LCMV-specific CD8 Research Articles

A Role for NOD2 in Intestinal Memory T cell Formation and Function

Abstract Aberrant resident memory T cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Genetic mutations in NOD2 are associated with the highest risk of CD development. NOD2 has been shown to be critical for initiating adaptive immune responses at systemic sites. However, the role of NOD2 in establishing memory T cell responses in the intestine remains unclear. Using an acute model of LCMV infection, we interrogated the role of NOD2 in intestinal memory T cell generation and function. Littermate wildtype or NOD2-deficient mice were infected intraperitoneally with LCMV-Armstrong, and the intestinal T cell priming, effector, and memory T cell responses were profiled in the mesenteric lymph nodes (mLN) and small intestinal lamina propria (SILP). Augmented NOD2 signalling at homeostasis and during infection increased endogenous and LCMV-specific CD4+ T cell numbers in the mLN, but not in the spleen. This effect was due to a NOD2-dependent increase in T cell homing to the mLN. Furthermore, NOD2-deficiency resulted in decreased effector and memory T cell numbers in the SILP. This effect was associated with decreased memory T cell functions in NOD2-deficient mice during ex vivo and in vivo recall experiments. Taken together, our experiments suggest a role for NOD2 in mediating optimal T cell priming in the mesenteric lymph nodes, which later affects effector and memory T cell functions in the intestine.

A197 A ROLE FOR NOD2 IN INTESTINAL T CELL MEMORY DEVELOPMENT AND FUNCTION

Abstract Background Aberrant resident memory T cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T cell responses. However, the role of NOD2 in establishing memory T cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Aims It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific intestinal T cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Methods Intraperitoneal LCMV-Armstrong infection results in long-lived CD4+ and CD8+ resident-memory T cell generation in the intestinal lamina propria of mice. Wildtype and NOD2 deficient littermate mice were infected with LCMV-Armstrong, and the effects on intestinal T cell priming, effector function, and memory T cell function were examined by flow-cytometry, ex vivo and in vivo antigen-recall experiments, qPCR, and viral plaque assays. Results Augmented NOD2 signalling during priming increased LCMV-specific CD4+ T cell numbers in the mesenteric lymph nodes - but not in the spleen, independent of proliferation or cell death. Furthermore, NOD2-deficiency resulted in decreased effector and memory T cell numbers in the small intestinal lamina propria. This effect was associated with decresead memory CD4+ and CD8+ T cell functions in NOD2-deficient mice during ex vivo and in vivo peptide recall experiments. Conclusions NOD2 affects T cell priming in the mesenteric lymph nodes, which later affects effector and memory T cell functions in the intestinal lamina propria. Funding Agencies CAG, CIHREmerging and Pandemic Infections Consortium

Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

Platinum-Based Chemotherapy Attenuates the Effector Response of CD8 T Cells to Concomitant PD-1 Blockade.

Combination of chemotherapy with programmed cell death 1 (PD-1) blockade is a front-line treatment for lung cancer. However, it remains unknown whether and how chemotherapy affects the response of exhausted CD8 T cells to PD-1 blockade. We used the well-established mouse model of T-cell exhaustion with chronic lymphocytic choriomeningitis virus (LCMV) infection to assess the effect of chemotherapy (cisplatin+pemetrexed) on T-cell response to PD-1 blockade, in the absence of the impact of chemotherapy on antigen release and presentation observed in tumor models. When concomitantly administered with PD-1 blockade, chemotherapy affected the differentiation path of LCMV-specific CD8 T cells from stem-like to transitory effector cells, thereby reducing their expansion and production of IFNγ. After combination treatment, these restrained effector responses resulted in impaired viral control, compared with PD-1 blockade alone. The sequential combination strategy, where PD-1 blockade followed chemotherapy, proved to be superior to the concomitant combination, preserving the proliferative response of exhausted CD8 T cells to PD-1 blockade. Our findings suggest that the stem-like CD8 T cells themselves are relatively unaffected by chemotherapy partly because they are quiescent and maintained by slow self-renewal at the steady state. However, upon the proliferative burst mediated by PD-1 blockade, the accelerated differentiation and self-renewal of stem-like cells may be curbed by concomitant chemotherapy, ultimately resulting in impaired overall CD8 T-cell effector functions. In a translational context, we provide a proof-of-concept to consider optimizing the timing of chemo-immunotherapy strategies for improved CD8 T-cell functions. See related commentary by Vignali and Luke, p. 1705.

Unraveling T-cell Exhaustion: Genetic Screening Meets In Vitro Modeling.

T-cell exhaustion poses a significant barrier to the efficacy of immunotherapies. In the past decade, immune checkpoint blockade (ICB) has been the leading strategy to prevent or reverse T-cell exhaustion. Although ICB yields promising clinical outcomes in patients with cancer, its impact on T-cell reinvigoration is often short-lived. High-throughput genomic tools, including CRISPR screening along with single-cell RNA and chromatin accessibility sequencing may point toward new therapeutic avenues. However, their utility in identifying key mediators of T-cell exhaustion is constrained by the restricted scalability of well-validated in vivo exhaustion models, like chronic LCMV infection. In a recent article in Science Immunology, Wu and colleagues introduce an in vitro exhaustion model that involves repetitive stimulation of T-cell receptor-transgenic, LCMV-specific P14 CD8 T cells. This approach enables a direct comparison of exhausted T (Tex) cells generated both in vivo and in vitro using the same antigen, adeptly pinpointing exhaustion features that can be recapitulated in vitro. Leveraging this efficient and scalable model alongside CRISPR screening, the authors highlight the transcription factor BHLHE40 as a pivotal element in promoting Tex-cell transition from progenitor to intermediate Tex cells.

PD-1 blockade increases the self-renewal of stem-like CD8 T cells to compensate for their accelerated differentiation into effectors.

PD-1+TCF-1+ stem-like CD8 T cells act as critical resource cells for maintaining T cell immunity in chronic viral infections and cancer. In addition, they provide the proliferative burst of effector CD8 T cells after programmed death protein 1 (PD-1)-directed immunotherapy. However, it is not known whether checkpoint blockade diminishes the number of these stem-like progenitor cells as effector cell differentiation increases. To investigate this, we used the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Treatment of chronically infected mice with either αPD-1 or αPD-L1 antibody not only increased effector cell differentiation from the virus-specific stem-like CD8 T cells but also increased their proliferation so their numbers were maintained. The increased self-renewal of LCMV-specific stem-like CD8 T cells was mTOR dependent. We used microscopy to understand the division of these progenitor cells and found that after PD-1 blockade, an individual dividing cell could give rise to a differentiated TCF-1- daughter cell alongside a self-renewing TCF-1+ sister cell. This asymmetric division helped to preserve the number of stem-like cells. Moreover, we found that the PD-1+TCF-1+ stem-like CD8 T cells retained their transcriptional program and their in vivo functionality in terms of responding to viral infection and to repeat PD-1 blockade. Together, our results demonstrate that PD-1 blockade does not deplete the stem-like population despite increasing effector differentiation. These findings have implications for PD-1-directed immunotherapy in humans.

IL-15 promotes self-renewal of progenitor exhausted CD8 T cells during persistent antigenic stimulation.

In chronic infections and cancer, exhausted CD8 T cells exhibit heterogeneous subpopulations. TCF1+PD-1+ progenitor exhausted CD8 T cells (Tpex) can self-renew and give rise to Tim-3+PD-1+ terminally differentiated CD8 T cells that retain their effector functions. Tpex cells are thus essential to maintaining a pool of antigen-specific CD8 T cells during persistent antigenic stimulation, and only they respond to PD-1-targeted therapy. Despite their potential as a crucial therapeutic target for immune interventions, the mechanisms controlling the maintenance of virus-specific Tpex cells remain to be determined. We observed approximately 10-fold fewer Tpex cells in the spleens of mice chronically infected with lymphocytic choriomeningitis virus (LCMV) one-year post-infection (p.i.) than at three months p.i. Similar to memory CD8 T cells, Tpex cells have been found to undergo self-renewal in the lymphoid organs, prominently the bone marrow, during chronic LCMV infection. Furthermore, ex vivo treatment with IL-15 preferentially induced the proliferation of Tpex cells rather than the terminally differentiated subsets. Interestingly, single-cell RNA sequencing analysis of LCMV-specific exhausted CD8 T cells after ex vivo IL-15 treatment compared with those before treatment revealed increased expression of ribosome-related genes and decreased expression of genes associated with the TCR signaling pathway and apoptosis in both Tpex and Ttex subsets. The exogenous administration of IL-15 to chronically LCMV-infected mice also significantly increased self-renewal of Tpex cells in the spleen and bone marrow. In addition, we assessed the responsiveness of CD8 tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma patients to IL-15. Similar to the data we obtained from chronic viral infection in mice, the expansion of the Tpex subset of PD-1+ CD8 TILs upon ex vivo IL-15 treatment was significantly higher than that of the terminally differentiated subset. These results show that IL-15 could promote self-renewal of Tpex cells, which has important therapeutic implications.

Tim-3 expression in Treg controls viral persistence and effector T cell response during chronic LCMV infection.

Abstract Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3 +Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10, compared to Tim-3 −Treg. Similarly, it was recently reported that there is an increase in Tim-3 +Treg in people with HIV regardless of viral suppression. However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in liver, and kidney at 30 days post-infection. Interestingly, we found that there was a decrease in the frequency of PD-1 +Tim-3 +and PD-1 +Tox +LCMV-specific CD8 T cells. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Our findings demonstrate that modulation of a single surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. These studies may also help to identify Tim-3 directed therapies for the management of persistent infections and other chronic illnesses. Supported by grants from NIH (T32GM008208, R01 AI138504)

Virus-infected mast cells activate virus-specific CD8+ T cells.

Efficient anti-viral responses of CD8+ T cells require signals that promote their effector cell differentiation, that are mainly provided by dendritic cells (DCs). Mast cells (MCs) are key drivers of DC maturation, but also influence their migration and antigen presenting properties and therefore indirectly mediate CD8+ T cell activation. MCs initiate innate immune responses at pathogen entry sites, promote the development of adaptive immune responses after infection, and release mediators including chemokines that recruit and activate immune cells including T cells during viral infections. However, whether MCs can directly activate virus-specific CD8+ T cells remains largely unknown. Here, we used an in vitro viral infection model with lymphocytic choriomeningitis virus (LCMV)-infected MCs or DCs co-cultured with either LCMV-specific CD8+ T cells or with WT (unspecific) CD8+ T cells. Similar to LCMV-infected DCs, LCMV-infected MCs clustered with virus-specific CD8+ T cells and induced their activation and production of antiviral cytokines. In addition, the co-stimulatory molecules CD86 and OX40L, but not CD80, were upregulated on MCs and an increased production of IL-6 and type I interferons after LCMV infection was shown. Our findings suggest that MCs can promote CD8+ T cell activation during viral infections. MC-mediated CD8+ T cell activation might be especially important within infected tissues where direct cellular interaction can take place. A better understanding of anti-viral functions of MCs may help developing new strategies to better treat viral infections.

Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy

Abstract CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (T EX). T EXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial T EXreinvigoration. Some T EXregain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of T EXreinvigoration are not well understood. Moreover, a key unknown question is why only some T EXgain reinvigoration. Here, we used proliferation as a surrogate for T EXreinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated T EXbefore first cell division and ask what molecular signals induce T EXreinvigoration. Thus, we found that T EXreinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated T EXupon anti-PD-L1 compared to untreated T EXat 48 hours post treatment. Therefore, we identified the molecular signatures of T EXthat have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy.

A173 ELUCIDATING THE EFFECTS OF NOD2-MEDIATED SIGNALLING ON INTESTINAL RESIDENT-MEMORY T-CELL FORMATION AND FUNCTION

Abstract Background Aberrant resident memory T-cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T-cell responses. However, the role of NOD2 in establishing memory T-cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Purpose It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific T-cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Method To address the effects of NOD2-signalling on intestinal T-cell priming in vivo, wildtype (WT) mice were adoptively transferred 50,000 naïve LCMV-specific (SMARTA) CD4+ T-cells. Mice were subsequently infected with LCMV-Armstrong in the presence or absence of the NOD2 agonist; MDP. 5-days following infection, the numbers and percentage of LCMV-specific T-cells in the mesenteric lymph nodes and spleen were examined. To examine the effects of NOD2 on intestinal TRM generation, littermate WT and NOD2 KO mice were infected with LCMV-Armstrong. Thirty-six-days following infection, the percentage and number of LCMV-specific CD4+ T-cells were profiled in the small and large intestinal lamina-propria by means of gp66-77 class-II MHC-tetramer staining. In another set of experiments, littermate WT and NOD2 KO mice were re-infected with LCMV-C13 30-days following LCMV-Armstrong immunization, and the interferon-response in the small intestine was profiled by quantitative PCR to assess the effect of NOD2-deficiency on antigen recall responses. Result(s) NOD2-stimulation by means of MDP injection increased the percentage and number of adoptively transferred SMARTA CD4+ T-cells in the mesenteric lymph nodes upon LCMV infection. Furthermore, NOD2-deficiency did not alter intestinal LCMV-specific CD4+ TRM seeding in the small and large intestinal lamina propria 36 days after infection. However, in vivo antigen recall experiments showed a decreased intestinal IFN response in NOD2 KO mice. Conclusion(s) Our findings reveal a potential role of NOD2 in the intestinal CD4+ T-cell priming and subsequent Ag-specific memory response. Disclosure of Interest None Declared

A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.

Manipulation of diacylglycerol and ERK-mediated signaling differentially controls CD8+ T cell responses during chronic viral infection.

Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen exposure is less understood. Here, we investigated the role of diacylglycerol (DAG)-mediated signaling downstream of the TCR during chronic lymphocytic choriomeningitis virus clone 13 (LCMV CL13) infection by blocking DAG kinase zeta (DGKζ), a negative regulator of DAG. We examined the activation, survival, expansion, and phenotype of virus-specific T cell in the acute and chronic phases of LCMV CL13-infected in mice after DGKζ blockade or selective activation of ERK. Upon LCMV CL13 infection, DGKζ deficiency promoted early short-lived effector cell (SLEC) differentiation of LCMV-specific CD8+ T cells, but this was followed by abrupt cell death. Short-term inhibition of DGKζ with ASP1570, a DGKζ-selective pharmacological inhibitor, augmented CD8+ T cell activation without causing cell death, which reduced virus titers both in the acute and chronic phases of LCMV CL13 infection. Unexpectedly, the selective enhancement of ERK, one key signaling pathway downstream of DAG, lowered viral titers and promoted expansion, survival, and a memory phenotype of LCMV-specific CD8+ T cells in the acute phase with fewer exhausted T cells in the chronic phase. The difference seen between DGKζ deficiency and selective ERK enhancement could be potentially explained by the activation of the AKT/mTOR pathway by DGKζ deficiency, since the mTOR inhibitor rapamycin rescued the abrupt cell death seen in virus-specific DGKζ KO CD8+ T cells. Thus, while ERK is downstream of DAG signaling, the two pathways lead to distinct outcomes in the context of chronic CD8+ T cell activation, whereby DAG promotes SLEC differentiation and ERK promotes a memory phenotype.

Core fucosylation of N-linked glycans: a novel player in memory CD8+ T cell differentiation following acute viral infection

Abstract Core fucosylation (CF) of N-linked glycans is indispensable for normal physiology, but whether CF regulates any aspect of T cell activation, differentiation, or function remains unknown. Fucosyltransferase 8 (Fut8) is the only enzyme in mammals that catalyzes CF and following activation, both expression of Fut8 and CF synthesis increases on CD8+ T cells. To test the hypothesis that CF of N-glycans regulates memory T cell differentiation following acute viral infection, we generated mice with a floxed Fut8 gene. WT (CD4-Cre−) and Fut8−/− (CD4-Cre+) mice were infected with LCMV Armstrong (LCMV-Arm) and the differentiation of LCMV-specific CD8+ T cells was analyzed by measuring the surface expression of killer-cell lectin like receptor G1 (KLRG1) and the α chain of interleukin 7 receptor (CD127) to identify short-lived effector and memory precursor T cells, respectively. LCMV-specific CD8+ T cells from both WT and Fut8−/− followed the same pattern of expansion and contraction without any significant difference in the overall frequency of LCMV-specific CD8+ T cells. However, Fut8−/− retained a higher percentage of CD127+KLRG1− and a lower percentage of KLRG1+CD127− CD8 T cells throughout the course of infection suggesting CF influences the progression to long-term memory. At day 42 post infection, mice were infected with virulent Listeria monocytogenes expressing the LCMV epitope GP33–41. Both WT and Fut8−/− GP33-specific CD8+ T cells expanded equally. However, Fut8−/− retained a higher frequency of GP33-specific CD8+ T cells and did not undergo contraction until &amp;gt;3 weeks post-2ry challenge. Taken together, our findings suggest that CF of N-linked glycans regulates memory CD8+ T cell differentiation following acute viral infection. Supported by grants from NIH (R21 AI159401)

Helpless CD8 T cell memory explained: prolonged antigen presentation drives a temporal rather than terminal defect

Abstract CD4 T cell help is critical for long-term antiviral CD8 T cell immunity. Despite seemingly normal effector properties, “helpless” CD8 memory T cells (TM) generated in the absence of CD4 T cells during initial priming exhibit a substantially blunted recall response. Yet, how lack of CD4 help specifically impairs CD8 TM formation in the context of acute viral infections remains elusive. Here, we demonstrate that in transiently and constitutively CD4 T cell-deficient mice “helpless” antiviral CD8 TM to acute lymphocytic choriomeningitis virus (LCMV) exhibit a wide array of homeostatic, phenotypic, and functional defects that are broadly consistent with delayed CD8 TM maturation. Mechanistically, “helpless” CD8 T cell memory is not grounded in altered CD8 effector T (TE) cell differentiation in the absence of CD4 T cells but in exposure of post-effector CD8 T cells to protracted viral antigen presentation. Conversely, efficient generation of “helped” CD8 T cell memory is contingent on CD40L-mediated activities by LCMV-specific CD4 T cells that promote timely clearance of viral antigen. Importantly, we show that “helpless” memory-precursor CD8 TE display comparable memory potential to their “helped” counterparts, and that, once viral antigen is cleared, complete resolution of “helpless” CD8 memory is achieved over a period of ~1.5 years during which “helpless” CD8 TM eventually acquire mature TM properties including fully restored recall capacities. Collectively, our data redefine “helpless” CD8 T cell memory as a temporal rather than terminal defect, with implications for our general conceptualization of CD8 T cell memory as well as for the development of preventive and therapeutic strategies to improve protective CD8 TM function. Supported by grant NIH R01AI093637.

Defining the extra-thymic role of HEB in the development of CD8+ T stem cell-like immunological memory

Abstract The transcription factor HEB is involved in multiple stages of intrathymic T cell development, complexing with stage-specific partners to regulate different genes in a context-specific manner. Remarkably, little is known about how HEB influences the differentiation of peripheral T cells, and even less so of how the transcriptional circuitry in early T cell development may be re-invoked to generate long-lasting immunity. While T cells are detected in the periphery of HEB-deficient mice (HEB cKO), the contribution of HEB to the function of T cells upon pathogen encounter is obscure. Thus, HEB cKO mice were challenged with LCMV to determine the impact of HEB in the activation and differentiation of CD8+ T cells into effector and memory lineages. Throughout the course of infection, HEB cKO mice exhibited critical differences in their antigen-specific CD8+ response and had enhanced levels of stem cell-like memory T cells (Tscm). This implicated a role for HEB downstream of T cell activation, which we further confirmed using well-established in vitro cultures supplemented with or without Tscm-inducing cytokines. Consistently, HEB transcripts were detected in publicly available scRNA datasets from P14 transgenic mice infected with LCMV. To greater define the transcriptional pattern of HEB, and of others that orchestrate thymic T cell development in the context of peripheral T cell immunity, we will conduct a scRNA-seq comparative bioinformatic analysis of developing thymocytes to splenic LCMV-specific CD8+ T cells. This will advance our understanding of how early transcriptional cascades, like those initiated by HEB, are propagated in downstream immunological reactions, which may lead to therapies that can revitalize T cell immunity. Supported by a grant from NIH (1P01AI102853-06 ) and the The AAI Intersect Fellowship Program for Computational Scientists and Immunologists

The ATP-Exporting Hemichannel Pannexin-1 Promotes CD8+ T Cell Memory Establishment

Abstract Release of extracellular ATP (eATP) by dying cells is often identified as a "danger signal" by immune cells. However, eATP export by healthy cells is common, and a major transmembrane ATP exporter is the Pannexin 1 (Panx1) molecule. CD8+ T cells express Panx1 during thymic development, at steady-state and during immune response. However, the role of Panx1 on CD8+ T cell-mediated immune response is not well defined. Here, we used genetic and pharmacological ablation tools to address this question. T cell-specific Panx1 deletion does not affect CD8+ T cell thymic development or effector responses to viral infection (LCMV). In contrast, memory CD8+ T cell establishment is impaired by either pharmacological inhibition or CD8-specific Panx1 deletion. Using a tamoxifen-controlled, conditional Panx1 knockout LCMV-specific (P14) CD8+ T cell system, we found that both memory CD8+ T cell generation and survival rely on Panx1. In vitro data further supports these findings: two Panx1 inhibitors, 10Panx and Probenecid, reduce the expression of the central memory marker CD62L. Panx1-deficient CD8+ T cells develop a memory-like phenotype in response to IL-15, but fail to engage mitochondrial respiration and present decreased mitochondrial membrane potential, suggesting Panx1 promotes optimal mitochondrial function. These defects are partially reverted with exogenous eATP. Collectively, our data indicates that cell-intrinsic Panx1-mediated eATP export favors memory CD8+ T cell differentiation. We are currently testing whether CD8-intrinsic Panx1 controls memory CD8+ T cell generation solely as an autocrine eATP source, or if Panx1 also serves as a “rheostat” controlling intracellular ATP levels in antigen-responding CD8+ T cells.

T Cell Receptor Diversity and Lineage Relationship between Virus-Specific CD8 T Cell Subsets during Chronic Lymphocytic Choriomeningitis Virus Infection.

Recent studies on chronic viral infections have defined a novel programmed cell death 1-positive (PD-1+) T cell factor 1-positive (TCF1+) stem-like CD8 T cell subset that gives rise to the terminally differentiated exhausted CD8 T cells. In this study, we performed T cell receptor beta (TCRβ) sequencing of virus-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection to examine the TCR diversity and lineage relationship of these two functionally distinct subsets. We found that >95% of the TCR repertoire of the exhausted CD8 T cell subset was shared with the stem-like CD8 T cells. The TCR repertoires of both CD8 T cell subsets were composed mostly of a few dominant clonotypes, but there was slightly more breadth and diversity in the stem-like CD8 T cells than their exhausted counterpart (∼40 versus ∼15 GP33+ clonotypes; ∼20 versus ∼7 GP276+ clonotypes). Interestingly, the breadth of the TCR repertoire was broader during the early stages (day 8) of the chronic infection than the later stages (days 45 to 60), showing that there was a narrowing of the TCR repertoire during chronic infection (∼2-fold GP33+ and GP276+ stem-like subset; ∼10-fold GP33+ and ∼5-fold GP276+ exhausted subset). In contrast, during acute LCMV infection, the TCR repertoire was much broader in both GP33-specific effector (∼160 clonotypes) and memory CD8 T cells (∼160 clonotypes). Overall, our data demonstrate that the virus-specific CD8 T cell TCR repertoire is broad and remains stable after acute LCMV infection, but it contracts and is narrower during chronic infection. Our study also shows that the repertoire of the exhausted CD8 T cell subset is almost completely derived from the stem-like CD8 T cell subset during established chronic LCMV infection.IMPORTANCE CD8 TCR repertoires responding to chronic viral infections (HIV, hepatitis C virus [HCV], Epstein-Barr virus [EBV], and cytomegalovirus [CMV]) have limited breadth and diversity. How these repertoires change and are maintained throughout the chronic infection are unknown. We thus characterized the LCMV-specific CD8 TCR repertoires of stem-like and terminally exhausted subsets generated during chronic LCMV infections. During chronic LCMV infections, the repertoires started as diverse but became more clonal at the late time point. Further, the exhausted subset was composed of dominant clonotypes that were shared with the stem-like subset. Together, we demonstrate that the TCR repertoire contracts over time and is almost exclusively derived from the stem-like subset late during the persistent viral infection. Our data suggest that dominant clonotypes in the exhausted subset are derived from a diverse pool of stem-like clonotypes, which may be contributing to the clonality observed during chronic viral infections.

CD160 Plays a Protective Role During Chronic Infection by Enhancing Both Functionalities and Proliferative Capacity of CD8+ T Cells.

The understanding of protective immunity during HIV infection remains elusive. Here we showed that CD160 defines a polyfunctional and proliferative CD8+ T cell subset with a protective role during chronic HIV-1 infection. CD160+ CD8+ T cells derived from HIV+ patients correlated with slow progressions both in a cross-sectional study and in a 60-month longitudinal cohort, displaying enhanced cytotoxicity and proliferative capacity in response to HIV Gag stimulation; triggering CD160 promoted their functionalities through MEK–ERK and PI3K–AKT pathways. These observations were corroborated by studying chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The genetic ablation of CD160 severely impaired LCMV-specific CD8+ T cell functionalities and thereby resulted in loss of virus control. Interestingly, transcriptional profiling showed multiple costimulatory and survival pathways likely to be involved in CD160+ T cell development. Our data demonstrated that CD160 acts as a costimulatory molecule positively regulating CD8+ T cells during chronic viral infections, thus representing a potential target for immune intervention.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ Tcells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NKcells and ILC1. Here, we demonstrate that these NK1.1+ cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ Tcells (TRM cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM cells by NK1.1+ cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NKcells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.