Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

PubMed

Shen, Chuanlai; Xu, Tao; Wu, You; Li, Xiaoe; Xia, Lingzhi; Wang, Wei; Shahzad, Khawar Ali; Zhang, Lei; Wan, Xin; Qiu, Jie

2017-11-27

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin 257-264 -specific CD8 + T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen 18-27 - and surface antigen 183-191 -specific CD8 + T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

Antigen-specific T cell therapies for cancer

PubMed Central

Manzo, Teresa; Heslop, Helen E.; Rooney, Cliona M.

2015-01-01

Adoptively transferred antigen-specific T cells that recognize tumor antigens through their native receptors have many potential benefits as treatment for virus-associated diseases and malignancies, due to their ability to selectively recognize tumor antigens, expand and persist to provide long-term protection. Infusions of T cells targeting Epstein–Barr virus (EBV) antigens have shown encouraging response rates in patients with post-transplant lymphoproliferative disease as well as EBV-positive lymphomas and nasopharyngeal cancer, although a recent study also showed that human papilloma virus-reactive T cells can induce complete regression of metastatic cervical cancer. This strategy is also being evaluated to target non-viral tumor-associated antigens. Targeting these less immunogenic antigens is more challenging, as tumor antigens are generally weak, and high avidity T cells specific for self-antigens are deleted in the thymus, but tumor responses have been reported. Current research focusses on defining factors that promote in vivo persistence of transferred cells and ameliorate the immunosuppressive microenvironment. To this end, investigators are evaluating the effects of combining adoptive transfer of antigen-specific T cells with other immunotherapy moieties such as checkpoint inhibitors. Genetic modification of infused T cells may also be used to overcome tumor evasion mechanisms, and vaccines may be used to promote in vivo proliferation. PMID:26160910

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

NASA Astrophysics Data System (ADS)

Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

1986-08-01

The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Nonclassical T Cells and Their Antigens in Tuberculosis

PubMed Central

De Libero, Gennaro; Singhal, Amit; Lepore, Marco; Mori, Lucia

2014-01-01

T cells that recognize nonpeptidic antigens, and thereby are identified as nonclassical, represent important yet poorly characterized effectors of the immune response. They are present in large numbers in circulating blood and tissues and are as abundant as T cells recognizing peptide antigens. Nonclassical T cells exert multiple functions including immunoregulation, tumor control, and protection against infections. They recognize complexes of nonpeptidic antigens such as lipid and glycolipid molecules, vitamin B2 precursors, and phosphorylated metabolites of the mevalonate pathway. Each of these antigens is presented by antigen-presenting molecules other than major histocompatibility complex (MHC), including CD1, MHC class I–related molecule 1 (MR1), and butyrophilin 3A1 (BTN3A1) molecules. Here, we discuss how nonclassical T cells participate in the recognition of mycobacterial antigens and in the mycobacterial-specific immune response. PMID:25059739

A Novel Method Linking Antigen Presentation by Human Monocyte-Derived Macrophages to CD8+ T Cell Polyfunctionality

PubMed Central

Short, Kirsty R.; Grant, Emma J.; Vissers, Marloes; Reading, Patrick C.; Diavatopoulos, Dimitri A.; Kedzierska, Katherine

2013-01-01

To understand the interactions between innate and adaptive immunity, and specifically how virally infected macrophages impact T cell function, novel assays examining the ability of macrophages to present antigen to CD8+ T cells are needed. In the present study, we have developed a robust in vitro assay to measure how antigen presentation by human monocyte-derived macrophages (MDMs) affects the functional capacity of autologous CD8+ T cells. The assay is based on the polyfunctional characteristics of antigen-specific CD8+ T cells, and is thus called a Mac-CD8 Polyfunctionality Assay. Following purification of monocytes and their maturation to MDMs, MDMs were pulsed with an antigenic peptide to be presented to CD8+ T cells. Peptide-pulsed MDMs were then incubated with antigen-specific CD8+ T cells in order to assess the efficacy of antigen presentation to T cells. CD8+ T cell polyfunctionality was assessed by staining with mAbs to IFN-γ, TNF-α, and CD107a in a multi-color intracellular cytokine staining assay. To highlight the utility of the Mac-CD8 Polyfunctionality Assay, we assessed the effects of influenza infection on the ability of human macrophages to present antigen to CD8+ T cells. We found that influenza infection of human MDMs can alter the effector efficacy of MDMs to activate more CD8+ T cells with cytotoxic capacity. This has important implications for understanding how the virus-infected macrophages affect adaptive immunity at the site of infection. PMID:24312096

Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

PubMed Central

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-01-01

Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the

Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8(+) T cell responses.

PubMed

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-10-10

Synthetic subunit vaccines need to induce CD8(+) cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8(+) cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8(+) T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25-30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8(+) T cell responses (0.4% IFN-γ(+) of CD8(+)) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

PubMed Central

Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

2014-01-01

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

PubMed Central

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

[Limbic encephalitis with antibodies against intracellular antigens].

PubMed

Morita, Akihiko; Kamei, Satoshi

2010-04-01

Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Presentation of lipid antigens to T cells.

PubMed

Mori, Lucia; De Libero, Gennaro

2008-04-15

T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma

PubMed Central

Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

2016-01-01

T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC. PMID:26958940

Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

PubMed

Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

2016-03-29

T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

Recovery of antigen-specific T cell responses from dogs infected with Leishmania (L.) infantum by use of vaccine associated TLR-agonist adjuvant.

PubMed

Schaut, Robert G; Grinnage-Pulley, Tara L; Esch, Kevin J; Toepp, Angela J; Duthie, Malcolm S; Howard, Randall F; Reed, Steven G; Petersen, Christine A

2016-10-17

Visceral leishmaniasis (VL), caused by infection with the obligate intracellular protozoan parasite Leishmania infantum, is a fatal disease of dogs and humans. Protection against VL requires a T helper 1 (Th1) skewed CD4 + T response, but despite this knowledge, there are currently no approved-to-market vaccines for humans and only three veterinary-use vaccines globally. As VL progresses from asymptomatic to symptomatic, L. infantum-specific interferon gamma (IFNγ) driven-Th1 responses become dampened and a state of immune exhaustion established. T cell exhaustion and other immunoregulatory processes, starting during asymptomatic disease, are likely to hinder vaccine-induced responses if vaccine is administered to infected, but asymptomatic and seronegative, individuals. In this study we evaluated how immune exhaustion, shown previously by our group to worsen in concert with VL progression, effected the capacity of vaccine candidate antigen/toll-like receptor (TLR) agonist combinations to promote protective CD4 + T cell responses during progressive VL. In conjunction with Th1 responses, we also evaluated concomitant stimulation of immune-balanced IL-10 regulatory cytokine production by these vaccine products in progressive VL canine T cells. Vaccine antigen L111f in combination with TLR agonists significantly recovered CD4 + T cell IFNγ intracellular production in T cells from asymptomatic VL dogs. Vaccine antigen NS with TLR agonists significantly recovered CD4 + T cell production in both endemic control and VL dogs. Combinations of TLR agonists and vaccine antigens overcame L. infantum induced cellular exhaustion, allowing robust Th1 CD4 + T cell responses from symptomatic dogs that previously had dampened responses to antigen alone. Antigen-agonist adjuvants can be utilized to promote more robust vaccine responses from infected hosts in endemic areas where vaccination of asymptomatic, L. infantum-infected animals is likely. Copyright © 2016. Published by

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

PubMed

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Molecular recognition of microbial lipid-based antigens by T cells.

PubMed

Gras, Stephanie; Van Rhijn, Ildiko; Shahine, Adam; Le Nours, Jérôme

2018-05-01

The immune system has evolved to protect hosts from pathogens. T cells represent a critical component of the immune system by their engagement in host defence mechanisms against microbial infections. Our knowledge of the molecular recognition by T cells of pathogen-derived peptidic antigens that are presented by the major histocompatibility complex glycoproteins is now well established. However, lipids represent an additional, distinct chemical class of molecules that when presented by the family of CD1 antigen-presenting molecules can serve as antigens, and be recognized by specialized subsets of T cells leading to antigen-specific activation. Over the past decades, numerous CD1-presented self- and bacterial lipid-based antigens have been isolated and characterized. However, our understanding at the molecular level of T cell immunity to CD1 molecules presenting microbial lipid-based antigens is still largely unexplored. Here, we review the insights and the molecular basis underpinning the recognition of microbial lipid-based antigens by T cells.

Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing

PubMed Central

1983-01-01

We examined the ability of a set of cloned chicken ovalbumin (cOVA)- specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I- region molecules by T cells. PMID:6193218

Peripheral neuropathies associated with antibodies directed to intracellular neural antigens.

PubMed

Antoine, J-C

2014-10-01

Antibodies directed to intracellular neural antigens have been mainly described in paraneoplastic peripheral neuropathies and mostly includes anti-Hu and anti-CV2/CRMP5 antibodies. These antibodies occur with different patterns of neuropathy. With anti-Hu antibody, the most frequent manifestation is sensory neuronopathy with frequent autonomic involvement. With anti-CV2/CRMP5 the neuropathy is more frequently sensory and motor with an axonal or mixed demyelinating and axonal electrophysiological pattern. The clinical pattern of these neuropathies is in keeping with the cellular distribution of HuD and CRMP5 in the peripheral nervous system. Although present in high titer, these antibodies are probably not directly responsible for the neuropathy. Pathological and experimental studies indicate that cytotoxic T-cells are probably the main effectors of the immune response. These disorders contrast with those in which antibodies recognize a cell surface antigen and are probably responsible for the disease. The neuronal cell death and axonal degeneration which result from T-cell mediated immunity explains why treating these disorders remains challenging. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression

PubMed Central

Santoro, Stephen P.; Kim, Soorin; Motz, Gregory T.; Alatzoglou, Dimitrios; Li, Chunsheng; Irving, Melita; Powell, Daniel J.; Coukos, George

2014-01-01

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of pro-tumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA+ endothelial targets in vitro, regardless of the signaling domain. T cells bearing the 3rd generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA+ vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. PMID:25358763

Tumor-targeting domains for chimeric antigen receptor T cells.

PubMed

Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

2017-01-01

Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

PubMed

Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen specific Interleukin 17 response

PubMed Central

Zeng, Xun; Wei, Yu-ling; Huang, Jun; Newell, Evan W.; Yu, Hongxiang; Kidd, Brian A.; Kuhns, Michael S.; Waters, Ray W.; Davis, Mark M.; Weaver, Casey T.; Chien, Yueh-hsiu

2012-01-01

Summary γδ T cells contribute uniquely to host immune defense. However, how they function remains an enigma. Although it is unclear what most γδ T cells recognize, common dogma asserts that they recognize self-antigens. While they are the major initial Interleukin-17 (IL-17) producers in infections, it is unclear what is required to trigger these cells to act. Here, we report that a noted B cell antigen, the algae protein-phycoerythrin (PE) is an antigen for murine and human γδ T cells. PE also stained specific bovine γδ T cells. Employing this specificity, we demonstrated that antigen recognition, but not extensive clonal expansion, was required to activate naïve γδ T cells to make IL-17. In this activated state, γδ T cells gained the ability to respond to cytokine signals that perpetuated the IL-17 production. These results underscore the adaptability of lymphocyte antigen receptors and suggest a previously unrecognized antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

Chimeric antigen receptor-engineered natural killer and natural killer T cells for cancer immunotherapy.

PubMed

Bollino, Dominique; Webb, Tonya J

2017-09-01

Natural killer (NK) cells of the innate immune system and natural killer T (NKT) cells, which have roles in both the innate and adaptive responses, are unique lymphocyte subsets that have similarities in their functions and phenotypes. Both cell types can rapidly respond to the presence of tumor cells and participate in immune surveillance and antitumor immune responses. This has incited interest in the development of novel cancer therapeutics based on NK and NKT cell manipulation. Chimeric antigen receptors (CARs), generated through the fusion of an antigen-binding region of a monoclonal antibody or other ligand to intracellular signaling domains, can enhance lymphocyte targeting and activation toward diverse malignancies. Most of the CAR studies have focused on their expression in T cells; however, the functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. CAR-modified NK and NKT cells are becoming more prevalent because they provide a method to direct these cells more specifically to target cancer cells, with less risk of adverse effects. This review will outline current NK and NKT cell CAR constructs and how they compare to conventional CAR T cells, and discuss future modifications that can be explored to advance adoptive cell transfer of NK and NKT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy.

PubMed

Wennhold, Kerstin; Thelen, Martin; Schlößer, Hans Anton; Haustein, Natalie; Reuter, Sabrina; Garcia-Marquez, Maria; Lechner, Axel; Kobold, Sebastian; Rataj, Felicitas; Utermöhlen, Olaf; Chakupurakal, Geothy; Theurich, Sebastian; Hallek, Michael; Abken, Hinrich; Shimabukuro-Vornhagen, Alexander; von Bergwelt-Baildon, Michael

2017-09-01

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730-43. ©2017 AACR . ©2017 American Association for Cancer Research.

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

A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.

PubMed

Mamonkin, Maksim; Rouce, Rayne H; Tashiro, Haruko; Brenner, Malcolm K

2015-08-20

Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms. © 2015 by The American Society of Hematology.

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

PubMed

Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

PubMed Central

Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

2014-01-01

Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

PubMed

Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

2013-04-18

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia

PubMed Central

Porter, David L.; Levine, Bruce L.; Kalos, Michael; Bagg, Adam; June, Carl H.

2012-01-01

SUMMARY We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×105 cells per kilogram of body weight) of autologous chimeric antigen receptor–modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect. PMID:21830940

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

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

Engineering Chimeric Antigen Receptor T cells to Treat Glioblastoma.

PubMed

Choi, Bryan D; O'Rourke, Donald M; Maus, Marcela V

2017-08-01

Immunotherapy has emerged as a promising strategy for glioblastoma (GBM), a disease that remains universally fatal despite currently available standard-of-care. Adoptive T cell therapy has been shown to produce potent antitumor immunity while obviating the need for traditional antigen presentation and primary immune responses. Chimeric antigen receptors (CARs) are specialized molecules that can be expressed on the surface of T cells allowing for redirected cytotoxicity against tumor antigens of interest. To date, the application of CAR T cells for GBM has been relatively limited, in large part due to a dearth of well-described tumor specific antigens that are both homogenously and frequently expressed. A mutated version of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase that is expressed on the surface of GBM and other common neoplasms, but completely absent from all normal tissues. We have recently generated CAR T cells directed against EGFRvIII and reported results from a Phase I clinical trial investigating this platform in patients with EGFRvIII-expressing GBM. Our study showed that despite conventional notions of central nervous system "immune-privilege," EGFRvIII CAR T cells trafficked to intracerebral tumors, leading to successful targeting and eradication of this antigen in the brain. Here, we review our experience with EGFRvIII CAR T cells and highlight important considerations for the clinical translation of this therapy in patients with GBM.

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

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

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke

2010-03-15

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal inmore » any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.« less

Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens.

PubMed Central

Gurney, E G; Harrison, R O; Fenno, J

1980-01-01

We have isolated three clones of hybrid cells which synthesize antibodies specific for determinants on simian virus 40 (SV40) T antigens. Mouse myeloma NS1 cells were fused with spleen cells from mice that had been immunized with SV40-transformed mouse cells. Hybrid cells were selected in HAT medium and cloned in soft agar. We used an enzyme-linked immunosorbent assay for detection and quantification of mouse antibodies against SV40 T antigens. Monoclonal antibodies from 3 of the 24 clones that scored as positive in the enzyme-linked immunosorbent assay were verified by immunoprecipitation to be specific for SV40 T antigens. Two clones (7 and 412) produced antibodies that recognized denaturation-sensitive antigenic determinants unique to large T antigen. Antibodies from clone 7 appeared to have a low affinity for large T antigen. Antibodies from clone 412 had a higher affinity for large T antigen but did not recognize a subclass of large T antigen that was recognized by tumor serum. Antibodies of the third clone, clone 122, recognized a denaturation-stable antigenic determinant of the 53,000-dalton mouse nonviral T antigen in SV40-transformed cells. Antibodies from clone 122 also recognized similar (51,000- to 56,000-dalton) nonviral T antigens in SV40-transormed or lytically infected cells from five mammalian species and in four uninfected mouse lines. From these observations, we have concluded that (i) the 94,000-dalton SV40 large T antigen may exist as immunologically distinguishable subclasses, and (ii) the nonviral T antigens of five mammalian species share at least one antigenic determinant. Images PMID:6155477

Carbohydrates as T-cell antigens with implications in health and disease.

PubMed

Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

2016-10-01

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response▿

PubMed Central

Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S.

2011-01-01

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols. PMID:21367979

The polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response.

PubMed

Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S

2011-05-01

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4(+) T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4(+) T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

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.

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells.

PubMed

Boutin, Lola; Scotet, Emmanuel

2018-01-01

Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg), such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277) molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset.

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells

PubMed Central

Boutin, Lola; Scotet, Emmanuel

2018-01-01

Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg), such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277) molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset. PMID:29731756

Chimeric antigen receptor T-cell therapy for glioblastoma.

PubMed

Rodriguez, Analiz; Brown, Christine; Badie, Behnam

2017-09-01

Chimeric antigen receptor (CAR) T-cell therapy has shown great promise in the treatment of hematological disease, and its utility for treatment of solid tumors is beginning to unfold. Glioblastoma continues to portend a grim prognosis and immunotherapeutic approaches are being explored as a potential treatment strategy. Identification of appropriate glioma-associated antigens, barriers to cell delivery, and presence of an immunosuppressive microenvironment are factors that make CAR T-cell therapy for glioblastoma particularly challenging. However, insights gained from preclinical studies and ongoing clinical trials indicate that CAR T-cell therapy will continue to evolve and likely become integrated with current therapeutic strategies for malignant glioma. Copyright © 2017 Elsevier Inc. All rights reserved.

ɣδ T cell receptor ligands and modes of antigen recognition

PubMed Central

Champagne, Eric

2011-01-01

T lymphocytes expressing the γδ-type of T cell receptors for antigens contribute to all aspects of immune responses, including defenses against viruses, bacteria, parasites and tumors, allergy and autoimmunity. Multiple subsets have been individualized in humans as well as in mice and they appear to recognize in a TCR-dependent manner antigens as diverse as small non-peptidic molecules, soluble or membrane-anchored polypeptides and molecules related to MHC antigens on cell surfaces, implying diverse modes of antigen recognition. We review here the γδ TCR ligands which have been identified along the years and their characteristics, with emphasis on a few systems which have been extensively studied such as human γδ T cells responding to phosphoantigens or murine γδ T cells activated by allogeneic MHC antigens. We discuss a speculative model of antigen recognition involving simultaneous TCR recognition of MHC-like and non-MHC ligands which could fit with most available data and shares many similarities with the classical model of MHC-restricted antigen recognition for peptides or lipids by T cells subsets with αβ-type TCRs. PMID:21298486

γδ T cell receptor ligands and modes of antigen recognition.

PubMed

Champagne, Eric

2011-04-01

T lymphocytes expressing the γδ-type of T cell receptors (TCRs) for antigens contribute to all aspects of immune responses, including defenses against viruses, bacteria, parasites and tumors, allergy and autoimmunity. Multiple subsets have been individualized in humans as well as in mice and they appear to recognize in a TCR-dependent manner antigens as diverse as small non-peptidic molecules, soluble or membrane-anchored polypeptides and molecules related to MHC antigens on cell surfaces, implying diverse modes of antigen recognition. We review here the γδ TCR ligands which have been identified along the years and their characteristics, with emphasis on a few systems which have been extensively studied such as human γδ T cells responding to phosphoantigens or murine γδ T cells activated by allogeneic MHC antigens. We discuss a speculative model of antigen recognition involving simultaneous TCR recognition of MHC-like and non-MHC ligands which could fit with most available data and shares many similarities with the classical model of MHC-restricted antigen recognition for peptides or lipids by T cells subsets with αβ-type TCRs.

CD8+ T-cell responses rapidly select for antigen-negative tumor cells in the prostate.

PubMed

Bak, S Peter; Barnkob, Mike Stein; Wittrup, K Dane; Chen, Jianzhu

2013-12-01

Stimulation of patients' immune systems for the treatment of solid tumors is an emerging therapeutic paradigm. The use of enriched autologous T cells for adoptive cell therapy or vaccination with antigen-loaded dendritic cells have shown clinical efficacy in melanoma and prostate cancer, respectively. However, the long-term effects of immune responses on selection and outgrowth of antigen-negative tumor cells in specific tumor types must be determined to understand and achieve long-term therapeutic effects. In this study, we have investigated the expression of a tumor-specific antigen in situ after treatment with tumor-specific CD8(+) T cells in an autochthonous mouse model of prostate cancer. After T-cell treatment, aggregates of dead antigen-positive tumor cells were concentrated in the lumen of the prostate gland and were eventually eliminated from the prostate tissue. Despite the elimination of antigen-positive tumor cells, prostate tumor continued to grow in T-cell-treated mice. Interestingly, the remaining tumor cells were antigen negative and downregulated MHC class I expression. These results show that CD8(+) T cells are effective in eliminating antigen-bearing prostate tumor cells but they also can select for the outgrowth of antigen-negative tumor cells. These findings provide insights into the requirements for an effective cancer immunotherapy within the prostate that not only induces potent immune responses but also avoids selection and outgrowth of antigen-negative tumor cells. ©2013 AACR.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

PubMed

Wu, Julie H; Simonette, Rebecca A; Nguyen, Harrison P; Rady, Peter L; Tyring, Stephen K

2016-06-01

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

PubMed Central

Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G.; Nghiem, Paul

2013-01-01

Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region. PMID:23514892

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva▿

PubMed Central

Graham, Simon P.; Pellé, Roger; Yamage, Mat; Mwangi, Duncan M.; Honda, Yoshikazu; Mwakubambanya, Ramadhan S.; de Villiers, Etienne P.; Abuya, Evelyne; Awino, Elias; Gachanja, James; Mbwika, Ferdinand; Muthiani, Anthony M.; Muriuki, Cecelia; Nyanjui, John K.; Onono, Fredrick O.; Osaso, Julius; Riitho, Victor; Saya, Rosemary M.; Ellis, Shirley A.; McKeever, Declan J.; MacHugh, Niall D.; Gilbert, Sarah C.; Audonnet, Jean-Christophe; Morrison, W. Ivan; van der Bruggen, Pierre; Taracha, Evans L. N.

2008-01-01

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes. PMID:18070892

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.

Chimaeric antigen receptor T-cell therapy for tumour immunotherapy

PubMed Central

Sha, Huan-huan; Wang, Dan-dan; Yan, Da-li; Hu, Yong; Yang, Su-jin; Liu, Si-wen

2017-01-01

Chimaeric antigen receptor (CAR) T-cell therapies, as one of the cancer immunotherapies, have heralded a new era of treating cancer. The accumulating data, especially about CAR-modified T cells against CD19 support that CAR T-cell therapy is a highly effective immune therapy for B-cell malignancies. Apart from CD19, there have been many trials of CAR T cells directed other tumour specific or associated antigens (TSAs/TAAs) in haematologic malignancies and solid tumours. This review will briefly summarize basic CAR structure, parts of reported TSAs/TAAs, results of the clinical trials of CAR T-cell therapies as well as two life-threatening side effects. Experiments in vivo or in vitro, ongoing clinical trials and the outlook for CAR T-cell therapies also be included. Our future efforts will focus on identification of more viable cancer targets and more strategies to make CAR T-cell therapy safer. PMID:28053197

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

PubMed Central

2012-01-01

Background Merkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT) encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT), as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the immunodominant LT epitope

T cell activation is determined by the number of presented antigens.

PubMed

Deeg, Janosch; Axmann, Markus; Matic, Jovana; Liapis, Anastasia; Depoil, David; Afrose, Jehan; Curado, Silvia; Dustin, Michael L; Spatz, Joachim P

2013-01-01

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

Chimeric antigen receptor T cells: a novel therapy for solid tumors.

PubMed

Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

2017-03-29

The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

Adoptive T-cell immunotherapy of cancer using chimeric antigen receptor-grafted T cells.

PubMed

Davies, David Marc; Maher, John

2010-06-01

Harnessing the power of the immune system to target cancer has long been a goal of tumor immunologists. One avenue under investigation is the modification of T cells to express a chimeric antigen receptor (CAR). Expression of such a receptor enables T-cell specificity to be redirected against a chosen tumor antigen. Substantial research in this field has been carried out, incorporating a wide variety of malignancies and tumor-associated antigens. Ongoing investigations will ensure this area continues to expand at a rapid pace. This review will explain the evolution of CAR technology over the last two decades in addition to detailing the associated benefits and disadvantages. The outcome of recent phase I clinical trials and the impact that these have had upon the direction of future research in this field will also be addressed.

T Cell Activation is Determined by the Number of Presented Antigens

PubMed Central

2013-01-01

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90–140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm2. We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density. PMID:24117051

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

NASA Astrophysics Data System (ADS)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

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

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

T-cell brain infiltration and immature antigen-presenting cells in transgenic models of Alzheimer's disease-like cerebral amyloidosis.

PubMed

Ferretti, M T; Merlini, M; Späni, C; Gericke, C; Schweizer, N; Enzmann, G; Engelhardt, B; Kulic, L; Suter, T; Nitsch, R M

2016-05-01

Cerebral beta-amyloidosis, one of the pathological hallmarks of Alzheimer's disease (AD), elicits a well-characterised, microglia-mediated local innate immune response. In contrast, it is not clear whether cells of the adaptive immune system, in particular T-cells, react to cerebral amyloidosis in AD. Even though parenchymal T-cells have been described in post-mortem brains of AD patients, it is not known whether infiltrating T-cells are specifically recruited to the extracellular deposits of beta-amyloid, and whether they are locally activated into proliferating, effector cells upon interaction with antigen-presenting cells (APCs). To address these issues we have analysed by confocal microscopy and flow-cytometry the localisation and activation status of both T-cells and APCs in transgenic (tg) mice models of AD-like cerebral amyloidosis. Increased numbers of infiltrating T-cells were found in amyloid-burdened brain regions of tg mice, with concomitant up-regulation of endothelial adhesion molecules ICAM-1 and VCAM-1, compared to non-tg littermates. The infiltrating T-cells in tg brains did not co-localise with amyloid plaques, produced less interferon-gamma than those in controls and did not proliferate locally. Bona-fide dendritic cells were virtually absent from the brain parenchyma of both non-tg and tg mice, and APCs from tg brains showed an immature phenotype, with accumulation of MHC-II in intracellular compartments. These results indicate that cerebral amyloidosis promotes T-cell infiltration but interferes with local antigen presentation and T-cell activation. The inability of the brain immune surveillance to orchestrate a protective immune response to amyloid-beta peptide might contribute to the accumulation of amyloid in the progression of the disease. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells.

PubMed

Chang, Sung Ho; Jung, Eun Jung; Park, Youn Hee; Lim, Dong Gyun; Ko, Na Young; Choi, Wahn Soo; Her, Erk; Kim, Soo Hyun; Choi, Kang Duk; Bae, Jae Ho; Kim, Sun Hee; Kang, Chi Dug; Han, Duck Jong; Kim, Song Cheol

2009-08-01

The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells. CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry. A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells

PubMed Central

Lichtenegger, Felix S.; Rothe, Maurine; Schnorfeil, Frauke M.; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus. PMID:29535740

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells.

PubMed

Lichtenegger, Felix S; Rothe, Maurine; Schnorfeil, Frauke M; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4 + and CD8 + T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

Antigen-Specific CD8+ T Cells Fail To Respond to Shigella flexneri ▿

PubMed Central

Jehl, Stephanie P.; Doling, Amy M.; Giddings, Kara S.; Phalipon, Armelle; Sansonetti, Philippe J.; Goldberg, Marcia B.; Starnbach, Michael N.

2011-01-01

CD8+ T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8+ T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8+ T cells. To determine why CD8+ T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8+ T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8+ T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8+ T-cell epitope via the Shigella type III secretion system and characterized the CD8+ T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8+ T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection. PMID:21357720

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

PubMed Central

Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

2013-01-01

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642

Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

PubMed

Hombach, Andreas A; Abken, Hinrich

2017-02-01

Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30 + hematopoietic stem and progenitor cells while eliminating CD30 + lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-β and CD123, is of risk to destruct tissue stem cells.

Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

PubMed Central

Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

2013-01-01

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

Discovery of a protective Rickettsia prowazekii antigen recognized by CD8+ T cells, RP884, using an in vivo screening platform.

PubMed

Gazi, Michal; Caro-Gomez, Erika; Goez, Yenny; Cespedes, Maria A; Hidalgo, Marylin; Correa, Paula; Valbuena, Gustavo

2013-01-01

Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8(+) T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.

Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

PubMed Central

Owens, Gemma L.; Sheard, Victoria E.; Kalaitsidou, Milena; Blount, Daniel; Lad, Yatish; Cheadle, Eleanor J.; Edmondson, Richard J.; Kooner, Gurdeep; Gilham, David E.

2018-01-01

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells. PMID:29239915

On-chip activation and subsequent detection of individual antigen-specific T cells

PubMed Central

Song, Qing; Han, Qing; Bradshaw, Elizabeth M.; Kent, Sally C.; Raddassi, Khadir; Nilsson, Björn; Nepom, Gerald T.; Hafler, David A.; Love, J. Christopher

2010-01-01

The frequencies of antigen-specific CD4+ T cells in samples of human tissue has been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or Type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC Class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples. PMID:20000848

How to train your T cell: genetically engineered chimeric antigen receptor T cells versus bispecific T-cell engagers to target CD19 in B acute lymphoblastic leukemia.

PubMed

Ruella, Marco; Gill, Saar

2015-06-01

Antigen-specific T cell-based immunotherapy is getting its day in the sun. The contemporaneous development of two potent CD19-specific immunotherapeutic modalities for the treatment of B-cell malignancies provides exciting opportunities for patients, physicians and scientists alike. Patients with relapsed, refractory or poor-risk B-cell acute lymphoblastic leukemia (ALL) previously had few therapeutic options and now have two potential new lifelines. Physicians will have the choice between two powerful modalities and indeed could potentially enroll some patients on trials exploring both modalities if needed. For scientists interested in tumor immunology, the advent of chimeric antigen receptor T-cell therapy and of bispecific T-cell engagers (BiTEs) provides unprecedented opportunities to explore the promise and limitations of antigen-specific T-cell therapy in the context of human leukemia. In this article, we compare chimeric antigen receptor T cells and BiTEs targeting CD19 in B-cell ALL in the setting of the available clinical literature.

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter.

PubMed

Mayer, Elisabeth; Bannert, Christina; Gruber, Saskia; Klunker, Sven; Spittler, Andreas; Akdis, Cezmi A; Szépfalusi, Zsolt; Eiwegger, Thomas

2012-01-01

Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these "excessive" responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself. Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([(3)H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4(+)CD25(high)FoxP3(+) T cells were characterized by mRNA analysis and flow cytometry. Cord blood derived CD4(+)CD25(high) cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4(+)CD25(high) cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3(+)CD4(+)CD25(high)cells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4(+)CD25(+)CD127(low)) is highly suppressive even without prior antigen exposure. Cord blood harbors a very small subset of CD4(+)CD25(high) Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.

Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors.

PubMed

Aldoss, I; Bargou, R C; Nagorsen, D; Friberg, G R; Baeuerle, P A; Forman, S J

2017-04-01

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r

T-Cell Receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

PubMed Central

David, Rachel; Ma, Liang; Ivetic, Aleksandar; Takesono, Aya; Ridley, Anne J.; Chai, Jian-Guo; Tybulewicz, Victor; Marelli-Berg, Federica M.

2016-01-01

Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into non-lymphoid antigen-rich tissue tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signalling pathways that regulate T cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1-/- T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1-/- T cells in vivo. In contrast, Vav1-/- T-cell retention into antigen-rich tissue was severely impaired, reflecting their inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T-cell-mediated tissue damage. PMID:19060239

A T-cell–directed chimeric antigen receptor for the selective treatment of T-cell malignancies

PubMed Central

Mamonkin, Maksim; Rouce, Rayne H.; Tashiro, Haruko

2015-01-01

Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms. PMID:26056165

How chimeric antigen receptor design affects adoptive T cell therapy

PubMed Central

Gacerez, Albert T.; Arellano, Benjamine; Sentman, Charles L.

2016-01-01

Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR’s function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. PMID:27163336

Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors.

PubMed

Jindal, Vishal; Arora, Ena; Gupta, Sorab

2018-05-05

Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

PubMed Central

Cartellieri, Marc; Bachmann, Michael; Feldmann, Anja; Bippes, Claudia; Stamova, Slava; Wehner, Rebekka; Temme, Achim; Schmitz, Marc

2010-01-01

CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells. PMID:20467460

Selectively Reduced Intracellular Proliferation of Salmonella enterica Serovar Typhimurium within APCs Limits Antigen Presentation and Development of a Rapid CD8 T Cell Response1

PubMed Central

Albaghdadi, Homam; Robinson, Nirmal; Finlay, Brett; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Ag presentation to CD8+ T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 × 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8+ T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8+ T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8+ T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intra-cellular proliferation of virulent pathogens may be an important mechanism of immune evasion. PMID:19692639

Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

PubMed

Chaudhary, Neelkamal; Staab, Janet F; Marr, Kieren A

2010-02-17

Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H)1-T(H)17) and destructive allergic (T(H)2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown. To determine whether Asp f proteins are strictly associated with T(H)2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H)1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals. Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy.

PubMed

Brown, Christine E; Alizadeh, Darya; Starr, Renate; Weng, Lihong; Wagner, Jamie R; Naranjo, Araceli; Ostberg, Julie R; Blanchard, M Suzette; Kilpatrick, Julie; Simpson, Jennifer; Kurien, Anita; Priceman, Saul J; Wang, Xiuli; Harshbarger, Todd L; D'Apuzzo, Massimo; Ressler, Julie A; Jensen, Michael C; Barish, Michael E; Chen, Mike; Portnow, Jana; Forman, Stephen J; Badie, Behnam

2016-12-29

A patient with recurrent multifocal glioblastoma received chimeric antigen receptor (CAR)-engineered T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2). Multiple infusions of CAR T cells were administered over 220 days through two intracranial delivery routes - infusions into the resected tumor cavity followed by infusions into the ventricular system. Intracranial infusions of IL13Rα2-targeted CAR T cells were not associated with any toxic effects of grade 3 or higher. After CAR T-cell treatment, regression of all intracranial and spinal tumors was observed, along with corresponding increases in levels of cytokines and immune cells in the cerebrospinal fluid. This clinical response continued for 7.5 months after the initiation of CAR T-cell therapy. (Funded by Gateway for Cancer Research and others; ClinicalTrials.gov number, NCT02208362 .).

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

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells.

PubMed

Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A

2018-05-01

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Role of interleukin 1 in antigen-specific T cell proliferation.

PubMed

Chu, E; Rosenwasser, L J; Dinarello, C A; Lareau, M; Geha, R S

1984-03-01

The role of interleukin 1 (IL 1) in human antigen-specific T cell proliferation was examined. Nylon wool-purified T cells proliferated in the presence of autologous monocytes (Mo.) pulsed for 18 h with tetanus toxoid (TT) antigen (Mo.TT). Irradiation of Mo.TT with ultraviolet (UV) light (72 J/m2) abolished their capacity to support T cell proliferation and drastically reduced their capacity to secrete IL 1 after stimulation with Staphylococcus albus. The defect in antigen presentation induced by UV irradiation of Mo.TT was reversed in a dose-dependent manner by the addition of two different preparations containing human interleukin 1 (IL 1). The first preparation consisted of supernatants of Mo. stimulated with Con A for 18 hr and in which Con A activity was blocked by alpha-D-methyl-mannoside (Mo.-Con A-Sup). The second preparation consisted of human IL 1 partially purified from supernatants of human peripheral blood mononuclear cells stimulated with S. albus. This IL 1 copurified with human leukocyte pyrogen (LP) and was termed IL 1/LP. Both IL 1-containing preparations enhanced the response of C57BL/6 mouse thymocytes to phytohemagglutinin. A rabbit antibody to human IL 1/LP inhibited the capacity of T cells to proliferate in response to Mo.TT and inhibited the capacity of Mo.-Con A-Sup to reconstitute the T cell response to UV-irradiated Mo.TT. IL 1/LP was not necessary for T cells to recognize the immunogenic moiety presented by Mo., because monolayers of UV-irradiated Mo.TT were equivalent to monolayers of unirradiated MO.TT in their capacity to adsorb TT-reactive T cells specifically. Furthermore, the addition of rabbit antibody to IL 1/LP did not interfere with the capacity of UV-irradiated Mo.TT to adsorb TT-reactive T cells. The results obtained in this study indicate that IL 1 is involved in optimal antigen-driven proliferation of human T lymphocytes.

CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells

PubMed Central

Myles, Ian A.; Zhao, Ming; Nardone, Glenn; Olano, Lisa R.; Reckhow, Jensen D.; Saleem, Danial; Break, Timothy J.; Lionakis, Michail S.; Myers, Timothy G.; Gardina, Paul J.; Kirkpatrick, Charles H.; Holland, Steven M.; Datta, Sandip K.

2017-01-01

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms "transfer factor" and "DLE" were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans. These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity. PMID:27515950

Chimeric Antigen Receptor T-Cell Therapy for the Community Oncologist

PubMed Central

Levine, Bruce L.

2016-01-01

The field of cancer immunotherapy has rapidly progressed in the past decade as several therapeutic modalities have entered into the clinic. One such immunotherapy that has shown promise in the treatment of cancer is the use of chimeric antigen receptor (CAR)-modified T lymphocytes. CARs are engineered receptors constructed from antigen recognition regions of antibodies fused to T-cell signaling and costimulatory domains that can be used to reprogram a patient’s T cells to specifically target tumor cells. CAR T-cell therapy has demonstrated sustained complete responses for some patients with advanced leukemia, and a number of CAR therapies are being evaluated in clinical studies. CAR T-cell therapy-associated toxicities, including cytokine release syndrome, macrophage activation syndrome, and tumor lysis syndrome, have been observed and effectively managed in the clinic. In patients with significant clinical responses, sustained B-cell aplasia has also been observed and is a marker of CAR T-cell persistence that might provide long-term disease control. Education on CAR T-cell therapy efficacy and safety management is critical for clinicians and patients who are considering this novel type of treatment. In the present report, the current landscape of CAR T-cell therapy, the effective management of patients undergoing treatment, and which patients are the most suitable candidates for current trials are discussed. Implications for Practice: The present report describes the current status of chimeric antigen receptor (CAR) T lymphocytes as an immunotherapy for patients with relapsed or refractory B-cell malignancies. CAR T cells targeting CD19, a protein expressed on many B-cell malignancies, typically induce high complete response rates in patients with B-cell leukemia or lymphoma who have very limited therapeutic options. Recent clinical trial results of CD19 CAR T-cell therapies and the management of CAR T-cell-associated adverse events are discussed. The present

How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

PubMed

Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

2016-12-01

Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Early postnatal exposure to cigarette smoke impairs the antigen-specific T-cell responses in the spleen.

PubMed

Singh, Shashi P; Razani-Boroujerdi, Seddigheh; Pena-Philippides, Juan C; Langley, Raymond J; Mishra, Neerad C; Sopori, Mohan L

2006-12-15

Annually, approximately two million babies are exposed to cigarette smoke in utero and postnatally through cigarette smoking of their mothers. Exposure to mainstream cigarette smoke is known to impair both innate and adaptive immunities, and it has been hypothesized that the effects of in utero exposure to cigarette smoke on children's health might primarily stem from the adverse effects of cigarette smoke on the immune system. To simulate the environment that babies from smoking mothers encounter, we examined the effects of prenatal mainstream and postnatal sidestream cigarette smoke on spleen cell responses. Results show that postnatal exposure of newborn Balb/c mouse pups to sidestream cigarette smoke through the first 6 weeks of life strongly suppresses the antibody response of spleen cells to the T-cell-dependent antigen, sheep red blood cells. The reduction in the antibody response seen within 6 weeks of postnatal smoke exposure is much quicker than the published data on the time 25 weeks) required to establish reproducible immunosuppression in adult rats and mice. Moreover, the immunosuppression is not associated with significant changes in T-cell numbers or subset distribution. While the postnatal exposure to cigarette smoke did not affect the mitogenic response of T and B cells, the exposure inhibited the T cell receptor-mediated rise in the intracellular calcium concentration. These results suggest that the early postnatal period is highly sensitive to the immunosuppressive effects of environmental tobacco smoke, and the effects are causally associated with impaired antigen-mediated signaling in T cells.

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

New Perspective on Dextran Sodium Sulfate Colitis: Antigen-Specific T Cell Development during Intestinal Inflammation

PubMed Central

Morgan, Mary E.; Zheng, Bin; Koelink, Pim J.; van de Kant, Hendrick J. G.; Haazen, Lizette C. J. M.; van Roest, Manon; Garssen, Johan; Folkerts, Gert; Kraneveld, Aletta D.

2013-01-01

CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens. PMID:23936123

Cross-Priming of Naive Cd8 T Cells against Melanoma Antigens Using Dendritic Cells Loaded with Killed Allogeneic Melanoma Cells

PubMed Central

Berard, Frederic; Blanco, Patrick; Davoust, Jean; Neidhart-Berard, Eve-Marie; Nouri-Shirazi, Mahyar; Taquet, Nicolas; Rimoldi, Donata; Cerottini, Jean Charles; Banchereau, Jacques; Palucka, A. Karolina

2000-01-01

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA+CD27+CD8+ T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201+ naive T cells primed by DCs loaded with HLA-A201− melanoma cells are able to kill several HLA-A201+ melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols. PMID:11104796

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

Chemokine programming dendritic cell antigen response: part II - programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype.

PubMed

Park, Jaehyung; Bryers, James D

2013-05-01

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency. © 2012 Blackwell Publishing Ltd.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PubMed

Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

2017-01-15

The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy

PubMed Central

Maus, Marcela V.; June, Carl H.

2016-01-01

Chimeric antigen receptors (CARs) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of ≈90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into mechanisms regulating the persistence of CAR T cells. Additionally, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy. PMID:27084741

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

PubMed Central

Zikherman, Julie; Parameswaran, Ramya; Weiss, Arthur

2012-01-01

In humans up to 75% of newly generated B cells and about 30% of mature B cells exhibit some degree of autoreactivity1. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model BCR transgenic systems have highlighted the critical role played by functional unresponsiveness or ‘anergy’2,3. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B cell tolerance4,5. However, it remains unclear whether the mature diverse B cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which B cell antigen receptor (BCR) signaling rapidly and robustly induces GFP expression under the control of the Nur77 regulatory region, antigen-dependent and – independent BCR signaling events in vivo during B cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure in turn tunes the responsiveness of BCR signaling in B cells at least partly by down-modulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or ‘anergy’ exists in the mature B cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease. PMID:22902503

Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

PubMed

Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

2015-08-12

A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated.

Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells

PubMed Central

Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

2015-01-01

A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

[Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

PubMed

Ozawa, Keiya

2014-03-01

Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

Co-ordination of incoming and outgoing traffic in antigen-presenting cells by pattern recognition receptors and T cells.

PubMed

Nair, Priyanka; Amsen, Derk; Blander, J Magarian

2011-12-01

Dendritic cells are innate sentinels of the immune system and potent activators of naÏve T cells. Mechanisms must exist to enable these cells to achieve maximal activation of T cells specific for microbial antigens, while avoiding activation of T cells specific for self-antigens. Here we discuss how a combination of signals from pattern recognition receptors and T cells co-ordinates subcellular trafficking of antigen with both major histocompatibility complex class I and class II molecules and T-cell costimulatory molecules, resulting in the preferential presentation of microbial peptides within a stimulatory context. © 2011 John Wiley & Sons A/S.

Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells.

PubMed

Park, Jae H; Brentjens, Renier J

2010-04-01

Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

PubMed

Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

2017-07-28

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll

Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy.

PubMed

Mirzaei, Hamid Reza; Mirzaei, Hamed; Lee, Sang Yun; Hadjati, Jamshid; Till, Brian G

2016-10-01

Excitement is growing for therapies that harness the power of patients' immune systems to combat their diseases. One approach to immunotherapy involves engineering patients' own T cells to express a chimeric antigen receptor (CAR) to treat advanced cancers, particularly those refractory to conventional therapeutic agents. Although these engineered immune cells have made remarkable strides in the treatment of patients with certain hematologic malignancies, success with solid tumors has been limited, probably due to immunosuppressive mechanisms in the tumor niche. In nearly all studies to date, T cells bearing αβ receptors have been used to generate CAR T cells. In this review, we highlight biological characteristics of γδ T cells that are distinct from those of αβ T cells, including homing to epithelial and mucosal tissues and unique functions such as direct antigen recognition, lack of alloreactivity, and ability to present antigens. We offer our perspective that these features make γδ T cells promising for use in cellular therapy against several types of solid tumors, including melanoma and gastrointestinal cancers. Engineered γδ T cells should be considered as a new platform for adoptive T cell cancer therapy for mucosal tumors. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

PubMed

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.

PubMed

Jordan, Kimberly R; McMahan, Rachel H; Kemmler, Charles B; Kappler, John W; Slansky, Jill E

2010-03-09

Peptide vaccines enhance the response of T cells toward tumor antigens and represent a strategy to augment antigen-independent immunotherapies of cancer. However, peptide vaccines that include native tumor antigens rarely prevent tumor growth. We have assembled a set of peptide variants for a mouse-colon tumor model to determine how to improve T-cell responses. These peptides have similar affinity for MHC molecules, but differ in the affinity of the peptide-MHC/T-cell receptor interaction with a tumor-specific T-cell clone. We systematically demonstrated that effective antitumor responses are generated after vaccination with variant peptides that stimulate the largest proportion of endogenous T cells specific for the native tumor antigen. Importantly, we found some variant peptides that strongly stimulated a specific T-cell clone in vitro, but elicited fewer tumor-specific T cells in vivo, and were not protective. The T cells expanded by the effective vaccines responded to the wild-type antigen by making cytokines and killing target cells, whereas most of the T cells expanded by the ineffective vaccines only responded to the peptide variants. We conclude that peptide-variant vaccines are most effective when the peptides react with a large responsive part of the tumor-specific T-cell repertoire.

T cell receptor–induced phosphoinositide-3-kinase p110δ activity is required for T cell localization to antigenic tissue in mice

PubMed Central

Jarmin, Sarah J.; David, Rachel; Ma, Liang; Chai, Jan-Guo; Dewchand, Hamlata; Takesono, Aya; Ridley, Anne J.; Okkenhaug, Klaus; Marelli-Berg, Federica M.

2008-01-01

The establishment of T cell–mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110δ contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110δ displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110δ activity. These observations suggest that pharmacological targeting of p110δ activity is a viable strategy for the therapy of T cell–mediated pathology. PMID:18259608

The Impact of T Cell Intrinsic Antigen Adaptation on Peripheral Immune Tolerance

PubMed Central

Singh, Nevil J; Chen, Chuan; Schwartz, Ronald H

2006-01-01

Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation) in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell–replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell–replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell–mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo. PMID:17048986

Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression.

PubMed

Furusawa, Chikara; Yamaguchi, Tomoyuki

The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.

Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression

PubMed Central

Furusawa, Chikara; Yamaguchi, Tomoyuki

2016-01-01

The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification. PMID:27668873

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

PubMed

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin; Wang, Lei

2017-11-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

PubMed Central

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin

2017-01-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy. PMID:29155896

Uniform cell-autonomous tumorigenesis of the choroid plexus by papovavirus large T antigens.

PubMed Central

Chen, J D; Van Dyke, T

1991-01-01

The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these tumors develop focally after several months, it has been suggested that secondary cellular alterations are required to induce a tumor in this tissue. In contrast to SV40, the related lymphotropic papovavirus early region induces rapid nonfocal choroid plexus neoplasia in transgenic mice. Here, using hybrid gene constructs, we showed that T antigen from either virus in in fact sufficient to induce these tumors. Their abilities to induce proliferative abnormalities in other tissues, such as kidney and thymus, were also indistinguishable. Differences in the rate of choroid plexus tumorigenesis reflected differences in the control regions of the two viruses, rather than differences in T antigen per se. Under SV40 regulation, expression was limited to a fraction of the choroid plexus cells prior to the formation of focal tumors. When SV40 T antigen was placed under lymphotropic papovavirus control, in contrast, expression was generally uniform in the choroid plexus and rapid expansion of the tissue ensued. We found a direct relationship between T-antigen expression, morphological transformation, and proliferation of the choroid plexus epithelial cells. Analysis of mosaic transgenic mice indicated further that T antigen exerts its mitogenic effect cell autonomously. These studies form the foundation for elucidating the role of various T-antigen subactivities in tumorigenesis. Images PMID:1658622

Adaptor protein-3 is required in dendritic cells for optimal Toll-like receptor signaling from phagosomes and antigen presentation to CD4+ T cells

PubMed Central

Mantegazza, Adriana R.; Guttentag, Susan H.; El-Benna, Jamel; Sasai, Miwa; Iwasaki, Akiko; Shen, Hao; Laufer, Terri M.; Marks, Michael S.

2012-01-01

SUMMARY Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4+ T cell activation and Th1 effector function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores. PMID:22560444

SYNTHESIS, INTRACELLULAR DISTRIBUTION, AND SECRETION OF IMMUNOGLOBULIN AND H-2 ANTIGEN IN MURINE SPLENOCYTES

PubMed Central

Wernet, Dorothee; Vitetta, Ellen S.; Uhr, Jonathan W.; Boyse, Edward A.

1973-01-01

A/J spleen cells were labeled with [3H]leucine and at intervals thereafter were homogenized and separated into microsomes and cell sap. Ig and H-2 antigens were assayed in the cell fractions and cell supernatants using immunoprecipitation. In addition, cells labeled by enzymatic radioiodination were incubated to determine the rates of release of Ig and H-2 antigens from the surface. The results indicate that the majority of Ig and H-2 antigens remain membrane bound throughout their intracellular life. In contrast to Ig, H-2 antigens are neither secreted nor shed from the cell surface. It is suggested that Ig is a peripheral protein of the cell membrane, whereas H-2 antigens are integral ones. The release of Ig on a fragment of plasma membrane could occur at fixed cell surface areas that contain no H-2 antigens or from which they have migrated before release. PMID:4200648

Chimeric Antigen Receptor (CAR) T Cell Therapy for Malignant Pleural Mesothelioma (MPM)

PubMed Central

Klampatsa, Astero; Haas, Andrew R.; Moon, Edmund K.; Albelda, Steven M.

2017-01-01

Cancer immunotherapy has now become a recognized approach to treating cancers. In addition to checkpoint blockade, adoptive T cell transfer (ACT) using chimeric antigen receptors (CARs) has shown impressive clinical outcomes in leukemias and is now being explored in solid tumors. CARs are engineered receptors, stably or transiently transduced into T cells, that aim to enhance T cell effector function by recognizing and binding to a specific tumor-associated antigen. In this review, we provide a summary of CAR T cell preclinical studies and clinical trials for malignant pleural mesothelioma (MPM), a rare, locally invasive pleural cancer with poor prognosis. We list other attractive potential targets for CAR T cell therapy for MPM, and discuss augmentation strategies of CAR T cell therapy with other forms of immunotherapy in this disease. PMID:28862644

Effects of cryopreservation on chimeric antigen receptor T cell functions.

PubMed

Xu, Hao; Cao, Wenyue; Huang, Liang; Xiao, Min; Cao, Yang; Zhao, Lei; Wang, Na; Zhou, Jianfeng

2018-06-14

Chimeric antigen receptor T (CART) cell therapy has emerged as a potentially curative "drug" for cancer treatment. Cryopreservation of CART cells is necessary for their clinical application. Systematic studies on the effects of cryopreservation on the antitumor function of CART cells are lacking. Therefore, we compared the phenotypes and functions of CART cells that were cryopreserved during ex vivo expansion with those of freshly isolated populations. T cells expressing an anti-B-cell-maturation-antigen (BCMA) chimeric antigen receptor (CAR) were expanded in vitro for 10 days and then cryopreserved. After one month, the cells were resuscitated, and their transduction rates, apoptosis rates and cell subsets were examined via flow cytometry. The results indicated no significant changes in transduction rates or cell subsets, and the survival rate of the resuscitated cells was approximately 90% Furthermore, similar tumoricidal effects and degranulation functions of the resuscitated cells compared with normally cultured cells were verified by calcein release and CD107a assays. A NOD/SCID mouse model was used to estimate the differences in the in vivo antitumor effects of the cryopreserved and normally cultured T cells, but no significant differences were observed. Following co-culture with several target cell types, the cytokines released by the cryopreserved and normally cultured T cells were measured via enzyme-linked immunosorbent assays (ELISAs). The results revealed that the release of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was significantly decreased. These data demonstrated that with the exception of a decrease in cytokine release, the cryopreserved CART cells retained their antitumor functions. Copyright © 2018. Published by Elsevier Inc.

Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

PubMed Central

Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

2016-01-01

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

Chimeric Antigen Receptor T Cell Therapy in Hematology.

PubMed

Ataca, Pınar; Arslan, Önder

2015-12-01

It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

PubMed Central

Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

2016-01-01

Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

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.

Toxicities of chimeric antigen receptor T cells: recognition and management

PubMed Central

Brudno, Jennifer N.

2016-01-01

Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

PubMed

Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

1989-01-01

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape

PubMed Central

Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A.; Wakefield, Amanda; Bielamowicz, Kevin; Chow, Kevin K.H.; Brawley, Vita S.; Byrd, Tiara T.; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S.; Baker, Matthew L.; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K.

2016-01-01

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape. PMID:27427982

Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation.

PubMed

Kopitar, A N; Ihan Hren, N; Ihan, A

2006-02-01

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

PubMed

Borchers, S; Maβlo, C; Müller, C A; Tahedl, A; Volkind, J; Nowak, Y; Umansky, V; Esterlechner, J; Frank, M H; Ganss, C; Kluth, M A; Utikal, J

2018-01-01

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8 + T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8 + T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma. © 2017 British Society for Immunology.

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

PubMed Central

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

2011-01-01

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

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation.

PubMed

Woodham, Andrew W; Yan, Lisa; Skeate, Joseph G; van der Veen, Daniel; Brand, Heike H; Wong, Michael K; Da Silva, Diane M; Kast, W Martin

2016-12-01

Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8 + T cells receiving signal 1 + 2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.

Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens

PubMed Central

Gao, Jie; Ochyl, Lukasz J; Yang, Ellen; Moon, James J

2017-01-01

Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials. PMID:28243087

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

PubMed Central

Zerrahn, J; Deppert, W

1993-01-01

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T. Images PMID:8382310

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.

Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo.

PubMed Central

Riedlinger, J; Grencis, R K; Wakelin, D

1986-01-01

T-cell lines specific for infective muscle larvae antigens of the intestinal nematode Trichinella spiralis have been generated in vitro. These antigen-specific T-cell lines express the L3T4+ Ly2- phenotype and secrete the lymphokines IL-2, IL-3 and gamma-IFN. They are stable in culture for up to 15 weeks and are protective when adoptively transferred into naive recipients. As few as 2 x 10(5) T. spiralis-specific tract. In addition, intestinal mastocytosis and peripheral blood eosinophilia were accelerated after adoptive transfer of T. spiralis-specific T-cell lines. PMID:2423438

Antigen Potency and Maximal Efficacy Reveal a Mechanism of Efficient T Cell Activation

PubMed Central

Wheeler, Richard J.; Zhang, Hao; Cordoba, Shaun-Paul; Peng, Yan-Chun; Chen, Ji-Li; Cerundolo, Vincenzo; Dong, Tao; Coombs, Daniel; van der Merwe, P. Anton

2014-01-01

T cell activation, a critical event in adaptive immune responses, follows productive interactions between T cell receptors (TCRs) and antigens, in the form of peptide-bound major histocompatibility complexes (pMHCs) on the surfaces of antigen-presenting-cells. Upon activation, T cells can lyse infected cells, secrete cytokines, such as interferon-γ (IFN-γ), and perform other effector functions with various efficiencies that directly depend on the binding parameters of the TCR-pMHC complex. The mechanism that relates binding parameters to the efficiency of activation of the T cell remains controversial; some studies suggest that the dissociation constant (KD) determines the response (the “affinity model”), whereas others suggest that the off-rate (koff) is critical (the “productive hit rate model”). Here, we used mathematical modeling to show that antigen potency, as determined by the EC50, the functional correlate that is used to support KD-based models, could not be used to discriminate between the affinity and productive hit rate models. Our theoretical work showed that both models predicted a correlation between antigen potency and KD, but only the productive hit rate model predicted a correlation between maximal efficacy (Emax) and koff. We confirmed the predictions made by the productive hit rate model in experiments with cytotoxic T cell clones and a panel of pMHC variants. Therefore, we suggest that the activity of an antigen is determined by both its potency and maximal efficacy. We discuss the implications of our findings to the practical evaluation of T cell activation, for example in adoptive immunotherapies, and relate our work to the pharmacological theory of dose-response. PMID:21653229

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

PubMed

Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

1982-01-01

A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T-cell activation.

PubMed

Kitzmüller, C; Wallner, M; Deifl, S; Mutschlechner, S; Walterskirchen, C; Zlabinger, G J; Ferreira, F; Bohle, B

2012-11-01

BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1-like fold and displays negligible IgE-binding but strong T cell-activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation. Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T-cell clones were compared. Fluorescently labeled BM4 and Bet v 1 were used to study surface binding, endocytosis, and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T-cell clones and the polarization of naïve T cells. BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 showed increased surface binding and internalization as well as faster endolysosomal degradation compared with Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T-cell clones and promoted less IL-5 production in T cells than Bet v 1-pulsed mdDC. The loss of the Bet v 1-fold changes the protein's interaction with the human immune system at the level of antigen-presenting cells resulting in altered T-cell responses. By combining low IgE-binding with strong and modulating T cell-activating capacity, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy. © 2012 John Wiley & Sons A/S.

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

PubMed

Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

2013-10-01

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel

PubMed Central

Irving, Melita; Vuillefroy de Silly, Romain; Scholten, Kirsten; Dilek, Nahzli; Coukos, George

2017-01-01

T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors. PMID:28421069

Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor

PubMed Central

Jena, Bipulendu; Dotti, Gianpietro

2010-01-01

Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. To broaden the clinical application of adoptive immunotherapy against malignancies, investigators have developed robust systems for the genetic modification and characterization of T cells expressing introduced chimeric antigen receptors (CARs) to redirect specificity. Human trials are under way in patients with aggressive malignancies to test the hypothesis that manipulating the recipient and reprogramming T cells before adoptive transfer may improve their therapeutic effect. These examples of personalized medicine infuse T cells designed to meet patients' needs by redirecting their specificity to target molecular determinants on the underlying malignancy. The generation of clinical grade CAR+ T cells is an example of bench-to-bedside translational science that has been accomplished using investigator-initiated trials operating largely without industry support. The next-generation trials will deliver designer T cells with improved homing, CAR-mediated signaling, and replicative potential, as investigators move from the bedside to the bench and back again. PMID:20439624

Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone.

PubMed Central

Leung, K N; Nash, A A; Sia, D Y; Wildy, P

1984-01-01

A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed. PMID:6209206

Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone.

PubMed

Leung, K N; Nash, A A; Sia, D Y; Wildy, P

1984-12-01

A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed.

Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

PubMed Central

Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

2014-01-01

Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

Effective antigen presentation to helper T cells by human eosinophils.

PubMed

Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

PubMed

Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

2013-01-01

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

Studies on B-cell memory. III. T-dependent aspect of B memory generation in mice immunized with T-independent type-2(TI-2) antigen.

PubMed

Hosokawa, T; Tanaka, Y; Aoike, A; Kawai, K; Muramatsu, S

1984-09-01

The time course of B-cell memory development to a dinitrophenyl (DNP) T-independent type-2 (TI-2) antigen was investigated by adoptive cell transfer. Strong IgM and IgG memory developed in BALB/c mice after immunization with DNP-dextran, to be recalled by challenge with either T-dependent (TD) antigen or TI-2 antigen. However, only weak IgM memory and very feeble IgG memory were detected in athymic nude mice receiving the same immunization as euthymic mice. Once memory was established under probable T cell influence, its recall by TI-2 antigen challenge seemed independent of T cell help and did not require sharing of carriers between priming and challenge antigens. The following may be concluded. (i) Long-term IgM and IgG memory is induced by TI-2 antigen priming in the presence of functional T cells. (ii) The class switch from IgM to IgG in the memory B cell pool is driven effectively by TI-2 antigen and is probably T cell-dependent.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

PubMed Central

Thiel, U; Pirson, S; Müller-Spahn, C; Conrad, H; Busch, D H; Bernhard, H; Burdach, S; Richter, G H S

2011-01-01

Background: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8+ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. Methods: Following repetitive CHM1319 (VIMPCSWWV) and EZH2666 (YMCSFLFNL) peptide-driven stimulations with HLA-A*0201+ dendritic cells (DC), allo-restricted HLA-A*0201− CD8+ T cells were stained with HLA-A*0201/peptide multimers, sorted and expanded by limiting dilution. Results: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A*0201 and killed HLA-A*0201+ ET lines expressing the antigen while HLA-A*0201– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2−/−γC−/− mice. Within this context, we identified the CHM1319 peptide as a new candidate target antigen for ET immunotherapy. Conclusion: These results clearly identify the ET-derived antigens, EZH2666 and CHM1319, as suitable targets for protective allo-restricted human CD8+ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation. PMID:21407224

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

PubMed Central

Wu, Chia-Yung; Roybal, Kole T.; Puchner, Elias M.; Onuffer, James; Lim, Wendell A.

2016-01-01

There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen specificity. In these split receptors, antigen binding and intracellular signaling components only assemble in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate both cell autonomous recognition and user control. PMID:26405231

Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice

PubMed Central

Olds, Peter; Park, Andrew; Schlesinger, Sarah J.

2011-01-01

Foxp3+ regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11chigh dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3+ cells. The induced CD4+CD25+Foxp3+ cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RBhi T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3+ T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease. PMID:22084406

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva.

PubMed

Morrison, W I

2007-08-19

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.

Chimeric-antigen receptor T (CAR-T) cell therapy for solid tumors: challenges and opportunities

PubMed Central

Xia, An-Liang; Wang, Xiao-Chen; Lu, Yi-Jun; Lu, Xiao-Jie; Sun, Beicheng

2017-01-01

Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells. However, CAR T-cell therapy for solid tumors currently is faced with numerous challenges such as physical barriers, the immunosuppressive tumor microenvironment and the specificity and safety. The clinical results in solid tumors have been much less encouraging, with multiple cases of toxicity and a lack of therapeutic response. In this review, we will discuss the current stats and challenges of CAR-T cell therapy for solid tumors, and propose possibl e solutions and future perspectives. PMID:29163850

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

PubMed

Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

2008-09-01

A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

PubMed

Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

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

Hara, T.; Jung, L.K.L.; FU, S.M.

1986-03-01

With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% ofmore » the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.« less

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Uptake routes of tumor-antigen MAGE-A3 by dendritic cells determine priming of naïve T-cell subtypes.

PubMed

Moeller, Ines; Spagnoli, Giulio C; Finke, Jürgen; Veelken, Hendrik; Houet, Leonora

2012-11-01

Induction of tumor-antigen-specific T cells in active cancer immunotherapy is generally difficult due to the very low anti-tumoral precursor cytotoxic T cells. By improving tumor-antigen uptake and presentation by dendritic cells (DCs), this problem can be overcome. Focusing on MAGE-A3 protein, frequently expressed in many types of tumors, we analyzed different DC-uptake routes after additional coating the recombinant MAGE-A3 protein with either a specific monoclonal antibody or an immune complex formulation. Opsonization of the protein with antibody resulted in increased DC-uptake compared to the uncoated rhMAGE-A3 protein. This was partly due to Fcγ receptor-dependent internalization. However, unspecific antigen internalization via macropinocytosis also played a role. When analyzing DC-uptake of MAGE-A3 antigen expressed in multiple myeloma cell line U266, pretreatment with proteasome inhibitor bortezomib resulted in increased apoptosis compared to γ-irradiation. Bortezomib-mediated immunogenic apoptosis, characterized by elevated surface expression of hsp90, triggered higher phagocytosis of U266 cells by DCs involving specific DC-derived receptors. We further investigated the impact of antigen delivery on T-cell priming. Induction of CD8(+) T-cell response was favored by stimulating naïve T cells with either antibody-opsonized MAGE-A3 protein or with the bortezomib-pretreated U266 cells, indicating that receptor-mediated uptake favors cross-presentation of antigens. In contrast, CD4(+) T cells were preferentially induced after stimulation with the uncoated protein or protein in the immune complex, both antigen formulations were preferentially internalized by DCs via macropinocytosis. In summary, receptor-mediated DC-uptake mechanisms favored the induction of CD8(+) T cells, relevant for clinical anti-tumor response.

Chimeric Antigen Receptors T Cell Therapy in Solid Tumor: Challenges and Clinical Applications.

PubMed

Mirzaei, Hamid R; Rodriguez, Analiz; Shepphird, Jennifer; Brown, Christine E; Badie, Behnam

2017-01-01

Adoptive cellular immunotherapy (ACT) employing engineered T lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated promising antitumor effects in advanced hematologic cancers, such as relapsed or refractory acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma, supporting the translation of ACT to non-hematological malignancies. Although CAR T cell therapy has made remarkable strides in the treatment of patients with certain hematological cancers, in solid tumors success has been limited likely due to heterogeneous antigen expression, immunosuppressive networks in the tumor microenvironment limiting CAR T cell function and persistence, and suboptimal trafficking to solid tumors. Here, we outline specific approaches to overcome barriers to CAR T cell effectiveness in the context of the tumor microenvironment and offer our perspective on how expanding the use of CAR T cells in solid tumors may require modifications in CAR T cell design. We anticipate these modifications will further expand CAR T cell therapy in clinical practice.

A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy.

PubMed

Zhang, Erhao; Xu, Hanmei

2017-01-03

Adoptive cell therapy using chimeric antigen receptor (CAR)-engineered T cells has emerged as a very promising approach to combating cancer. Despite its ability to eliminate tumors shown in some clinical trials, CAR-T cell therapy involves some significant safety challenges, such as cytokine release syndrome (CRS) and "on-target, off-tumor" toxicity, which is related to poor control of the dose, location, and timing of T cell activity. In the past few years, some strategies to avoid the side effects of CAR-T cell therapy have been reported, including suicide gene, inhibitory CAR, dual-antigen receptor, and the use of exogenous molecules as switches to control the CAR-T cell functions. Because of the advances of the CAR paradigm and other forms of cancer immunotherapy, the most effective means of defeating the cancer has become the integration therapy with the combinatorial control system of switchable dual-receptor CAR-T cell and immune checkpoint blockade.

Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

PubMed

Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

2018-05-01

Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition.

PubMed

Riddell, Stanley R; Sommermeyer, Daniel; Berger, Carolina; Liu, Lingfeng Steven; Balakrishnan, Ashwini; Salter, Alex; Hudecek, Michael; Maloney, David G; Turtle, Cameron J

2014-01-01

The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in targeting CD19 on B-cell malignancies. The clinical trials of CD19 chimeric antigen receptor therapy have thus far not attempted to select defined subsets before transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to using adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.

T Cells with Low Avidity for a Tissue-Restricted Antigen Routinely Evade Central and Peripheral Tolerance and Cause Autoimmunity

PubMed Central

Zehn, Dietmar; Bevan, Michael J.

2009-01-01

Summary T cells causing autoimmunity must escape tolerance. We observed that CD8+ T cells with high avidity for an antigen expressed in the pancreas, kidney, and thymic medulla were efficiently removed from a polyclonal repertoire by central and peripheral tolerance mechanisms. However, both mechanisms spared low-avidity T cells from elimination. Neither the introduction of activated, self-antigen-specific CD4+ helper T cells nor a global inflammatory stimulus were sufficient to activate the low-avidity CD8+ T cells and did not break tolerance. In contrast, challenge with a recombinant bacterium expressing the self antigen primed the low-avidity T cells, and the animals rapidly developed autoimmune diabetes. We suggest that whereas thymic and peripheral tolerance mechanisms remove cells that can be primed by endogenous amounts of self antigen, they do not guard against tissue destruction by low-avidity effector T cells, which have been primed by higher amounts of self antigen or by crossreactive antigens. PMID:16879996

Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-12-15

Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as "cellular drugs". As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

PubMed

Smith, Mark L; Mason, Hugh S; Shuler, Michael L

2002-12-30

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed. Copyright 2002 Wiley Periodicals, Inc.

Impairment of T Cell Function in Parasitic Infections

PubMed Central

Rodrigues, Vasco; Cordeiro-da-Silva, Anabela; Laforge, Mireille; Ouaissi, Ali; Akharid, Khadija; Silvestre, Ricardo; Estaquier, Jérôme

2014-01-01

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence. PMID:24551250

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.

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

Chimeric Antigen Receptor T cells for B Cell Neoplasms: Choose the Right CAR for You.

PubMed

Ruella, Marco; June, Carl H

2016-10-01

Genetic redirection of T lymphocytes allows us to unleash these potent cellular immune effectors against cancer. Chimeric antigen receptor (CAR) T cells are the best-in-class example that genetic engineering of T cells can lead to deep and durable responses, as has been shown in several clinical trials for CD19+ B cell malignancies. As a consequence, in the last few years, several academic institutions and commercial partners have started developing anti-CD19 CAR T cell products. Although most of these T cell products are highly effective in vivo, basic differences among them can generate different performance characteristics and thereby impact their long-term clinical outcome. Several strategies are being implemented in order to solve the current open issues of CART19 therapy: (i) increasing efficacy against indolent B cell leukemias and lymphomas, (ii) avoiding or preventing antigen-loss relapses, (iii) reducing and managing toxicity, and (iv) bringing this CART therapy to routine clinical practice. The field of CART therapies is thriving, and exciting new avenues are opening for both scientists and patients.

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.

PubMed

Linnemann, Carsten; van Buuren, Marit M; Bies, Laura; Verdegaal, Els M E; Schotte, Remko; Calis, Jorg J A; Behjati, Sam; Velds, Arno; Hilkmann, Henk; Atmioui, Dris El; Visser, Marten; Stratton, Michael R; Haanen, John B A G; Spits, Hergen; van der Burg, Sjoerd H; Schumacher, Ton N M

2015-01-01

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen-specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen-specific CD4+ T-cell reactivity is a common property in human melanoma.

ErbB-targeted CAR T-cell immunotherapy of cancer.

PubMed

Whilding, Lynsey M; Maher, John

2015-01-01

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

Lack of Original Antigenic Sin in Recall CD8+ T Cell Responses

PubMed Central

Zehn, Dietmar; Turner, Michael J.; Lefrançois, Leo; Bevan, Michael J.

2010-01-01

In the real world, mice and men are not immunologically naive, having been exposed to numerous antigenic challenges. Prior infections sometimes negatively impact the response to a subsequent infection. This can occur in serial infections with pathogens sharing cross-reactive Ags. At the T cell level it has been proposed that preformed memory T cells, which cross-react with low avidity to epitopes presented in subsequent infections, dampen the response of high-avidity T cells. We investigated this with a series of related MHC class-I restricted Ags expressed by bacterial and viral pathogens. In all cases, we find that high-avidity CD8+ T cell precursors, either naive or memory, massively expand in secondary cross-reactive infections to dominate the response over low-avidity memory T cells. This holds true even when >10% of the CD8+ T cell compartment consists of memory T cells that cross-react weakly with the rechallenge ligand. Occasionally, memory cells generated by low-avidity stimulation in a primary infection recognize a cross-reactive epitope with high avidity and contribute positively to the response to a second infection. Taken together, our data show that the phenomenon of original antigenic sin does not occur in all heterologous infections. PMID:20439913

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

PubMed

Wu, Chia-Yung; Roybal, Kole T; Puchner, Elias M; Onuffer, James; Lim, Wendell A

2015-10-16

There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control. Copyright © 2015, American Association for the Advancement of Science.

The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies.

PubMed

Neal, Lillian R; Bailey, Stefanie R; Wyatt, Megan M; Bowers, Jacob S; Majchrzak, Kinga; Nelson, Michelle H; Haupt, Carl; Paulos, Chrystal M; Varela, Juan C

2017-01-01

Adoptive T cell transfer (ACT) can mediate objective responses in patients with advanced malignancies. There have been major advances in this field, including the optimization of the ex vivo generation of tumor-reactive lymphocytes to ample numbers for effective ACT therapy via the use of natural and artificial antigen presenting cells (APCs). Herein we review the basic properties of APCs and how they have been manufactured through the years to augment vaccine and T cell-based cancer therapies. We then discuss how these novel APCs impact the function and memory properties of T cells. Finally, we propose new ways to synthesize aAPCs to augment the therapeutic effectiveness of antitumor T cells for ACT therapy.

The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies

PubMed Central

Neal, Lillian R.; Bailey, Stefanie R.; Wyatt, Megan M.; Bowers, Jacob S.; Majchrzak, Kinga; Nelson, Michelle H.; Haupt, Carl; Paulos, Chrystal M.; Varela, Juan C.

2017-01-01

Adoptive T cell transfer (ACT) can mediate objective responses in patients with advanced malignancies. There have been major advances in this field, including the optimization of the ex vivo generation of tumor-reactive lymphocytes to ample numbers for effective ACT therapy via the use of natural and artificial antigen presenting cells (APCs). Herein we review the basic properties of APCs and how they have been manufactured through the years to augment vaccine and T cell-based cancer therapies. We then discuss how these novel APCs impact the function and memory properties of T cells. Finally, we propose new ways to synthesize aAPCs to augment the therapeutic effectiveness of antitumor T cells for ACT therapy. PMID:28825053

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

PubMed

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling.

PubMed

Bendz, Henriette; Ruhland, Sibylle C; Pandya, Maya J; Hainzl, Otmar; Riegelsberger, Stefan; Braüchle, Christoph; Mayer, Matthias P; Buchner, Johannes; Issels, Rolf D; Noessner, Elfriede

2007-10-26

Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance.

At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

PubMed

Daniyan, Anthony F O; Brentjens, Renier J

2016-12-01

The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic. © Society for Leukocyte Biology.

Endogenous antigen processing drives the primary CD4+ T cell response to influenza

PubMed Central

Miller, Michael A.; Ganesan, Asha Purnima V.; Luckashenak, Nancy; Mendonca, Mark; Eisenlohr, Laurence C.

2015-01-01

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with MHC class II molecules. Alternative pathways of epitope production have been identified but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome, and gamma-interferon inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses. PMID:26413780

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines.

PubMed

Burger, C; Fanning, E

1983-04-15

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.

The molecular bases of δ/αβ T cell-mediated antigen recognition.

PubMed

Pellicci, Daniel G; Uldrich, Adam P; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B G; de Boer, Renate; Lim, Ricky T; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H M; Gras, Stephanie; Rossjohn, Jamie; Godfrey, Dale I

2014-12-15

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. © 2014 Pellicci et al.

Chimeric antigen receptor T cells: power tools to wipe out leukemia and lymphoma.

PubMed

Riet, Tobias; Abken, Hinrich

2015-08-01

Adoptive cell therapy for malignant diseases is showing promise in recent early-phase trials in the treatment of B cell leukemia/lymphoma. Genetically engineered with a tumor-specific chimeric antigen receptor, patient's T cells produce lasting and complete leukemia regression. However, treatment is associated with some toxicity which needs our attention and the field still faces some hurdles at the scientific, technologic and clinical levels. Surmounting these obstacles will establish chimeric antigen receptor T cell therapy as a powerful approach to cure hematologic malignancies, paving the way for the treatment of other common types of cancer in the future.

T suppressor cells are required for the maintenance of the antigen-induced B-cell unresponsive state in humans

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

Benveniste, E.; Stevens, R.H.

1983-04-01

Tetanus toxoid immunization of humans generates circulating B cells which secrete IgG anti-tetanus toxoid antibodies (IgG-Tet) when stimulated in vitro with T cells and pokeweed mitogen (PWM). A unique property of these cells is the inhibition of maturation into antibody-secreting plasma cells following a 1-hr in vitro pulse with tetanus toxoid. Studies were undertaken to determine if different T-cell subsets could modulate the in vitro generated B-cell unresponsive state. The addition of OKT4+/OKT8- cells to antigen-treated B cells resulted in a partial reversal of the antigen-induced inhibition of IgG-Tet synthesis. The addition of OKT4-/OKT8+ cells to the treated B cellsmore » caused a suppression of IgG-Tet synthesis comparable to that seen in cultures containing unfractionated T cells. These results indicate that (1) the B-cell unresponsive state generated by antigen treatment is not absolute, (2) the degree of B-cell unresponsiveness results from a balance of suppressor and helper signals, and (3) T-suppressor cells need to be present to induce and maintain the B-cell unresponsive state.« less

Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation

PubMed Central

Chakera, A; Bennett, S; Lawrence, S; Morteau, O; Mason, P D; O'Callaghan, C A; Cornall, R J

2011-01-01

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein–Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured. PMID:21671906

Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii.

PubMed

John, Beena; Harris, Tajie H; Tait, Elia D; Wilson, Emma H; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A

2009-07-01

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

PubMed

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

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

Han, Hui; Peng, Ji-Run, E-mail: pengjr@medmail.com.cn; Chen, Peng-Cheng

Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparationmore » of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.« less

Use of Engineered Exosomes Expressing HLA and Costimulatory Molecules to Generate Antigen-specific CD8+ T Cells for Adoptive Cell Therapy.

PubMed

Kim, Sueon; Sohn, Hyun-Jung; Lee, Hyun-Joo; Sohn, Dae-Hee; Hyun, Seung-Joo; Cho, Hyun-Il; Kim, Tai-Gyu

2017-04-01

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.

Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment.

PubMed

Wang, Zhenguang; Guo, Yelei; Han, Weidong

2017-12-01

Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

PubMed

Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

2003-12-01

Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells

PubMed Central

Hunger, Robert E.; Sieling, Peter A.; Ochoa, Maria Teresa; Sugaya, Makoto; Burdick, Anne E.; Rea, Thomas H.; Brennan, Patrick J.; Belisle, John T.; Blauvelt, Andrew; Porcelli, Steven A.; Modlin, Robert L.

2004-01-01

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. PMID:14991068

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.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

PubMed

Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

2011-05-01

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of regulated secretion on anti-parasitic CD8 T cell responses

PubMed Central

Grover, Harshita Satija; Chu, H. Hamlet; Kelly, Felice D.; Yang, Soo Jung; Reese, Michael L.; Blanchard, Nicolas; Gonzalez, Federico; Chan, Shiao Wei; Boothroyd, John C.; Shastri, Nilabh; Robey, Ellen A.

2014-01-01

Summary CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen ROP5 that elicits a modest CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense granule antigen, GRA6, is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion impacts the efficacy of parasite-specific CD8 T cell responses. PMID:24857659

Cloning and expansion of antigen-specific T cells using iPS cell technology: development of "off-the-shelf" T cells for the use in allogeneic transfusion settings.

PubMed

Kawamoto, Hiroshi; Masuda, Kyoko; Nagano, Seiji; Maeda, Takuya

2018-03-01

Recent advances in adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) have led to moderate therapeutic anti-cancer effects in clinical trials. However, a critical issue, namely that CTLs collected from patients are easily exhausted during expansion culture, has yet to be solved. To address this issue, we have been developing a strategy which utilizes induced pluripotent stem cell (iPSC) technology. This strategy is based on the idea that when iPSCs are produced from antigen-specific CTLs, CTLs regenerated from such iPSCs should show the same antigen specificity as the original CTLs. Pursuing this idea, we previously succeeded in regenerating melanoma antigen MART1-specific CTLs, and more recently in producing potent CTLs expressing CD8αβ heterodimer. We are now developing a novel method by which non-T derived iPSCs are transduced with exogenous T cell receptor genes. If this method is applied to Human Leukocyte Antigen (HLA) haplotype-homozygous iPSC stock, it will be possible to prepare "off-the-shelf" T cells. As a first-in-human trial, we are planning to apply our strategy to relapsed acute myeloid leukemia patients by targeting the WT1 antigen.

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

PubMed Central

McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

2010-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4+ T cell responses in cattle. Monocytes and CD4+ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4+ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4+ T cell responses in vitro for an important pathogen of livestock. PMID:20167197

Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects.

PubMed

Di, Shengmeng; Li, Zonghai

2016-04-01

Chimeric antigen receptors (CARs) are artificial recombinant receptors that generally combine the antigen-recognition domain of a monoclonal antibody with T cell activation domains. Recent years have seen great success in clinical trials employing CD19-specific CAR-T cell therapy for B cell leukemia. Nevertheless, solid tumors remain a major challenge for CAR-T cell therapy. This review summarizes the preclinical and clinical studies on the treatment of solid tumors with CAR-T cells. The major hurdles for the success of CAR-T and the novel strategies to address these hurdles have also been described and discussed.

Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes

NASA Astrophysics Data System (ADS)

Marañón, Concepción; Desoutter, Jean-François; Hoeffel, Guillaume; Cohen, William; Hanau, Daniel; Hosmalin, Anne

2004-04-01

A better understanding of the antigen presentation pathways that lead to CD8+ T cell recognition of HIV epitopes in vivo is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4+ T lymphocytes by dendritic cells to CD8+ T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4+ T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4+ T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFN immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.

Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells.

PubMed

Jensen, Michael C; Riddell, Stanley R

2014-01-01

A major advance in adoptive T-cell therapy (ACT) is the ability to efficiently endow patient's T cells with reactivity for tumor antigens through the stable or regulated introduction of genes that encode high affinity tumor-targeting T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). Case reports and small series of patients treated with TCR- or CAR-modified T cells have shown durable responses in a subset of patients, particularly with B-cell malignancies treated with T cells modified to express a CAR that targets the CD19 molecule. However, many patients do not respond to therapy and serious on and off-target toxicities have been observed with TCR- and CAR-modified T cells. Thus, challenges remain to make ACT with gene-modified T cells a reproducibly effective and safe therapy and to expand the breadth of patients that can be treated to include those with common epithelial malignancies. This review discusses research topics in our laboratories that focus on the design and implementation of ACT with CAR-modified T cells. These include cell intrinsic properties of distinct T-cell subsets that may facilitate preparing therapeutic T-cell products of defined composition for reproducible efficacy and safety, the design of tumor targeting receptors that optimize signaling of T-cell effector functions and facilitate tracking of migration of CAR-modified T cells in vivo, and novel CAR designs that have alternative ligand binding domains or confer regulated function and/or survival of transduced T cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

PubMed

Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

2016-05-01

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors.

PubMed

Raiber, Eun-Ang; Tulone, Calogero; Zhang, Yanjing; Martinez-Pomares, Luisa; Steed, Emily; Sponaas, Anna M; Langhorne, Jean; Noursadeghi, Mahdad; Chain, Benjamin M; Tabor, Alethea B

2010-05-21

Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope-specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells. This does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Preliminary results suggest that access to nonlymphoid tissues is very limited in the steady state but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.

Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation

PubMed Central

den Brok, Martijn H.; Büll, Christian; Wassink, Melissa; de Graaf, Annemarie M.; Wagenaars, Jori A.; Minderman, Marthe; Thakur, Mayank; Amigorena, Sebastian; Rijke, Eric O.; Schrier, Carla C.; Adema, Gosse J.

2016-01-01

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity. PMID:27819292

Caspase-3 Is Transiently Activated without Cell Death during Early Antigen Driven Expansion of CD8+ T Cells In Vivo

PubMed Central

McComb, Scott; Mulligan, Rebecca; Sad, Subash

2010-01-01

Background CD8+ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8+ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8+ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8+ T cell responses has yet to be examined. Methods and Findings We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8+ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8+ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3hi and caspase-3low CD8+ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62Llow) CD8+ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8+ cells remained active caspase-3low throughout the contraction phase. Conclusions Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8+ T cells. Furthermore, the contraction of CD8+ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3. PMID:21203525

Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression

PubMed Central

DuPage, Michel; Cheung, Ann; Mazumdar, Claire; Winslow, Monte M.; Bronson, Roderick; Schmidt, Leah M.; Crowley, Denise; Chen, Jianzhu; Jacks, Tyler

2010-01-01

SUMMARY Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors. PMID:21251614

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C.

PubMed

Pan, C-H; Yang, P-M; Hwang, L-H; Kao, Shing-F; Chen, P-J; Chiang, B-L; Chen, D-S

2002-07-01

The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH2-related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH0 or TH2-related cytokines, which might play a critical role in the chronicity of HCV infection.

Cytokine Release Syndrome After Chimeric Antigen Receptor T Cell Therapy for Acute Lymphoblastic Leukemia.

PubMed

Fitzgerald, Julie C; Weiss, Scott L; Maude, Shannon L; Barrett, David M; Lacey, Simon F; Melenhorst, J Joseph; Shaw, Pamela; Berg, Robert A; June, Carl H; Porter, David L; Frey, Noelle V; Grupp, Stephan A; Teachey, David T

2017-02-01

Initial success with chimeric antigen receptor-modified T cell therapy for relapsed/refractory acute lymphoblastic leukemia is leading to expanded use through multicenter trials. Cytokine release syndrome, the most severe toxicity, presents a novel critical illness syndrome with limited data regarding diagnosis, prognosis, and therapy. We sought to characterize the timing, severity, and intensive care management of cytokine release syndrome after chimeric antigen receptor-modified T cell therapy. Retrospective cohort study. Academic children's hospital. Thirty-nine subjects with relapsed/refractory acute lymphoblastic leukemia treated with chimeric antigen receptor-modified T cell therapy on a phase I/IIa clinical trial (ClinicalTrials.gov number NCT01626495). All subjects received chimeric antigen receptor-modified T cell therapy. Thirteen subjects with cardiovascular dysfunction were treated with the interleukin-6 receptor antibody tocilizumab. Eighteen subjects (46%) developed grade 3-4 cytokine release syndrome, with prolonged fever (median, 6.5 d), hyperferritinemia (median peak ferritin, 60,214 ng/mL), and organ dysfunction. Fourteen (36%) developed cardiovascular dysfunction treated with vasoactive infusions a median of 5 days after T cell therapy. Six (15%) developed acute respiratory failure treated with invasive mechanical ventilation a median of 6 days after T cell therapy; five met criteria for acute respiratory distress syndrome. Encephalopathy, hepatic, and renal dysfunction manifested later than cardiovascular and respiratory dysfunction. Subjects had a median of 15 organ dysfunction days (interquartile range, 8-20). Treatment with tocilizumab in 13 subjects resulted in rapid defervescence (median, 4 hr) and clinical improvement. Grade 3-4 cytokine release syndrome occurred in 46% of patients following T cell therapy for relapsed/refractory acute lymphoblastic leukemia. Clinicians should be aware of expanding use of this breakthrough therapy and

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

PubMed Central

Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

PubMed

Blachère, Nathalie E; Orange, Dana E; Santomasso, Bianca D; Doerner, Jessica; Foo, Patricia K; Herre, Margaret; Fak, John; Monette, Sébastien; Gantman, Emily C; Frank, Mayu O; Darnell, Robert B

2014-11-01

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

PubMed Central

Eris, J M; Basten, A; Brink, R; Doherty, K; Kehry, M R; Hodgkin, P D

1994-01-01

B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses. Images PMID:7514304

Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen

DOE PAGES

Visperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; ...

2016-12-13

ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. Finally, the screen is based on a fluorescence polarizationmore » assay for peptide binding to ZAP-70.« less

Antigen challenge leads to in vivo activation and elimination of highly polarized TH1 memory T cells

PubMed Central

Hayashi, Nobuki; Liu, Dacai; Min, Booki; Ben-Sasson, Shlomo Z.; Paul, William E.

2002-01-01

TH1 memory T cells derived from T cell receptor transgenic mice, in which the T cell antigen receptor is specific for a cytochrome C peptide in association with I-Ek, were transferred into normal B10.A mice and allowed to adopt a resting phenotype. When challenged, 30–60 days after transfer, with i.v. cytochrome C, the transgenic cells rapidly became activated, expressed mRNA for IFNγ, and began to divide. However, after 48 h, the frequency of the cells fell progressively, reaching levels only slightly above the limit of detection by day 8 and thereafter remain depressed for up to 90 days. The remaining cells were anergic as shown by limitation in proliferation and IFNγ production in response to in vitro antigen stimulation. Even if challenged with antigen emulsified in complete Freund's adjuvant, the overall pattern was similar, except that in the draining lymph nodes, the surviving antigen-specific cells were not anergic, although spleen cells were still strikingly anergic. Thus, antigenic challenge of mice possessing resting memory TH1 CD4 T cells leads to the unanticipated loss of most of the specific cells and an apparent depletion rather than enhancement of immunologic memory. PMID:11959916

Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

PubMed

O'Neill, F J; Hu, Y; Chen, T; Carney, H

1997-02-27

In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

PubMed Central

Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

2017-01-01

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

PubMed Central

John, Beena; Harris, Tajie H.; Tait, Elia D.; Wilson, Emma H.; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S.; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A.

2009-01-01

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis. PMID:19578440

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Chimeric antigen receptor T cell therapy in pancreatic cancer: from research to practice.

PubMed

Jindal, Vishal; Arora, Ena; Masab, Muhammad; Gupta, Sorab

2018-05-04

Chimeric antigen receptor (CAR) T cell therapy is genetically engineered tumor antigen-specific anticancer immunotherapy, which after showing great success in hematological malignancies is currently being tried in advanced solid tumors like pancreatic cancer. Immunosuppressive tumor microenvironment and dense fibrous stroma are some of the limitation in the success of this novel therapy. However, genetic modifications and combination therapy is the topic of the research to improve its efficacy. In this article, we summarize the current state of knowledge, limitations, and future prospects for CAR T cell therapy in pancreatic cancer.

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

PubMed Central

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

2008-01-01

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

Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

PubMed Central

Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

2014-01-01

Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112