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Summary CD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1−/− mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function., Graphical abstract, Highlights • PAR1 signaling in human CD8 T cells accelerates TCR-induced calcium mobilization • PAR1 participates in the repositioning of the MTOC at the immunological synapse • PAR1 facilitates polarized secretion of cytotoxic granules at the immunological synapse • PAR1−/− Gp33-specific CD8 T cells show reduced expansion and effector function, Molecular biology; Immunology; Immune response

The programed death-1 (PD1)/programed death-ligand 1 (PD-L1) pathway plays a critical role in regulating immune responses against pathogens by controlling the balance between immunity and limiting tissue damage. In chronic viral infections such as HIV/SIV, hepatitis B virus, and hepatitis C virus, failure to completely eliminate virus leads to a sustained inflammatory/activated environment accompanied by the accumulation of T cells with diminished in-vitro effector function, ‘exhausted T cells’ [1–3]. In HIV/SIV infections, chronic T-cell immune activation is reflected by increased expression of a variety of immunomodulatory receptors including, PD1, CTLA-4, LAG3, CD244/2B4, CD160, and others [4–11]. In-vitro and in-vivo studies of HIV/SIV infection have shown that blockade of PD1 is associated with enhanced virus-specific responses and improved viral control [12–15]. These observations support the hypothesis that PD1/PD-L1 interactions contribute to the functional dysregulation, exhaustion, and ineffective viral control [8,16–18]. This evidence suggests that the PD1/PD-L1 pathway may be a potential target for immune intervention in patients with HIV infection. PD1 is expressed upon activation by T, B, and natural killer (NK) cells, dendritic cells, and activated monocytes, although its function in the latter is not well defined [19]. PD1 interacts with two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed by hematopoietic cells, such as T and B cells, dendritic cells, macrophages, and some epithelial cells (lung and vascular endothelium) [19–22]. In contrast, PD-L2 expression is more restricted and is inducible in dendritic cells, macrophages, bone marrow–derived mast cells, and some B cells [19,23]. PD1 interaction with its ligands regulates T cell receptor signaling by distinct mechanisms leading to diminished effector function, including cytokine secretion, proliferation, cytotoxicity, motility, and cell survival [24–27]. In addition, engagement of PD1 with its ligands induces reverse signaling on PD-L1/PD-L2 expressing cells [19,28,29]. Lymphoid organs are the primary targets of HIV/SIV infection [30–33]. In HIV/SIV-infected lymph nodes, the germinal center (GC) T follicular helper CD4+ T cells (CD4+ Tfh) exhibit higher expression of PD1 compared with extra follicular CD4+ and CD8+ T cells. PD-L1 expression has been observed inside and outside germinal centers [34,35]. To obtain further insights as to the potential role of PD-L1 blockade in the treatment of HIV infection, we analyzed the expression of PD1 and PD-L1 in human lymph node from 23 patients infected with HIV. In addition, we have evaluated the safety and the effect on SIV viral load of in-vivo PD1/PD-L1 blockade using a fully human anti-PD-L1 mAb (MSB0010718C, Avelumab, EMD-Serono, Inc., Rockland, Massachusetts, USA) in rhesus (Rh) macaques chronically infected with SIVmac239, that had previously received IL-15 [36].

Summary Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that under steady state conditions, IL-7 signaling is principally mediated by activation of Signal Transducers and Activators of Transcription 5 (STAT5). In contrast, under lymphopenic conditions there is a modulation of STAT1 expression resulting in an IL-7-dependent STAT1 and STAT5 activation. Consequently, the IL-7-induced transcriptome is altered with enrichment of interferon (IFN) stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4 T cells undergoing Lymphopenia-Induced Proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7-dependent program. This mechanism could be a physiological process to “sense space” regulating the size of the CD4 T cell pool. During HIV infection the virus could exploit this pathway leading to the homeostatic dysregulation of the T cell pools observed in these patients.

HIV immune activation plays an important role in the immunopathogenesis of the disease. The mechanisms driving this immune activation are partially defined and likely are the result of multiple factors. The introduction of combination antiretroviral therapy (cART) has improved the life expectancy of HIV infected individuals, however there is evidence that in the setting of "undetectable" HIV-RNA plasma levels, there is some level of persistent immune activation in these patients. A better understanding of the immune activation pathways should be of value in developing complementary therapies to restore the immune systems of patients with HIV infection. This review discusses the cytokine mediated pathways of immune activation of the CD4 and CD8 T cell pools during HIV infection.

The onset of autoimmunity in experimental rodent models and patients frequently correlates with a lymphopenic state. In this condition, the immune system has evolved compensatory homeostatic mechanisms that induce quiescent naive T cells to proliferate and differentiate into memory-like lymphocytes even in the apparent absence of antigenic stimulation. Because memory T cells have less stringent requirements for activation than naive cells, we hypothesized that autoreactive T cells that arrive to secondary lymphoid organs in a lymphopenic environment could differentiate and bypass the mechanisms of peripheral tolerance such as those mediated by self-antigen cross-presentation. Here, we show that lymphopenia-driven proliferation and differentiation of potentially autoreactive CD8+T cells into memory-like cells is not sufficient to induce self-reactivity against a pancreatic antigen. Induction of an organ-specific autoimmunity required antigen-specific CD4+T cell help. Notably, we found that this function could be accomplished by memory-like CD4+T cells generated in vivo through lymphopenia-induced proliferation. These helper cells promoted the further differentiation of memory-like CD8+T cells into effectors in response to antigen cross-presentation, resulting in their migration to the tissue of antigen expression where autoimmunity ensued. Thus, the cooperation of self-reactive memory-like CD4+and CD8+T cells under lymphopenic conditions overcomes cross-tolerance resulting in autoimmunity.

HIV infection and its associated chronic immune activation alter T cell homeostasis leading to CD4 T cell depletion and CD8 T cell expansion. The mechanisms behind these outcomes are not totally defined and only partially explained by the direct cytopathic effect of the virus. We established a murine model of immunpoathogenesis in which adoptive T cell transfer into lymphopenic hosts chronically treated with IFN-α leads to CD4 T cell depletion and CD8 T cell expansion, recapitulating the alteration of the T cell pools observed in HIV infected patients. In these conditions, CD4 T cells undergoing IL-7/lymphopenia induced-proliferation upregulate their level of the Signal Transducer and Activator of Transcription 1 (STAT1) resulting in enhanced CD4 T cell responsiveness to IFN-α. In this setting, CD4 T cell depletion resulted from an indirect effect of IFN-α driven inflammatory environment, since animals with IFNAR deficient T cells underwent the same degree of CD4 T cell depletion than wild type animals. In contrast, direct IFN-α signaling through IFNAR on CD8 T cells was necessary for their expansion induced by chronic exposure to Type-I IFN under lymphopenic conditions. These findings suggest that direct and indirect effects of IFN-α signaling may play a role in the pathogenesis of HIV infection. Further analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection.

HIV infection is associated with chronic immune activation and alteration of T cell homeostasis (CD4 T cell depletion and CD8 T cell expansion). While the acute CD4 depletion observed in the initial phase of HIV infection is likely due to direct cytopatic effects of the virus, the mechanism/s underlying the steady decline of the CD4 T cell pool during the chronic phase of infection are unclear and are felt to be associated with “immune activation”. In the current study, we addressed the impact of the combination of two distinct forces: homeostatic (CD4 T cell depletion) and inflammatory (HIV-driven IFN-α) on T cell homeostasis. We present evidences that IL-7 and lymphopenia enhanced CD4 T cell responsiveness to IFN-α by modulating expression of the Signal Transducers and Activators of Transcription (STAT) 1, 2 and 3. In a murine model, CD4 T cell depletion and CD8 T cell expansion were observed in a lymphopenic host chronically treated with IFN-α. These findings suggest that a synergistic interaction between lymphopenia and IFN-α may play a role in the pathogenesis of HIV infection. The analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection.

To maintain the size of T cell pools throughout life, homeostatic mechanisms regulate both survival and proliferation, which is achieved by IL-7 and TCR signaling. IL-7 (through lymphopenia-induced proliferation/LIP) can also mediate expansion of autoreactive clones leading to the onset of autoimmune disease or enhance effector function against tumors and viruses. In the setting of chronic HIV infection, CD4 T cell depletion and the increased availability of IL-7 triggers LIP that occurs in an inflammatory environment rich in Type-I IFNs; we hypothesized that this pathway could contribute to the immune activation leading to CD4 T cell depletion and CD8 T cell pool expansion. Accordantly, we found that genes associated with γ c cytokines and Type-I IFN signaling were differentially expressed in CD4 and CD8 T cells from chronic HIV infected patients. Using a lymphopenic murine model, we present new evidence that, in vivo, IL-7 differentially regulated the expression of the total-Signal Transducer and Activator of Transcription 1 (t-STAT1) in CD4 and CD8 T cells undergoing LIP and in so doing enhanced CD4 T cell responsiveness to Type-I IFN. In this setting, chronic treatment with IFN- α led to decreased CD4 T cell counts and CD8 T cell expansion. This interplay between IL-7 and Type-I IFN might be advantageous in immunity against pathogens, however chronic stimulation of this pathway could be deleterious for CD4 T cell homeostasis and may contribute to the aberrant immune activation and eventual CD4 T cell depletion observed during HIV infection.

To maintain the size of T cell pools throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 (through lymphopenia-induced proliferation/LIP) can also mediate expansion of autoreactive clones leading to the onset of autoimmune disease or enhance effector function against tumors and viruses. In the present study, we hypothesized that in a setting of chronic inflammatory environment, such as HIV infection, LIP could contribute to the immune activation leading to CD4 T cell depletion and CD8 T cell expansion. Type-I IFN is an essential cytokine in host defense during viral infection. In HIV infected patients, the genes associated with γc cytokines and Type-I IFN signaling were differentially expressed in CD4 and CD8 T cells. Using a lymphopenic murine model, we present evidence that, in vivo, IL-7 differentially regulated the expression of the total-Signal Transducer and Activator of Transcription 1 (t-STAT1) in CD4 and CD8 T cells undergoing LIP and in so doing enhanced CD4 T cell responsiveness to Type-I IFN. In this setting, chronic treatment with IFN-α led to decreased CD4 T cell counts and CD8 T cell expansion. This interplay between IL-7 and Type-I IFN might be advantageous in immunity against pathogens, however chronic stimulation of this pathway could be deleterious for CD4 T cell homeostasis and may contribute to the aberrant immune activation and eventual CD4 T cell depletion observed during HIV infection.

After acute viral infection a small population of effector CD8 T cells upregulate CD127 expression and develop into long lived resting memory T cells. During chronic infections, such as HIV, there is an accumulation of CD8 T cells with effector/memory-like phenotype. These observations led to the hypothesis that there is a developmental impairment in the long lived resting memory T cells within that setting. The transcription factors RUNX3 and Eomesodermin (EOMES) are important in the development of long lived memory CD8 T cells. We studied the dynamics of CD127 expression, RUNX3 and EOMES in patients with HIV infection and different levels of viremia. Consistent with other reports, CD127 expression was decreased in the memory CD8 T cell subset. The CD8 memory CD127low T cells showed increased expression of RUNX3 and EOMES mRNA, as well as effector molecules perforin and granzyme B. In patients with suppressed viremia, we could identify two populations of CD8 T cells expressing EOMEShigh and EOMESint. The EOMESint cells showed a short-lived effector phenotype (perforin+, granzyme B+, CD127low) and the EOMEShigh cells showed a memory-like phenotype (perforin-, CD127high). These results suggest that in human CD8 T cells, EOMES is necessary for the maintenance of long lived memory CD8 T cells and its expression was influenced by HIV-RNA levels and the associated inflammatory environment (R=0.4, p=0.01). These findings provide new insights into the pathogenesis of HIV infection.

International audience; BACKGROUND: Lymphopenia results in the proliferation and differentiation of naive T cells into memory-like cells in the apparent absence of antigenic stimulation. This response, at least in part due to a greater availability of cytokines, is thought to promote anti-self responses. Although potentially autoreactive memory-like CD8(+) T cells generated in a lymphopenic environment are subject to the mechanisms of peripheral tolerance, they can induce autoimmunity in the presence of antigen-specific memory-like CD4(+) T helper cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we studied the mechanisms underlying CD4 help under lymphopenic conditions in transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that the self-reactivity mediated by the cooperation of memory-like CD8(+) and CD4(+) T cells was not abrogated by CD40L blockade. In contrast, treatment with anti-IL-2 antibodies inhibited the onset of autoimmunity. IL-2 neutralization prevented the CD4-mediated differentiation of memory-like CD8(+) T cells into pathogenic effectors in response to self-antigen cross-presentation. Furthermore, in the absence of helper cells, induction of IL-2 signaling by an IL-2 immune complex was sufficient to promote memory-like CD8(+) T cell self-reactivity. CONCLUSIONS/SIGNIFICANCE: IL-2 mediates the cooperation of memory-like CD4(+) and CD8(+) T cells in the breakdown of cross-tolerance, resulting in effector cytotoxic T lymphocyte differentiation and the induction of autoimmune disease.

To preserve the size and diversity of the T cell repertoire throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 signals through a heterodimer receptor composed of the IL-7Rα and the γc chains. Binding of IL-7 to its receptor leads to activation of STAT5 and induction of target genes implicated in survival and proliferation signals. IL-7/IL-7R signaling can also activate STAT1 and STAT3 but the function of IL-7-dependent activation of these transcription factors is largely unknown. Under lymphopenic conditions, IL-7 modulates the expression levels of total STAT1 (t-STAT1) leading to an IL-7-dependent STAT1 signaling in addition to STAT5. We generated a transgenic mouse that overexpress STAT1 in CD4 and CD8 T cells to understand the contribution of IL-7-dependent STAT1 activation on T cell homeostasis in the setting of lymphopenia. We found that under lymphopenia-induced proliferation T cells from the STAT1 transgenic mouse upregulates t-STAT1 leading to diminish CD4 T cell survival. This mechanism could be a physiological mechanism to regulate the expansion of the CD4 T cell pool under lymphopenic conditions.