whereas blockade of PD1 and CD244 increased the proliferation of CD4+ and CD8+ T lymphocytes of AML patients but IFN-γ production was not significantly increased. In conclusion, our data indicate that CD4+ and CD8+ T cells from AML patients appeared to be exhausted and blockade of some immune checkpoints can improve the proliferation of those cells.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

PubMed Central

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. PMID:24146866

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

PubMed

Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

2010-05-15

T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (<2 and 2-6 years) (p<0.001) and fewer %CD4+CD25+FoxP3+ Tregs in the tumor draining lymph nodes of OSA patients compared to the unrelated lymph node (p=0.049). However, there was no significant difference in % Tregs in the peripheral blood or lymph nodes between the control dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration.

PubMed

Aydin, Iraz T; Tokcaer, Zeynep; Dalgic, Aydin; Konu, Ozlen; Akcali, Kamil C

2007-12-01

The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration.

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Genetically engineered T cells to target EGFRvIII expressing glioblastoma.

PubMed

Bullain, Szofia S; Sahin, Ayguen; Szentirmai, Oszkar; Sanchez, Carlos; Lin, Ning; Baratta, Elizabeth; Waterman, Peter; Weissleder, Ralph; Mulligan, Richard C; Carter, Bob S

2009-09-01

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

CD4+ T cells defined by their Vβ T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis

PubMed Central

Keesen, T S L; Antonelli, L R V; Faria, D R; Guimarães, L H; Bacellar, O; Carvalho, E M; Dutra, W O; Gollob, K J

2011-01-01

Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vβ region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vβ region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4+ T cells expressing Vβ 5·2 and Vβ 24 in CL compared to controls; (2) a Leishmania antigen-induced increase in CD4+ T cells expressing Vβ 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4+ Vβ 5·2-, 11- and 24-positive T cells, with higher expression of CD45RO, HLA-DR, interferon-γ, tumour necrosis factor-α and interleukin-10 compared to other Vβ-expressing subpopulations; (4) a positive correlation between higher frequencies of CD4+Vβ5·2+ T cells and larger lesions; and (5) biased homing of CD4+ T cells expressing Vβ 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long-lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology. PMID:21726211

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

PubMed

Rouhani, Sherin J; Eccles, Jacob D; Riccardi, Priscila; Peske, J David; Tewalt, Eric F; Cohen, Jarish N; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H

2015-04-10

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction

PubMed Central

Rouhani, Sherin J.; Eccles, Jacob D.; Riccardi, Priscila; Peske, J. David; Tewalt, Eric F.; Cohen, Jarish N.; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H.

2015-01-01

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. PMID:25857745

Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets

PubMed Central

2011-01-01

Background It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells. Results We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells. Conclusion Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response. PMID:21914188

Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

PubMed

Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

2018-05-01

Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

PubMed

Kochenderfer, James N; Rosenberg, Steven A

2013-05-01

Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

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

Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano

The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.« less

Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells.

PubMed

Yee, Karen K; Sukumaran, Sunil K; Kotha, Ramana; Gilbertson, Timothy A; Margolskee, Robert F

2011-03-29

Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium-glucose cotransporter (SGLT1), and two components of the ATP-gated K(+) (K(ATP)) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K(ATP) channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K(ATP) channels constitute a major portion of K(+) channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K(ATP) may serve as mediators of the T1r-independent sweet taste of sugars.

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

USGS Publications Warehouse

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations.

PubMed

Peerbolte, R; Leenhouts, K; Hooykaas-van Slogteren, G M; Hoge, J H; Wullems, G J; Schilperoort, R A

1986-07-01

Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having a Tn1831 insertion in the auxin locus, were investigated for their T-DNA structure and expression. In addition to clones with the expected phenotype, i.e. phytohormone autonomy, regeneration of non-rooting shoots and octopine synthesis (Aut(+)Reg(+)Ocs(+) 'type I' clones), clones were obtained with an aberrant phenotype. Among these were the Aut(-)Reg(-)Ocs(+) 'type II' clones. Two shooty type I clones and three type II callus clones (all randomly chosen) as well as a rooting shoot regenerated from a type II clone via a high kinetin treatment, all had a T-DNA structure which differed significantly from 'regular' T-DNA structures. No Tn1831 DNA sequences were detected in these clones. The two type I clones were identical: they both contained the same highly truncated T-DNA segments. One TL-DNA segment of approximately 0.7 kb, originating form the left part of the TL-region, was present at one copy per diploid tobacco genome. Another segment with a maximum size of about 7 kb was derived from the right hand part of the TL-region and was present at minimally two copies. Three copies of a truncated TR-DNA segment were detected, probably starting at the right TR-DNA border repeat and ending halfway the regular TR-region. Indications have been obtained that at least some of the T-DNA segments are closely linked, sometimes via intervening plant DNA sequences. The type I clones harbored TL-DNA transcripts 4, 6a/b and 3 as well as TR-DNA transcript 0'. The type II clones harbored three to six highly truncated T-DNA segments, originating from the right part of the TL-region. In addition they had TR-DNA segments, similar to those of the type I clones. On Northern blots TR-DNA transcripts 0' and 1' were detected as well as the TL-DNA transcripts 3 and 6a/b and an 1800 bp hybrid transcript (tr.Y) containing gene 6b sequences. Possible origins of the observed

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

PubMed Central

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F.; Shao, Wei; Cyktor, Joshua C.; Cillo, Anthony R.; Halvas, Elias K.; Coffin, John M.; Mellors, John W.; Kearney, Mary F.

2017-01-01

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression. PMID:28416661

Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis.

PubMed

Hernández-Chirlaque, Cristina; Gámez-Belmonte, Reyes; Ocón, Borja; Martínez-Moya, Patricia; Wirtz, Stefan; Sánchez de Medina, Fermín; Martínez-Augustin, Olga

2017-07-01

Two alkaline phosphatase isoforms, intestinal [IAP] and tissue non-specific alkaline phosphatase [TNAP], are coexpressed in mouse colon, with the latter predominating in colitis. We aimed to examine the role of TNAP in T lymphocytes, using heterozygous TNAP+/- mice [as TNAP-/- mice are non-viable]. In vitro primary cultures and in vivo T cell models using TNAP+/- mice were used. Stimulated splenocytes [lipopolysaccharide and concanavalin A] and T lymphocytes [concanavalin A and a-CD3/a-CD28] showed a decreased cytokine production and expression when compared with wild-type [WT] cells. Decreased T cell activation was reproduced by the TNAP inhibitors levamisole, theophylline, and phenylalanine in WT cells. Intraperitoneal administration of anti-CD3 in vivo resulted in reduced plasma cytokine levels, and decreased activation of splenocytes and T cells ex vivo in TNAP+/- mice. We further tested the hypothesis that TNAP expressed in T lymphocytes is involved in T cell activation and inflammation, using the lymphocyte transfer model of colitis. Rag1-/- mice were transferred with T naïve cells [CD4+ CD62L+] from TNAP+/- or WT mice and developed colitis, which was attenuated in the group receiving TNAP+/- cells. Compared with WT, T cells from TNAP+/- mice showed a decreased capacity for proliferation, with no change in differentiation. Our results offer clear evidence that TNAP modulates T lymphocyte function and specifically T cell-dependent colitis. This was associated with distinct changes in the type of TNAP expressed, probably because of changes in glycosylation. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene

PubMed Central

Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars

2012-01-01

Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479

Effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation

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

Sheu, C.W.; Moreland, F.M.; Dunkel, V.C.

1987-01-01

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO/sub 2/ incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14more » had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.« less

Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

PubMed

Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

2000-12-15

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone.

PubMed

Ben Zakour, Nouri L; Davies, Mark R; You, Yuanhai; Chen, Jonathan H K; Forde, Brian M; Stanton-Cook, Mitchell; Yang, Ruifu; Cui, Yujun; Barnett, Timothy C; Venturini, Carola; Ong, Cheryl-lynn Y; Tse, Herman; Dougan, Gordon; Zhang, Jianzhong; Yuen, Kwok-Yung; Beatson, Scott A; Walker, Mark J

2015-11-02

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone, with bacteriophage-encoded determinants DNase Sda1 and superantigen SpeA2 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS, led to our investigation of the next most common cause of scarlet fever, emm1 GAS. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone

PubMed Central

Ben Zakour, Nouri L.; Davies, Mark R.; You, Yuanhai; Chen, Jonathan H. K.; Forde, Brian M.; Stanton-Cook, Mitchell; Yang, Ruifu; Cui, Yujun; Barnett, Timothy C.; Venturini, Carola; Ong, Cheryl-lynn Y.; Tse, Herman; Dougan, Gordon; Zhang, Jianzhong; Yuen, Kwok-Yung; Beatson, Scott A.; Walker, Mark J.

2015-01-01

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome123. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone45, with bacteriophage-encoded determinants DNase Sda16 and superantigen SpeA27 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS8910, led to our investigation of the next most common cause of scarlet fever, emm1 GAS89. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones1011 in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements. PMID:26522788

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

PubMed

Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual

2017-02-01

It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by

CD40L Expression Allows CD8+ T Cells to Promote Their Own Expansion and Differentiation through Dendritic Cells

PubMed Central

Tay, Neil Q.; Lee, Debbie C. P.; Chua, Yen Leong; Prabhu, Nayana; Gascoigne, Nicholas R. J.; Kemeny, David M.

2017-01-01

CD8+ T cells play an important role in providing protective immunity against a wide range of pathogens, and a number of different factors control their activation. Although CD40L-mediated CD40 licensing of dendritic cells (DCs) by CD4+ T cells is known to be necessary for the generation of a robust CD8+ T cell response, the contribution of CD8+ T cell-expressed CD40L on DC licensing is less clear. We have previously shown that CD8+ T cells are able to induce the production of IL-12 p70 by DCs in a CD40L-dependent manner, providing some evidence that CD8+ T cell-mediated activation of DCs is possible. To better understand the role of CD40L on CD8+ T cell responses, we generated and characterized CD40L-expressing CD8+ T cells both in vitro and in vivo. We found that CD40L was expressed on 30–50% of effector CD8+ T cells when stimulated and that this expression was transient. The expression of CD40L on CD8+ T cells promoted the proliferation and differentiation of both the CD40L-expressing CD8+ T cells and the bystander effector CD8+ T cells. This process occurred via a cell-extrinsic manner and was mediated by DCs. These data demonstrate the existence of a mechanism where CD8+ T cells and DCs cooperate to maximize CD8+ T cell responses. PMID:29163545

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

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

PubMed

Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

2011-10-15

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

Identification of an inducible regulator of c-myb expression during T-cell activation.

PubMed Central

Phan, S C; Feeley, B; Withers, D; Boxer, L M

1996-01-01

Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation. PMID:8628306

Towards depersonalized abacavir therapy: chemical modification eliminates HLA-B*57 : 01-restricted CD8+ T-cell activation.

PubMed

Naisbitt, Dean J; Yang, Emma L; Alhaidari, Mohammad; Berry, Neil G; Lawrenson, Alexandre S; Farrell, John; Martin, Philip; Strebel, Klaus; Owen, Andrew; Pye, Matthew; French, Neil S; Clarke, Stephen E; O'Neill, Paul M; Park, B Kevin

2015-11-28

Exposure to abacavir is associated with T-cell-mediated hypersensitivity reactions in individuals carrying human leukocyte antigen (HLA)-B57 : 01. To activate T cells, abacavir interacts directly with endogenous HLA-B57 : 01 and HLA-B57 : 01 expressed on the surface of antigen presenting cells. We have investigated whether chemical modification of abacavir can produce a molecule with antiviral activity that does not bind to HLA-B57 : 01 and activate T cells. An interdisciplinary laboratory study using samples from human donors expressing HLA-B57 : 01. Researchers were blinded to the analogue structures and modelling data. Sixteen 6-amino substituted abacavir analogues were synthesized. Computational docking studies were completed to predict capacity for analogue binding within HLA-B57 : 01. Abacavir-responsive CD8 clones were generated to study the association between HLA-B57 : 01 analogue binding and T-cell activation. Antiviral activity and the direct inhibitory effect of analogues on proliferation were assessed. Major histocompatibility complex class I-restricted CD8 clones proliferated and secreted IFNγ following abacavir binding to surface and endogenous HLA-B57 : 01. Several analogues retained antiviral activity and showed no overt inhibitory effect on proliferation, but displayed highly divergent antigen-driven T-cell responses. For example, abacavir and N-propyl abacavir were equally potent at activating clones, whereas the closely related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity. Docking abacavir analogues to HLA-B57 : 01 revealed a quantitative relationship between drug-protein binding and the T-cell response. These studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favourable antiviral profile. The in-silico model provides a tool to aid the design of safer antiviral agents that may not require a personalized medicines approach to therapy.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

PubMed

Zhang, Xiaoyang; Wang, Dongxu; Han, Yang; Duan, Feifei; Lv, Qinyan; Li, Zhanjun

2014-11-01

To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Toward eliminating HLA class I expression to generate universal cells from allogeneic donors

PubMed Central

Torikai, Hiroki; Reik, Andreas; Soldner, Frank; Warren, Edus H.; Yuen, Carrie; Zhou, Yuanyue; Crossland, Denise L.; Huls, Helen; Littman, Nicholas; Zhang, Ziying; Tykodi, Scott S.; Kebriaei, Partow; Lee, Dean A.; Miller, Jeffrey C.; Rebar, Edward J.; Holmes, Michael C.; Jaenisch, Rudolf; Champlin, Richard E.; Gregory, Philip D.

2013-01-01

Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as “non-self” by the recipient. To this end, we developed designer zinc finger nucleases and employed a “hit-and-run” approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T cells, including CD19-specific T cells. The HLA-Aneg T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by natural killer cells of cells that had lost HLA expression was circumvented by enforced expression of nonclassical HLA molecules. Furthermore, we demonstrate that zinc finger nucleases can eliminate HLA-A expression from embryonic stem cells, which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from a single donor can be administered to multiple recipients. PMID:23741009

Cloning and Expression of cDNA for Rat Heme Oxygenase

NASA Astrophysics Data System (ADS)

Shibahara, Shigeki; Muller, Rita; Taguchi, Hayao; Yoshida, Tadashi

1985-12-01

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in λ gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

PubMed Central

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-01-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

Cloned ferrets produced by somatic cell nuclear transfer

PubMed Central

Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M.; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H.; Engelhardt, John F.

2007-01-01

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (~3–4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. PMID:16584722

Cloned ferrets produced by somatic cell nuclear transfer.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H; Engelhardt, John F

2006-05-15

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.

Constitutively expressing cell lines that secrete a truncated bovine herpes virus-1 glycoprotein (gpI) stimulate T-lymphocyte responsiveness.

PubMed

Leary, T P; Gao, Y; Splitter, G A

1992-07-01

The desire to obtain authentically glycosylated viral protein products in sufficient quantity for immunological study has led to the use of eucaryotic expression vectors for protein production. An additional advantage is that these protein products can be studied individually in the absence of their native viral environment. We have cloned a complementary DNA (cDNA) encoding bovine herpes virus-1 (BHV-1) glycoprotein 1 (gpI) into the eucaryotic expression vector, pZipNeo SVX1. Since this protein is normally embedded within the membrane of BHV-1 infected cells, we removed sequences encoding the transmembrane domain of the native protein. After transfection of the plasmid construct into the canine osteosarcoma cell line, D17, or Madin-Darby bovine kidney (MDBK) cells, a truncated BHV-1 (gpI) was secreted into the culture medium as demonstrated by radioimmunoprecipitation and SDS-PAGE. Both a CD4+ T-lymphocyte line specific for BHV-1 and freshly isolated T lymphocytes could recognize and respond to the secreted recombinant gpI. Further, recombinant gpI could elicit both antibody and cellular responses in cattle when used as an immunogen. Having established constitutively glycoprotein producing cell lines, future studies in vaccine evaluation of gpI will be facilitated.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

PubMed

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-12-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Tolerance without clonal expansion: self-antigen-expressing B cells program self-reactive T cells for future deletion.

PubMed

Frommer, Friederike; Heinen, Tobias J A J; Wunderlich, F Thomas; Yogev, Nir; Buch, Thorsten; Roers, Axel; Bettelli, Estelle; Müller, Werner; Anderton, Stephen M; Waisman, Ari

2008-10-15

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

PubMed

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

2016-11-17

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

PubMed Central

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

2016-01-01

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

T-bet expression by Th cells promotes type 1 inflammation but is dispensable for colitis.

PubMed

Zimmermann, J; Kühl, A A; Weber, M; Grün, J R; Löffler, J; Haftmann, C; Riedel, R; Maschmeyer, P; Lehmann, K; Westendorf, K; Mashreghi, M-F; Löhning, M; Mack, M; Radbruch, A; Chang, H D

2016-11-01

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

PubMed Central

Dialynas, D P; Murre, C; Quertermous, T; Boss, J M; Leiden, J M; Seidman, J G; Strominger, J L

1986-01-01

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation. Images PMID:3458221

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Singh, Surender; Kumar, Sudarshan; Kaushik, Jai K; Mohanty, Ashok K; Malakar, Dhruba

2015-12-01

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas.

PubMed

Poropatich, Kate; Fontanarosa, Joel; Swaminathan, Suchitra; Dittmann, Dave; Chen, Siqi; Samant, Sandeep; Zhang, Bin

2017-11-01

The success of programmed cell death 1 (PD-1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)-positive; 14 HPV-negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4 + and CD8 + T-cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD-1, TIM-3 and CTLA-4. Next-generation sequencing of the T-cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8 + T cells expressing CTLA-4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV-positive patients, as well as higher expression levels of TIM-3 + PD-1 + CD8 + T cells (P = 0.0072) than controls. For all patients, PD-1 expression on CD8 + T cells - particularly in HPV-negative HNSCC cases - strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8 + TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic - and potentially more immunogenic - than those from HPV-negative patients. Collectively, our findings demonstrate, for the first time, that high-stage HPV-negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA-4 + CD8 + T-cell levels, that tumour-familiar CD8 + T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV-positive HNSCC patients potentially recognize

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.

Development of tumor-reactive T cells after nonmyeloablative allogeneic hematopoietic stem cell transplant for chronic lymphocytic leukemia.

PubMed

Nishida, Tetsuya; Hudecek, Michael; Kostic, Ana; Bleakley, Marie; Warren, Edus H; Maloney, David; Storb, Rainer; Riddell, Stanley R

2009-07-15

Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy. Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8(+) T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes. Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8(+) and CD4(+) T cells specific for antigens expressed by CLL. A clonal analysis of the CD8(+) T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8(+) T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease. The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

PubMed

Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

2006-09-01

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene.

PubMed

Bogado, A L G; Martins, G F; Sasse, J P; Guimarães, J da S; Garcia, J L

2017-03-15

Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

PubMed Central

Haussmann, C; Rohdich, F; Lottspeich, F; Eberhardt, S; Scheuring, J; Mackamul, S; Bacher, A

1997-01-01

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome. PMID:9006053

Epidermal Cadm1 expression promotes autoimmune alopecia via enhanced T cell adhesion and cytotoxicity.

PubMed

Giangreco, Adam; Hoste, Esther; Takai, Yoshimi; Rosewell, Ian; Watt, Fiona M

2012-02-01

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

2002-01-01

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

PubMed

Stephen, Tom L; Payne, Kyle K; Chaurio, Ricardo A; Allegrezza, Michael J; Zhu, Hengrui; Perez-Sanz, Jairo; Perales-Puchalt, Alfredo; Nguyen, Jenny M; Vara-Ailor, Ana E; Eruslanov, Evgeniy B; Borowsky, Mark E; Zhang, Rugang; Laufer, Terri M; Conejo-Garcia, Jose R

2017-01-17

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity. Copyright © 2017 Elsevier Inc. All rights reserved.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.