LCMV-specific CD8 Research Articles

Functional restoration of exhausted CD4(+) and CD8(+) T cells in chronic viral infection by vinegar-processed flos of Daphne genkwa.

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers.

Bone Marrow Memory CD8+ T Cells Play a Supporting Role in Hematopoiesis

The bone marrow (BM) has an important function as primary lymphoid organ through the process of hematopoiesis. This process is sustained by hematopoietic stem cells (HSCs) and their life-long production of new blood cells, which is made possible by their unique ability to self-renew. Next to this, BM also functions as a secondary lymphoid organ, as it can mediate primary T cell responses against invading pathogens. We and others have shown that activated T cells can influence the hematopoietic process through the production of pro-inflammatory cytokines. This indicates that adaptive immune responses and hematopoiesis are intertwined in the BM, though most of the cellular and molecular interactions that occur during this crosstalk are yet unknown.Based on previous observations that T cell deficient mice have altered hematopoiesis and that depletion of T cells from allogeneic BM grafts compromises HSC engraftment, we questioned to what extent BM T cells can directly affect the function of HSCs. To test this, we sorted and co-cultured murine BM T cells with HSCs (Lin-Sca-1+c-Kit+CD48-CD150+). We found that particularly BM CD8+ central memory (CD44+CD62L+) T cells (Tcm) enhance the capacity of HSCs to self-renew. Furthermore, we found that TCR-transgenic mice, which do not have memory T cells, have lower numbers of HSCs, which could subsequently be increased by transferring BM CD8+ Tcm. Remarkably, an increase in HSC numbers was also observed when HSCs were cultured with only supernatant derived from BM CD8+ Tcm. Moreover, HSCs cultured with supernatant from BM CD8+ Tcm and later transplanted in myeloablated hosts displayed a strongly enhanced ability to restore hematopoiesis. Importantly, the strong impact of BM T cells on HSCs was not only apparent in the steady state situation, but also following a viral infection with either acute or chronic lymphocytic choriomeningitis virus (LCMV). We could establish that both acute and chronic LCMV-specific CD8+ T cells or supernatant from these cells could increase the HSC self-renewal capacity.In conclusion, our findings demonstrate that BM memory CD8+ T cells can positively influence the function of HSC through soluble mediators. We postulate that this process is particularly relevant after immune activation, in order to protect and/or restore the HSC pool and the subsequent hematopoietic recovery. We are currently using a proteomics approach to identify these soluble mediator produced by CD8+ T cells. DisclosuresNo relevant conflicts of interest to declare.

Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downregulation of costimulatory GITR ligand on APC.

The costimulatory TNFR family member GITR can provide important survival signals for CD8 T cells. However, little is known about the regulation of this pathway during a chronic infection. In this study, we show that GITR ligand (GITRL) is maximally induced on APCs at day 2 post-lymphocytic choriomeningitis virus (LCMV) clone 13 infection, but is downregulated to below baseline levels by day 8 postinfection (p.i.), and remains so at the chronic stage of infection. At its peak, GITRL expression is highest on macrophages, with lower expression on conventional and plasmacytoid dendritic cells. GITR expression was highest on T regulatory cells but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 p.i. and was maintained at low, but above baseline levels at the chronic stage of LCMV infection. As GITRL was limiting at the chronic stage of infection, we investigated the potential of therapeutic stimulation of GITR at this stage using agonistic anti-GITR Ab. Anti-GITR treatment at day 21 p.i. increased the frequency and number of LCMV-specific CD8 T cells, resulting in increased in vivo CTL activity and a concomitant decrease in viral load, despite the persistence of PD-1 expression. These effects of anti-GITR were CD8 T cell intrinsic, with no detectable effects on Th1 or T regulatory cells. In contrast to other TNFR agonists, such as anti-4-1BB, which can cause immune pathology, a single therapeutic dose of anti-GITR did not induce splenomegaly or increase serum alanine transaminase. These studies identify GITR as a promising therapeutic target for chronic infection.

Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution.

Vaccines are desired that maintain abundant memory T cells at nonlymphoid sites of microbial exposure, where they may be anatomically positioned for immediate pathogen interception. Here, we test the impact of antigen persistence on mouse CD8 and CD4 T cell distribution and differentiation by comparing responses to infections with different strains of LCMV that cause either acute or chronic infections. We used in vivo labeling techniques that discriminate between T cells present within tissues and abundant populations that fail to be removed from vascular compartments, despite perfusion. LCMV persistence caused up to ∼30-fold more virus-specific CD8 T cells to distribute to the lung compared with acute infection. Persistent infection also maintained mucosal-homing α4β7 integrin expression, higher granzyme B expression, alterations in the expression of the TRM markers CD69 and CD103, and greater accumulation of virus-specific CD8 T cells in the large intestine, liver, kidney, and female reproductive tract. Persistent infection also increased LCMV-specific CD4 T cell quantity in mucosal tissues and induced maintenance of CXCR4, an HIV coreceptor. This study clarifies the relationship between viral persistence and CD4 and CD8 T cell distribution and mucosal phenotype, indicating that chronic LCMV infection magnifies T cell migration to nonlymphoid tissues.

45: Functional restoration of exhausted CD4+ and CD8+ T cells in chronic viral infection by daphnane diterpene ester derived from Daphne genkwa flos buds via negative regulatory Tim-3 molecule

CD4+ and CD8+ T-cell exhaustion developed in chronic viral infection has been important issue because exhausted antigen-specific CD4+ and CD8+ T cells showed impaired ability to eradicate persistently infected virus and produce effector cytokines such as IFN-γ and TNF-α. Hence, strategies that either restore endogenous exhausted T-cell responses or provide functional CD4+ and CD8+ T cells need to be urgently developed for therapeutics of human chronic infection. In spite of promising development using antibody and cell immunotherapy, there were no attempts to restore exhausted CD4+ and CD8+ T cells using treatment of small bioactive molecules, especially derived from natural resources. Here, using mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found for the first time that genkwadaphnin isolated from dried flower buds of Daphne genkwa (Thymelaeceae) significantly restored exhausted CD4+ and CD8+ T cells, as corroborated by evidences that genkwadaphnin treatment enhanced functional LCMV-specific CD4+ and CD8+ T cells in both quantitative and qualitative. Repeated administration of lower genkwadaphnin doses also induced recovery of exhausted LCMV-specific CD4+ and CD8+ T cells. Furthermore, pretreatment of genkwadaphnin prior to LCMV infection prevent the generation of exhausted LCMV-specific CD8+ T cells. Such restoration of exhausted LCMV-specific CD4+ and CD8+ T cells by genkwadaphnin was closely associated with reduction of viral burden in sera as well as tissues. More intriguingly, genkwadaphnin treatment induced down-regulation of negative regulatory molecules such as PD-1 and Tim-3 in exhausted LCMV-specific CD4+ and CD8+ T cells with more apparent down-regulation of Tim-3, which suggesting that Tim-3 molecule is main target to restore exhausted T-cell responses with genkwadaphnin. Collectively, these results provide new valuable insight into the use of genkwadaphnin isolated form flos buds of Daphne genkwa to develop therapeutic strategy of chronic human diseases, such as hepatitis B and C virus (HBV and HCV), human immunodeficiency virus (HIV) as well as cancers.

Gammaherpesvirus latency differentially impacts the generation of primary versus secondary memory CD8+ T cells during subsequent infection.

Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8(+) T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4(+) T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8(+) T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8(+) T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8(+) T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. CD8(+) T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8(+) T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection.

Regulatory T (T reg) cells are critical for preventing autoimmunity mediated by self-reactive T cells, but their role in modulating immune responses during chronic viral infection is not well defined. To address this question and to investigate a role for T reg cells in exhaustion of virus-specific CD8 T cells, we depleted T reg cells in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). T reg cell ablation resulted in 10-100-fold expansion of functional LCMV-specific CD8 T cells. Rescue of exhausted CD8 T cells was dependent on cognate antigen, B7 costimulation, and conventional CD4 T cells. Despite the striking recovery of LCMV-specific CD8 T cell responses, T reg cell depletion failed to diminish viral load. Interestingly, T reg cell ablation triggered up-regulation of the molecule programmed cell death ligand-1 (PD-L1), which upon binding PD-1 on T cells delivers inhibitory signals. Increased PD-L1 expression was observed especially on LCMV-infected cells, and combining T reg cell depletion with PD-L1 blockade resulted in a significant reduction in viral titers, which was more pronounced than that upon PD-L1 blockade alone. These results suggest that T reg cells effectively maintain CD8 T cell exhaustion, but blockade of the PD-1 inhibitory pathway is critical for elimination of infected cells.

Enforced viral replication, a mechanism for immune activation

Since many decades, it is known that viral infection can lead to a strong immune activation. This partially explains the strong association of viral infection with autoimmunity, immunopathology during chronic virus infections and complications during transplantation. One explanation for the strong immune stimulatory effects of viruses is the expression of specific patterns (e.g. viral RNA). Pathogen-specific patterns can be recognized by endogenous receptors (e.g.: Toll -like receptors), which are known to induce a strong innate immune activation. Whether further immunological mechanisms contribute to virus-specific immune activation is current topic of research. In the recently published work [1,2], we identified the mechanism of enforced viral replication. We found that specialized cells in the spleen and lymph nodes (CD169+ macrophages and conventional dendritic cells) selectively allowed replication of viruses. This enforced viral replication led to an exorbitant propagation of viral antigens and viral RNA. Viral antigen led to massive activation of the adaptive immune system, whilst viral RNA activated innate immunity. Therefore the mechanism of enforced viral replication partially explains the association of strong immune activation with virus infection. First, we analyzed the role of enforced viral replication during infection with cytopathic vesicular stomatitis virus (VSV). We demonstrated that CD169+ macrophages express the protein UBP43 (gene name: Usp18). UBP43 is a cellular interferon type I (IFN-I) inhibitor. Therefore, due to expression of UBP43, IFN-I could not induce an antiviral status in CD169+ macrophages. Thus VSV, once it infected CD169+ macrophages, showed strong replication in this particular cell type. Other types of macrophages (Kupffer cells and red pulp macrophages) did not show VSV replication after infection. The enhanced replication of VSV in CD169+ macrophages induced a strong immune activation. Genetic depletion of Usp18 or depletion of CD169+ macrophages limited enforced viral replication and resulted in a reduced activation of the adaptive and innate immune response. As a consequence virus could not be controlled resulting in fast death after infection. Together our data show that replication of VSV in specific splenic niches is essential for innate and adaptive immune activation and for surviving an infection with cytopathic VSV. To investigate the contribution of enforced viral replication to autoimmune disorders we tested its role in the induction of autoimmune diabetes using the RIP-GP mouse model. RIP-GP mice express the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) under the rat insulin promoter. LCMV-specific CD8+ T cells, which usually develop during LCMV infection, show cross-reactivity to pancreatic beta cells of RIP-GP mice. Therefore RIP-GP mice develop diabetes during infection with LCMV. We found that induction of LCMV-specific CD8+ T cells as well as LCMV induced innate immune response depended on enforced viral replication. Genetic knockdown of Usp18, depletion of CD169+ macrophages and conventional dendritic cells or pharmacological inhibition of viral life cycle, blunted viral replication in the spleen and limited innate and adaptive immune activation against LCMV. Thereby the development of autoimmune diabetes was prevented. In summary, professional innate immune cells purposely allow virus replication due to over-expression of UBP43. UBP43 expression and accelerated virus replication was essential to activate the antiviral innate and adaptive immune response. At the one hand this mechanism is absolutely required to protect from fatal virus infection, at the other hand it also strongly contributes to induction of virus induced autoimmune diabetes. In the future, it will be investigated in which other diseases this mechanism could play a role, and whether it may also have clinical implications.

Sepsis increases susceptibility to chronic infection and exacerbates CD8 T cell exhaustion (VIR4P.1008)

Abstract Sepsis survivors often display impairment in innate and adaptive immune responses and become highly susceptible to ‘secondary’ infections with intracellular pathogens that are normally controlled by CD8 T cells. It is unknown if or when this observed immunoparalysis of CD8 T cell immunity recovers, and the long-term consequences of sepsis on the ability of naïve CD8 T cells to respond to subsequent infections are poorly understood. Previously, we demonstrated that sepsis has sustained detrimental effects on the capacity of CD8 T cells to respond to acute viral and bacterial infections. Here, using the cecal ligation and puncture (CLP) mouse model, we show that mice recovering from a septic event are more susceptible to chronic LCMV Clone-13 infection demonstrated by increased morbidity and mortality. LCMV-specific CD8 T cells from chronically infected CLP mice display an increase in inhibitory molecule expression (e.g., PD-1) and diminished Ag-driven cytokine production, suggesting that sepsis exacerbates CD8 T cell exhaustion following chronic viral infection. Furthermore, this exhausted phenotype of virus-specific CD8 T cells is observed earlier in LCMV Clone-13 infected septic mice compared to control mice. Together, these results illustrate that sepsis increases the vulnerability to chronic infections and can result in substantial and long-lasting changes in the CD8 T cell compartment affecting the ability of the host to respond to newly introduced antigens.

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.

The co-stimulatory molecule GITR intrinsically enhances type 1 and follicular helper CD4 T cell responses to establish early control of a persistent viral infection and potentiate the late humoral response (IRC9P.700)

Abstract During persistent infection, the immune system must be tightly regulated to balance immune control against pathology. The glucocorticoid-induced TNFR (GITR) is an important co-stimulatory molecule that plays a critical CD4 T cell-intrinsic role in the control of a mouse model of chronic viral infection, LCMV clone 13. 8 days post-infection (dpi) of wild-type (WT) and GITR-/- mice revealed that GITR-/- mice have 2-fold fewer LCMV-specific CD8 T cells, with higher PD-1 and Tim-3 levels. These defects became more striking at 45 dpi, when GITR-/- mice have a ~9-fold deficit in IFNγ+CD107+TNF+ CD8 T cells. GITR-/- mice had impaired viral control, with 35- and 10- fold higher viral load in the kidney at 8 and 45 dpi, respectively. Depletion of CD4 cells largely abrogated the differences between WT and GITR-/- mice at 8 dpi, suggesting that CD4 T cells underscore the impaired T cell responses of GITR-/- mice. GITR-/- mice have 3-4-fold fewer IFNγ+ and IL-2+ Th1 cells at 8 dpi. Using DEREG mice, we found that the compromised immunity and viral control in GITR-/- mice are independent of Foxp3+ Tregs. GITR-/- mice also had 2-fold fewer follicular helper (Tfh) cells at 21-45 dpi, with concomitant impairment in LCMV-specific IgG titres. Mixed bone marrow chimeras revealed a CD4 T cell-intrinsic role for GITR in potentiating Th1 and Tfh responses. Our findings reveal a critical role for GITR in co-stimulation of CD4 helper T cell subsets for optimal cell-mediated and humoral immunity.

Direct Infection of Dendritic Cells during Chronic Viral Infection Suppresses Antiviral T Cell Proliferation and Induces IL-10 Expression in CD4 T Cells

Elevated levels of systemic IL-10 have been associated with several chronic viral infections, including HCV, EBV, HCMV and LCMV. In the chronic LCMV infection model, both elevated IL-10 and enhanced infection of dendritic cells (DCs) are important for viral persistence. This report highlights the relationship between enhanced viral tropism for DCs and the induction of IL-10 in CD4 T cells, which we identify as the most frequent IL-10-expressing cell type in chronic LCMV infection. Here we report that infected CD8αneg DCs express elevated IL-10, induce IL-10 expression in LCMV specific CD4 T cells, and suppress LCMV-specific T cell proliferation. DCs exposed in vivo to persistent LCMV retain the capacity to stimulate CD4 T cell proliferation but induce IL-10 production by both polyclonal and LCMV-specific CD4 T cells. Our study delineates the unique effects of direct infection versus viral exposure on DCs. Collectively these data point to enhanced infection of DCs as a key trigger of the IL-10 induction cascade resulting in maintenance of elevated IL-10 expression in CD4 T cells and inhibition of LCMV-specific CD4 and CD8 T cell proliferation.

Functional limitations of plasmacytoid dendritic cells limit type I interferon, T cell responses and virus control in early life.

Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8+ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

Bone Marrow Memory CD8 T Cells Positively Influence Hematopoietic Stem Cell Function

The bone marrow (BM) not only serves as a primary, but also as a secondary lymphoid organ, since it can mediate primary T cell responses against blood-borne antigens and it harbors a significant portion of the memory T cell compartment. Yet, it remains unclear to what extent BM T cells affect the local hematopoietic process. This is important from a clinical perspective, since the development of BM failure and anemia is frequently associated with (chronic) T cell-mediated inflammatory diseases, such as rheumatoid arthritis and viral infections. We postulate that particularly hematopoietic stem cells (HSCs) may be susceptible to T cell activity, since HSCs are localized in endothelial BM niches and are thus in close vicinity of where T cells enter the BM parenchyma and get activated. In support of this, we have previously shown that IFNγ, one of the key cytokines produced by activated T cells, strongly impairs HSC self-renewal and enhances their differentiation towards monoctyes, at the expense of neutrophils and erythrocytes.To examine the impact of T cells on HSC function, we performed co-culture assays and found that T cells from murine BM actually have a positive impact on HSC function, as they enhance both their differentiation and self-renewal capacity. This feature is restricted to a subset of memory CD8 T cells in the BM, since neither naïve T cells from BM nor memory CD8 T cells from the spleen showed the same effect. Correspondingly, transgenic mice with only naïve and no memory T cells have fewer HSC numbers than control mice, which can be transiently restored when memory CD8 BM T cells are injected. To test the relevance of these findings in an inflammatory setting, we infected mice with the Armstrong-strain of lymphocytic choriomeningitis virus (LCMV), which induces an acute infection that leads to a strong influx of antigen-experienced T cells in the BM. We found that LCMV-specific memory CD8 T cells isolated from BM 12 days after infection increased both the differentiation and self-renewal capacity of HSCs. Interestingly, HSCs isolated from infected mice also displayed an enhanced propensity to differentiate towards myeloid cells compared to HSCs from non-infected control mice, whereas their self-renewal capacity was not altered. To test whether chronically stimulated T cells are also able to influence HSC function, we infected mice with LCMV clone 13, which leads to a chronic infection and induces exhaustion of the virus-specific T cells. Interestingly, virus-specific T cells isolated from BM 27 days after infection, which were exhausted based on phenotype and function, did not influence HSC differentiation but they were still able to enhance the self-renewal of HSCs from non-infected control mice, although to a lesser extent than in the acute infection. These data illustrate that antigen-experienced memory CD8 T cells in BM have a positive impact on the function of HSCs. Although cytokines produced by activated T cells, such as IFNγ and TNFα, can dramatically impair HSC maintenance, it is intriguing that memory T cells can actually fulfill a positive function on the HSC compartment. We speculate that homing of memory T cells to the BM after viral infection may play an important role in restoring the damage on the hematopoietic compartment that is inflicted by the infection itself and the ensuing cytokine storm. Enhancement of such a positive feedback mechanism may be a promising new strategy for treatment of patients with BM failure. Disclosures:No relevant conflicts of interest to declare.

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8(+) T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4(+) Foxp3(+) T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8(+) T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8(+) T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.

54: Role of transforming growth factor-beta signaling in CD8+ T cells in mucosal anti-viral immune responses

Under steady-state, the gut balances regulatory and inflammatory processes in order to preserve membrane integrity while preventing autoimmunity or inappropriate inflammation to commensal bacteria and innocuous food antigens. However, the vast majority of sexually transmitted infections also occur via a tolerogenic mucosal barrier. In such infections, the cytotoxic T lymphocyte (CTL) response is often “too late and too little” to induce protective immunity in most individuals. Transforming growth factor (TGF)β is highly abundant in the intestinal milieu. The decision to break tolerance in favor of immunity in mucosal tissues is likely regulated by a balance between levels of TGFβ and inflammatory cytokines. Our work demonstrates that immunosuppressive effects of TGFβ are subdued in the presence of high inflammatory signals; however, in the absence of strong inflammation and co-stimulatory signals, TGFβ inhibits proper T cell activation. The innate and adaptive immune responses are most likely dampened during sexual transmission of pathogens, in part due to high concentrations of TGFβ in mucosal tissues. To study the role of TGFβ signaling in mucosal anti-viral immunity, we established a rectal viral transmission model in mice using lymphocytic choriomeningitis virus (LCMV). Compared to systemic infections, the kinetics and the magnitude of the CD8 T cell response are much reduced upon rectal LCMV infection. To assess the role of TGFβ in mucosal immunity generated during rectal LCMV infection, we adoptively co-transferred LCMV-specific CD8 T cells (P14), carrying wildtype TGFβ receptor II (TGFβRII WT) and those deficient in this receptor (TGFβRII KO), into wild type hosts. The animals were then infected rectally with LCMV. We will present data on the mechanism by which TGFβ affects activation and expansion of effector CD8 T cells, their migration into the gut tissues, as well as their ability to perform cytolytic function and become tissue-resident memory T cells.

Diverging role for coronin 1 in antiviral CD4+ and CD8+ T cell responses

Coronin 1 is a member of the evolutionary conserved WD repeat protein family and is highly expressed in hematopoietic cells. Coronin 1 is essential for Ca2+ mobilization upon T cell receptor (TCR) stimulation providing a pro-survival signal for naïve peripheral T cells. Both in mouse and in human, coronin 1 deficiency is associated with severe T cell lymphopenia. In this work, we have analyzed antiviral T cell-mediated immunity in the presence and absence of coronin 1 in vivo after infection with lymphocytic choriomenigitis virus (LCMV) and vesicular stomatitis virus (VSV) in mice. Despite low peripheral T cell numbers we found that LCMV-specific CD8+ T cell responses were normal in the absence of coronin 1 and kinetics of LCMV-clearance were similar compared to wild type mice. In contrast, CD4+ T cell responses were profoundly decreased after LCMV- and VSV-infection. We propose that coronin 1 plays a differential role in CD8+ versus CD4+ T cell responses and activation.

Herpesvirus latency differentially impacts primary versus secondary memory T cell pools (P6119)

Abstract Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of herpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were infected with murine Gammaherpesvirus 68 (MHV68), a model of Epstein-Barr infection, and then after latency was established, infected with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). This resulted in increased LCMV-specific effector CD4+ T cells compared to mice infected only with LCMV. Although the number of LCMV-specific effector CD8+ T cells was not altered, they were skewed to a memory phenotype. When the memory phase was reached, mice that were infected with both viruses had a memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8+ T cells had altered CD27 and KLRG1 expression. Upon secondary challenge the LCMV-specific effector CD8+ and CD4+ T cell pools expanded and cleared the infection. When the secondary memory pool was established, mice that were also infected with MHV68 had decreased numbers of LCMV-specific CD8+ T cells and these cells were skewed to an effector-memory phenotype. Taken together these results demonstrate that acute viral infection in the context of ongoing latency affects the number and phenotype of primary versus secondary memory CD8+ T cells.

Viral infection alters subnuclear localization of PD-1 and CD62L loci in LCMV-specific CD8 T-cells (P6369)

Abstract During chronic viral infection, CD8 T-cell differentiation yields a unique transcriptional profile: T-cell exhaustion. The suboptimal effector function of exhausted cells is partly due to sustained expression of PD-1. Using the LCMV mouse model of infection, we examined pdcd1 (PD-1) and sell (CD62L) gene regulation at different stages of T-cell differentiation. We hypothesized that epigenetic programming and nuclear localization of these genes were coupled to viral persistence and T-cell differentiation. Changes in gene expression are linked to locus positioning in specific compartments such as nuclear lamina (silencing). Using DNA-FISH, we report a significant decrease in pdcd1 alleles associated to nuclear lamina in exhausted cells relative to other CD8 subsets. Dissociation from lamin was consistent with upregulated expression of PD-1 mRNA and protein, and with loss of PD-1 DNA methylation in exhausted cells. In contrast, we observed a significant increase in sell recruitment to lamin with exhaustion, mirroring changes in sell gene expression. CD8 T-cells from Blimp-1-deficient chronically infected mice exhibited an impaired ability to regulate pdcd1 and sell association to lamin, thus implicating Blimp-1 in modulating expression of both genes during chronic infection. Our findings contribute to the understanding of the molecular pathways mediating T-cell memory and exhaustion, and may provide insight into implementing therapeutic strategies to resolve chronic infections.

GITR potentiates anti-viral T cell immunity by limiting regulatory T cells (P1015)

Abstract The immune system must be tightly balanced to control infection while avoiding immune pathology. We investigated the role of the glucocorticoid-induced tumour necrosis factor receptor (GITR) during infection of mice with persistent lymphocytic choriomeningitis virus (LCMV) clone 13 infection. Infection of wild-type (WT) and GITR-/- mice with LCMV revealed that GITR plays a critical role in T cell responses and viral control. At 8 days post-infection (dpi), GITR-/- mice showed 3-fold fewer LCMV-specific CD8+ T cells, and these cells expressed significantly higher levels of inhibitory receptors Tim-3 and PD-1. These defects became more striking at 45 dpi, when there was also a substantial deficit in multifunctional (IFNγ+CD107a+TNFα+) CD8+ T cells in GITR-/- relative to WT mice. GITR-/- mice had 35-fold higher viral load in the kidney 8 dpi and 10-fold higher at day 45. Depletion of CD4+ T cells ameliorated the immune defects observed in GITR-/- mice, suggesting that a CD4+ population largely contributed to the GITR-/- phenotype. Strikingly, there were 4- to 5-fold more CD4+Foxp3+ regulatory T cells (Tregs) in the spleen and lymph nodes of GITR-/- mice 8 dpi, and only slightly more at 45 dpi. The dramatic increase in Treg numbers in GITR-/- mice 8 dpi is accompanied by a more suppressive phenotype. These studies demonstrate that GITR plays an indirect, yet critical role in CD8+ T cell responses and viral control by limiting the accumulation and function of Foxp3+ Tregs.