Cognate CD4 Research Articles

DN Treg Cells Specifically Suppress Cognate Alloreactive Memory T Cell By Granzyme B, and Prolong Allograft Survival.

[Background] Since the emphasis of clinical organ transplant survival moved from short term to long term, memory T (Tm) cell, by mediating several key-mechanisms such as transplant vasculopathy and allograft nephropathy, has been recognized as an essential culprit of long term organ rejection. It is inherently resistant to conventional therapies such as apoptosis-induction and co-stimulation-blockade, yet indispensable to the recipient's protective immunity against environmental pathogens. In this study, we show that double-negative (DN) Treg cell might be a delicate solution to this dilemma. [Methods and Results] DN Tregs specifically suppress cognate B6-anti-Balb/c CD8+ Tm both in vitro (suppression rate: 53.9±1.2%), and in vivo, with the CD8+ Tm cell-mediated graft rejection delayed from (21.5±1.7)-day to (63.0±4.7)-day in a BALB/c to B6Rag1-/- skin transplantation, but with no effect on anti-C3H Tm. Both CD8+Tem and Tcm compartments are significantly suppressed by DN Treg. We further identified the suppression as Granzyme B (GzmB)- and perforin (PFN)-dependent. Intriguingly, CD4+ Tm cells resist DN Treg both in vitro and in vivo. For the first time, we found CD4+ Tm highly express Serine Protease Inhibitor-6 (Spi6) ((5.03±1.15)-fold of CD4+ Teff, protein level), an inhibitor of GzmB. The deficiency of Spi6 sensitized CD4+ Tm both in vitro (suppression rate: 10.5±5.8% to 37.5±6.2%) and in vivo (skin-allograft survival from (28.3±5.7)-day to (75.5±7.9)-day) to killing by DN Tregs. Whereas the restored sensitivity can be again abrogated in vitro (suppression rate: 4.5±3.2%) and in vivo (skin-allograft survival (22.5±2.4)-day) by PFN-deficiency in DN Tregs. [Conclusion] In summary, DN Tregs specifically suppress cognate allo-reactive CD8+ Tm cells by GzmB-PFN pathway, and effectively delay CD8+ Tm cell-mediated graft rejection without compromising Tm cell-dependent protective immunity. The fact that CD4+ Tm resists DN Treg by highly expressing Spi6 and can be sensitized by Spi6-deficiency underlines the essential role of Spi6 in CD4+ Tm cell fate, which also shed light to the possibility of combining DN Treg and Spi6-targeting therapy to even further improve long-term allograft survival.

Steady-state antigen scavenging, cross-presentation, and CD8+ T cell priming: a new role for lymphatic endothelial cells.

Until recently, the known roles of lymphatic endothelial cells (LECs) in immune modulation were limited to directing immune cell trafficking and passively transporting peripheral Ags to lymph nodes. Recent studies demonstrated that LECs can directly suppress dendritic cell maturation and present peripheral tissue and tumor Ags for autoreactive T cell deletion. We asked whether LECs play a constitutive role in T cell deletion under homeostatic conditions. In this study, we demonstrate that murine LECs under noninflamed conditions actively scavenge and cross-present foreign exogenous Ags to cognate CD8(+) T cells. This cross-presentation was sensitive to inhibitors of lysosomal acidification and endoplasmic reticulum-golgi transport and was TAP1 dependent. Furthermore, LECs upregulated MHC class I and the PD-1 ligand PD-L1, but not the costimulatory molecules CD40, CD80, or CD86, upon Ag-specific interactions with CD8(+) T cells. Finally, Ag-specific CD8(+) T cells that were activated by LECs underwent proliferation, with early-generation apoptosis and dysfunctionally activated phenotypes that could not be reversed by exogenous IL-2. These findings help to establish LECs as APCs that are capable of scavenging and cross-presenting exogenous Ags, in turn causing dysfunctional activation of CD8(+) T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph node-resident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags.

INKT-cell help to B cells: a cooperative job between innate and adaptive immune responses.

T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4(+) T follicular helper (T(FH)) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody (Ab)-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens (Ags) that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells, and T cells. A decade ago, we discovered that iNKT cells can help B cells to proliferate and to produce IgG Abs in vitro and in vivo. This adjuvant-like function of Ag-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T-cell-B-cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations.

Differential requirement for IRF4 in CD8+ T cell responses to acute versus chronic LCMV infection (IRM4P.498)

Abstract The magnitude of the CD8+ T cell response to acute infection is regulated by the strength of TCR signaling. A key component to this is the level of expression of the transcription factor IRF4, which controls CD8+ T cell expansion in a dose-dependent manner. During an acute response to LCMV-Armstrong, a modest reduction in IRF4 levels, such as that achieved by a haplo-deficiency at the irf4 locus, leads to a 2-4 fold reduction in the number of LCMV specific CD8+ T cells without impacting viral clearance (RN manuscript submitted). These data indicate that acute LCMV infections are associated with an expansion of cognate CD8+ T cells that is in excess of that needed to clear the virus. In contrast, during LCMV-clone 13 infection, the modestly-reduced CD8+ T cell response of irf4+/- T cells fails to control the infection, resulting in long-term viral persistence. In this system, loss of one allele of irf4 results in reduced CD8+ T cell function, increased exhaustion due to loss of PD1 low Tbet high precursors, and less weight loss than in LCMV-clone 13-infected WT mice. These data indicate differential requirements for high levels of IRF4 in generating functional CD8+ T cell responses capable of clearing acute versus chronic virus infections.

Perforin Positive Regulatory DCs In Immune Tolerance: Novel Anti-Inflammatory Players In Metabolic Syndrome

Immature dendritic cells (imDCs) can have a tolerizing effect in the steady state or following transplantation. However, due to the significant heterogeneity of this cell population it is difficult to study the mechanisms of their tolerance induction. We previously described the generation of a highly defined population of imDCs expressing perforin and granzyme A (Perf-DCs) from hematopoietic progenitors; using TCR transgenic T cells we monitored their ability to delete cognate CD4 and CD8 T cells. While the former are deleted via an MHC-independent mechanism through the nitric oxide system, CD8+ T cell deletion occurs through a unique MHC-dependent perforin-based killing mechanism. This involves activation of Toll-like receptors 7, and signaling through Triggering Receptor Expressed on Myeloid cells -1. Importantly, this novel subpopulation of Perf-DCs was also detected in various lymphoid tissues in normal animals, and its frequency is markedly enhanced upon GM-CSF administration (Zangi et al, Blood 2012).Here, we investigated the potential regulatory role of Perf-DCs in steady state in-vivo by selectively knocking out the expression of perforin in these cells. To this end, we generated BM chimeras using a 1:1 mixture of BM from perforin KO mice and from BM of mice ablated of CD11chigh DCs using diphtheria toxin expression under the CD11c promoter (Birnberg et al, Immunity 2008). In the resulting PKO-DTA chimeras, perforin expression was completely lost in conventional CD11c+ DCs, while 50% of the T and NK cell populations still expressed perforin. At 6 months post transplant, DTA-PKO chimeric mice spontaneously gained more weight than chimeras created using a mixture of normal BM with BM from perforin KO mice (WT-PKO). The increased weight gain observed in DTA-PKO mice prompted us to test whether this phenomenon was accompanied by other metabolic alterations. Indeed, DTA-PKO mice exhibited elevated serum cholesterol and triglyceride levels compared to control WT-PKO chimeras (140±3.5 vs. 115±8.6, 125±31vs. 88±9.8 mg/dl, N≥5). Total body fat percent as measured by body composition MRI was significantly higher in DTA-PKO mice (30.3%±2.2 vs. 14.5%±2.3), along with highly elevated levels of leptin (37±10.5 vs. 9.8±3 ng/ml).In addition, DTA-PKO chimeric mice exhibited glucose intolerance (p=0.034) and reduced insulin sensitivity (p=6.07x10-6). Immunohistological evaluation revealed a significant reduction in the percentage of insulin expressing pancreatic β cell- tissue (2.2%±0.54 vs. 5.75%±1.98). Importantly, the visceral adipose tissue (VAT) of DTA-PKO chimeras contained more crown-like structures typically formed when macrophages within inflamed AT surround dead adipocytes.Based on these characteristics of metabolic syndrome that develop in DTA-PKO chimeras over 6 months, we tested whether high-fat diet (HFD) enhances the rate of disease development. Indeed, DTA-PKO chimeras maintained on HFD displayed more pronounced weight gain compared to their HFD-maintained WT counterparts when tested 6 weeks after HFD initiation. Likewise, cholesterol and triglycerides as well as leptin and IL-1b in the serum, and TNF-α and IL-6 in the AT were elevated in DTA-PKO mice compared to the WT-PKO animals. Importantly, analysis of immune cell populations in collagenase-digested VAT revealed more CD8+ and CD4+ T cells in DTA-PKO mice compared to control chimeras (78.3x103±17.5x103 vs. 24.9x103±3.2x103 and 113x103±21x103 vs. 43x103±4.4x103respectively). Thus, triggering of inflammation in the AT previously shown to be mediated by T cells (Winer et al, Nat.Med 2009; Nishimura et al, Nat.Med 2009), is not effectively regulated in mice lacking Perf-DCs. Interestingly, a similar enhanced rate of metabolic imbalance was found in regular diet-fed DTA-PKO chimeras using RIP-mOVA mice expressing ovalbumin in the thymus, pancreas and kidneys, and known to be more prone to autoimmunity. Moreover, a significantly larger fraction of dividing cells was observed when CD8 T cells, isolated from AT of DTA-PKO chimeric RIP-mOVA mice were stimulated against splenocytes of mice expressing ovalbumin in all tissues (wOVA mice). Taken together, our results demonstrate that Perf-DCs have a unique immune regulatory role under steady state, controlling unwanted inflammatory processes in adipose tissue. Further studies of the role of Perf-DCs in other models of autoimmunity are warranted.▪ Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Systemic minor histocompatibility antigen expression in blood endothelial cells prevents T cell‐mediated vascular immunopathology

Attenuation of T cell-mediated damage of blood endothelial cells (BECs) in transplanted organs is important to prevent transplant vasculopathy (TV) and chronic rejection. Here, we assessed the importance of minor histocompatibility antigen (mHA) distribution and different coinhibitory molecules for T cell-BEC interaction. A transgenic mHA was directed specifically to BECs using the Tie2 promoter and cellular interactions were assessed in graft-versus-host disease-like and heterotopic heart transplantation settings. We found that cognate CD4(+) T-cell help was critical for the activation of BEC-specific CD8(+) T cells. However, systemic mHA expression on BECs efficiently attenuated adoptively transferred, BEC-specific CD4(+) and CD8(+) T cells and hence prevented tissue damage, whereas restriction of mHA expression to heart BECs precipitated the development of TV. Importantly, the lack of the coinhibitory molecules programmed death-1 (PD-1) and B and T lymphocyte attenuator fostered the initial activation of BEC-specific CD4(+) T cells, but did not affect development of TV. In contrast, TV was significantly augmented in the absence of PD-1 on BEC-specific CD8(+) T cells. Taken together, these results indicate that antigen distribution in the vascular bed determines the impact of coinhibition and, as a consequence, critically impinges on T cell-mediated vascular immunopathology.

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

Abstract 1259: Murine HLA-restricted CD4+ T cell lines as source of high affinity TCRs specific for the human breast cancer-associated tumor antigen NY-BR-1.

Abstract Out of all malignancies, breast cancer is the second most common cancer worldwide and the leading cause of cancer death in females. Passive immunization approaches such as adoptive transfer of TCR-transduced autologous T cells specific for tumor associated antigens, provide an innovative strategy for cancer immunotherapy. The differentiation antigen NY-BR-1 has been described to be expressed in 60% of all invasive mammary carcinomas. Since NY-BR-1 protein levels are highly elevated in malignant breast tissues compared to healthy breast tissue, NY-BR-1 might represent a suitable target antigen for T cell based immunotherapy approaches against breast cancer. Successful immunological eradication of tumors depends on the presence of activated tumor antigen-specific CD4+ effector T cells as documented by numerous reports. Therefore, NY-BR-1 was screened for the presence of CD4+ T cell epitopes using HLA-transgenic mice immunized with a NY-BR-1-encoding expression plasmid and subsequent ex vivo analysis of the NY-BR-1-specific T cell responses against a synthetic peptide library covering the entire NY-BR-1 protein. Stable CD4+ T cell lines specific for six newly identified epitopes could be established from peptide-immunized DRB1*0301 and HLA-DRB1*0401-transgenic mice and HLA-DR-restriction of the cell lines was confirmed on peptide loaded T2/DR3 and T2/DR4 target cells. Next, natural processing of the epitopes identified in HLA transgenic mice will be tested in the human system using human HLA-matched cells lines infected with NY-BR-1 encoding adenovirus as targets for recognition by the murine NY-BR-1-specific CD4+ T cell lines. Clinical relevance of the newly identified NY-BR-1-specific epitopes will be investigated by screening of blood samples from NY-BR-1+, HLA-matched breast cancer patients for the presence of cognate CD4+ T cells. High affinity TCRs from NY-BR-1 specific CD4+ T cells will be sequenced and subsequently used to generate autologous TCR-transduced T cell lines ready for adoptive transfer of breast cancer patients Citation Format: Adriane Gardyan, Wolfram Osen, Stefan B. Eichmüller, Inka Zoernig, Dirk Jaeger. Murine HLA-restricted CD4+ T cell lines as source of high affinity TCRs specific for the human breast cancer-associated tumor antigen NY-BR-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1259. doi:10.1158/1538-7445.AM2013-1259

Antigen Presentation by Dendritic Cells in Rheumatoid Arthritis

Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disease that affects largely synovial joints. It has been postulated that activated autoreactive CD4 T cells play a key role in triggering and/or maintaining the chronic inflammatory process in RA. Dendritic cells (DCs) are antigen-presenting cells that activate cognate clonal CD4 T cells in the lymph nodes. The activation process involves the formation of a molecular structure at the DC-CD4 T cell contact zone called immunological synapse (IS). In RA, the synovium, a thin layer of tissue below the capsule in the joints, shows a massive infiltration of DCs and CD4 T cells. Subjects bearing HLA-DRB1 alleles of the Major Histocompatibility Complex II gene displaying a motif called RA "shared epitope (SE)", have an enhanced susceptibility to suffer RA. Interestingly, the SE-containing HLA-DRB1 molecules display a pocket with a high affinity for citrullinated antigens, which are found at higher levels in subjects prone to develop RA. Thus, it is possible that the DCs of susceptible individuals may form IS with particular features that may present citrullinated peptides to autorreactive naïve CD4 T clones that, after being activated, contribute to the initiation or development of the disease. Herein I put forward a model of RA initiation based on current information on the immune response and RA.

Experimental human pneumococcal carriage augments IL-17A-dependent T-cell defence of the lung.

Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4+ T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4+ T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4+ T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A+ CD4+ T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF+/IL-17A+ co-producing CD4+ memory T-cells (p<0.01); IFNγ+ CD4+ memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4+ memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = −0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4+ memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

Deletion of cognate CD8 T cells by immature dendritic cells: a novel role for perforin, granzyme A, TREM-1, and TLR7

AbstractImmature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4+ and CD8+ T cells after encountering cognate or noncognate imDCs. Whereas CD4+ T cells were deleted via an MHC-independent mechanism through the NO system, CD8+ T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforin-expressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.

Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with naïve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.

Release of dendritic cells from cognate CD4+T-cell recognition results in impaired peripheral tolerance and fatal cytotoxic T-cell mediated autoimmunity

Resting dendritic cells (DCs) induce tolerance of peripheral T cells that have escaped thymic negative selection and thus contribute significantly to protection against autoimmunity. We recently showed that CD4(+)Foxp3(+) regulatory T cells (Tregs) are important for maintaining the steady-state phenotype of DCs and their tolerizing capacity in vivo. We now provide evidence that DC activation in the absence of Tregs is a direct consequence of missing DC-Treg interactions rather than being secondary to generalized autoimmunity in Treg-less mice. We show that DCs that lack MHC class II and thus cannot make cognate interactions with CD4(+) T cells are completely unable to induce peripheral CD8(+) T-cell tolerance. Consequently, mice in which interactions between DC and CD4(+) T cells are not possible develop spontaneous and fatal cytotoxic T lymphocyte-mediated autoimmunity.

DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of cognate CD8+ T cells independently of the PD-1 pathway (123.19)

Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Cytotoxic CD8+ T cells specific for beta cell antigens are a main contributor to T1D development. CD8+ T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes have been implicated in T1D development in both patients and NOD mice. Targeted delivery to steady-state dendritic cells (DCs) of a beta cell peptide mimotope linked to anti-DEC-205 leads to deletional tolerance of transferred cognate CD8+ T cells in NOD mice. Treatment with anti-DEC-205 linked to NRP-V7, a superagonist mimotope of IGRP206-214, selectively deleted endogenous IGRP206-214-specific T cells, a highly prevalent specificity in NOD mice, and showed promise in T1D prevention. Simultaneous treatments with anti-DEC-205/InsI9, a mimotope peptide of the CD8+ T cell insulin epitope InsB15-23, and anti DEC-205/NRP-V7 are being held to study the effect of targeting multiple CD8+ T cell specificities on T1D prevention. Investigation of the operative tolerance mechanisms revealed that antibody blockade of the PD-1 pathway, often implicated in DC- mediated induction of peripheral tolerance, could not inhibit the deletion of transferred T cells by DEC-205-mediated targeting of their antigen to steady- state DC. Involvement of the CTLA-4 or Fas pathways will be probed to further study the molecular mechanism of tolerance induction. .

Examining the role of T cell co-receptors CD4 and CD8 in T cell activation by using high-affinity T cell receptors (121.28)

Abstract The recognition of foreign antigens by T cells is accomplished through the binding of the T cell receptor (TCR) in collaboration with co-receptor (CD4 or CD8) recognition of MHC. It is clear that CD4 and CD8 function by binding to their cognate MHCs and by delivering the kinase Lck to the TCR complex for initiation of signaling, however, the mechanistic details of this process remain unclear. For example, it remains unknown if co-receptors must engage the same MHC as the TCR, or if engagement of a nearby non-cognate MHC molecule is sufficient to initiate signaling. To examine the details of co-receptor function, we used a class II MHC specific TCR system consisting of a wild-type (KD 20 μM) and high-affinity variant (KD 25 nM). A panel of T cell hybridomas was generated expressing either the wild-type or high-affinity TCR paired with either the CD4 or CD8 co-receptor. The low-affinity, but not the high-affinity, TCR required cognate CD4 for activity. Unexpectedly, non-cognate CD8 was able to enhance the activity of the low-affinity TCR, and CD8:class I MHC interactions were required for activity mediated by the high-affinity TCR. Unexpectedly, CD8 also operated by preventing apoptosis of the antigen presenting cells (APCs). These data show that CD8 can act by binding to non-cognate MHC, and that its functions extend to the prevention of apoptosis of APCs. The findings thus provide a framework for a new model of co-receptor function.

Single chain MHC I trimer-based DNA vaccines for protection against Listeria monocytogenes infection

To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8(+) T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8(+) T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2K(d) and the immunodominant peptide LLO 91-99 generated functional primary and memory peptide-specific CD8(+) T cells that confer partial protection against L. monocytogenes infection. DNA immunization of K(d)/LLO(91-99) SCTs generated functional memory CD8(+) T cells independently of CD4(+) T cells, although the expression of cognate or non-cognate CD4(+) helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases.

Functional profile of cytomegalovirus (CMV)‐specific CD8+ T cells and kinetics of NKG2C+ NK Cells associated with the resolution of CMV DNAemia in allogeneic stem cell transplant recipients

Immune mechanisms involved in control of cytomegalovirus (CMV) infection in the allogeneic stem cell transplantation setting have not been fully disclosed. CMV pp65 and IE-1-specific CD8(+) T cells expressing IFN-γ, TNF-α, and CD107a, alone or in combination, and NKG2C(+) NK cells were prospectively enumerated during 13 episodes of CMV DNAemia. The expansion of monofunctional and polyfunctional CD8(+) T cells was associated with CMV DNAemia clearance. The size and functional diversity of the expanding CD8(+) T-cell population was greater in self-resolved episodes than in episodes treated with antivirals. These differences were related to the magnitude of expansion of cognate antigen IFN-γ CD4(+) T cells. The resolution of CMV DNAemia was associated frequently with a marked expansion of both CD56(dim) /CD16(+) NK cells and NKG2C(+) CD56(bright) /CD16(-) NK cells. The data lend support to the role of polyfunctional CD8(+) T cells in controlling CMV replication in the allogeneic stem cell transplantation setting, and suggest that NKG2C(+) NK cells may be involved critically in the resolution of CMV DNAemia episodes.

S59 Chemo-immunotherapy of mesothelioma: depletion of established, suppressive CD4 T cells is critical to achieving cures in a murine model

<h3>Introduction</h3> Mesothelioma is incurable and new treatment strategies are urgently needed. It has long been recognised that malignant cells can be attacked by the immune system but immunotherapy is only starting to emerge as an additional modality of treatment for cancer. The crucial role that helper CD4 T cells play in orchestrating CD8 T cell and other anti-tumour responses has only recently been appreciated. However the CD4 population contains effector and suppressive subsets with diverse and opposing functions. Although the effects of pemetrexed on tumour immunity are still being defined, gemcitabine is known to synergise with immunotherapy by causing tumour cell death, releasing tumour antigen and depleting suppressive immune cells. We investigated the role that different types of CD4 cell play in CD8 anti-tumour immune responses and how the CD4 response could be best manipulated to optimise outcomes to chemo-immunotherapy. <h3>Methods</h3> Thy1.2+ve balb/c mice bearing mesothelioma tumours expressing the neoantigen haemagglutinin (HA), were treated with gemcitabine and adoptive transfer of tumour antigen (HA) specific thy1.1+ve CD8 T cells and thy1.1+ve CD4 cells which had been differentiated in vitro into Th1s, Th2s, Th17s or regulatory T cells (Tregs). The activity of the adoptively transferred cells in vivo was tracked by identifying recovered thy1.1+ve cells by flow cytometry. <h3>Results</h3> In the prophylactic treatment setting, in vitro differentiated tumour antigen specific Th1s led to rejection of tumours inoculated 1 day later in around 40% of mice. However there was minimal activity against established, gemcitabine-treated tumours. Recovery of adoptively transferred CD8 T cells revealed that in established tumours, CD4s were unable to help cognate CD8 cells expand and infiltrate tumours. However if endogenous CD4s were depleted prior to treatment then CD8 tumour infiltration was significantly increased and cures were seen in around two-thirds of mice. <h3>Conclusions</h3> These results suggest that once a tumour is established, an over-riding endogenous suppressive CD4 response prevents effector CD4s from helping CD8 T cells to eradicate tumours. Removal of suppressive CD4 cells was critical to achieving cures with chemo-immunotherapy.

Structural and functional characterization of a single-chain peptide-MHC molecule that modulates both naive and activated CD8+ T cells.

Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8(+) T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8(+) T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK(d).IGRP) by using the class I MHC molecule H-2K(d) and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK(d).IGRP tetramers bound specifically to cognate CD8(+) T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-γ response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8(+) T cells makes them a potential intervention strategy in early and late stages of disease.