Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).
ABSTRACT: INTRODUCTION:Previous work from our laboratory has demonstrated in vivo persistence of CD103+ CD69+ brain resident memory CD8+ T-cells (bTRM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these TRM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-?-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. METHODS:We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8+ T-cells obtained from wild type mice to investigate the role of glial cells in the development of bTRM . RESULTS:In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8+ T-cells promote development of CD103+ CD69+ CD8+ T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8+ T-cells obtained from either WT or PD-1 KO mice. We found that ?CD3 Ab-stimulated CD8+ T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8+ T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69+ CD8+ T-cells, which promotes development of TRM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69+ CD8+ cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69+ CD8+ T-cells. CONCLUSIONS:Taken together, these results demonstrate a role for activated glia in promoting development of bTRM through the PD-1: PD-L1 pathway.
Project description:<h4>Background</h4>Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bT<sub>RM</sub>) following murine cytomegalovirus (MCMV) infection.<h4>Methods</h4>Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8<sup>+</sup> T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bT<sub>RMs</sub>.<h4>Results</h4>In wild-type (WT) animals, we observed a switch in the phenotype of brain-infiltrating CD8<sup>+</sup> T cell populations from KLRG1<sup>+</sup> CD127<sup>-</sup> (SLEC) to KLRG1<sup>-</sup> CD127<sup>+</sup> (MPEC) during transition from acute through chronic phases of infection. At 14 and 30 dpi, the majority of CD8<sup>+</sup> T cells expressed CD127, a marker of memory cells. In contrast, fewer CD8<sup>+</sup> T cells expressed CD127 within brains of infected, PD-L1 knockout (KO) animals. Notably, in WT mice, a large population of CD8<sup>+</sup> T cells was phenotyped as CD103<sup>+</sup> CD69<sup>+</sup>, markers of bT<sub>RM</sub>, and differences were observed in the numbers of these cells when compared to PD-L1 KOs. Immunohistochemical studies revealed that brain-resident CD103<sup>+</sup> bT<sub>RM</sub> cells were localized to the parenchyma. Higher frequencies of CXCR3 were also observed among WT animals in contrast to PD-L1 KOs.<h4>Conclusions</h4>Taken together, our results indicate that bT<sub>RMs</sub> are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population.
Project description:Infection of the central nervous system (CNS) by murine polyomavirus (MuPyV), a persistent natural mouse pathogen, establishes brain-resident memory CD8 T cells (bTRM) that uniformly and chronically express programmed cell death protein 1 (PD-1) irrespective of the expression of ?E integrin CD103, a TRM cell marker. In contrast, memory antiviral CD8 T cells in the spleen are PD-1-, despite viral loads being similar in both the brain and spleen during persistent infection. Repetitive antigen engagement is central to sustained PD-1 expression by T cells in chronic viral infections; however, recent evidence indicates that expression of inhibitory receptors, including PD-1, is part of the TRM differentiation program. Here we asked whether PD-1 expression by CD8 bTRM cells during persistent MuPyV encephalitis is antigen dependent. By transferring MuPyV-specific CD8 bTRM cells into the brains of naive mice and mice infected with cognate epitope-sufficient and -deficient MuPyVs, we demonstrate that antigen and inflammation are dispensable for PD-1 maintenance. In vitro and direct ex vivo analyses indicate that CD103- MuPyV-specific CD8 bTRM retain functional competence. We further show that the Pdcd-1 promoter of anti-MuPyV bTRM cells is epigenetically fixed in a demethylated state in the brain. In contrast, the PD-1 promoter of splenic antiviral memory CD8 T cells undergoes remethylation after being demethylated during acute infection. These data show that PD-1 expression is an intrinsic property of brain TRM cells in a persistent CNS viral infection.
Project description:Although the milestone discovery of immune checkpoint blockade (ICB) has been translated into clinical practice, only a fraction of patients can benefit from it with durable responses and subsequent long-term survival. Here, we tested the anti-tumor effect of combining PD-L1 blockade with 4-1BB costimulation in 3LL and 4T1.2 murine tumor models. Dual treatment induced further tumor regression and enhanced survival in tumor-bearing mice more so than PD-L1 and 4-1BB mAb alone. It was demonstrated that dual anti-PD-L1/anti-4-1BB immunotherapy increased the number of intratumoral CD103+CD8+ T cells and altered their distribution. Phenotypically, CD103+CD8+ T cells expressed a higher level of 4-1BB and PD-1 than their CD103- counterparts. Administration of PD-L1 mAb and 4-1BB mAb further increased the cytolytic capacity of CD103+CD8+ T cells. In vivo, CD103-CD8+ T cells could differentiate into CD103+CD8+ progeny cells. In a human setting, more CD8+ T cells differentiated into CD103+CD8+ T cells in the peripheral tumor region of lung cancer tissues than in the central tumor region. Collectively, infiltrated CD103+CD8+ T cells served as a potential effector T cell population. Combining 4-1BB agonism with PD-L1 blockade could increase tumor-infiltrated CD103+CD8+T cells, thereby facilitating tumor regression.
Project description:Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-? and IFN-?, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.
Project description:Accumulation of CD103+CD8+ resident memory T (TRM) cells in human lung tumors has been associated with a favorable prognosis. However, the contribution of TRM to anti-tumor immunity and to the response to immune checkpoint blockade has not been clearly established. Using quantitative multiplex immunofluorescence on cohorts of non-small cell lung cancer patients treated with anti-PD-(L)1, we show that an increased density of CD103+CD8+ lymphocytes in immunotherapy-naive tumors is associated with greatly improved outcomes. The density of CD103+CD8+ cells increases during immunotherapy in most responder, but not in non-responder, patients. CD103+CD8+ cells co-express CD49a and CD69 and display a molecular profile characterized by the expression of PD-1 and CD39. CD103+CD8+ tumor TRM, but not CD103-CD8+ tumor-infiltrating counterparts, express Aiolos, phosphorylated STAT-3, and IL-17; demonstrate enhanced proliferation and cytotoxicity toward autologous cancer cells; and frequently display oligoclonal expansion of TCR-? clonotypes. These results explain why CD103+CD8+ TRM are associated with better outcomes in anti-PD-(L)1-treated patients.
Project description:HIV-associated neurocognitive disorders (HAND) persist even during effective combination antiretroviral therapy (cART). Although the cause of HAND is unknown, studies link chronic immune activation, neuroinflammation, and cerebrospinal fluid viral escape to disease progression. In this study, we tested the hypothesis that specific, recall immune responses from brain-resident memory T cells (bTRM) could activate glia and induce neurotoxic mediators. To address this question, we developed a heterologous prime-central nervous system (CNS) boost strategy in mice. We observed that the murine brain became populated with long-lived CD8+ bTRM, some being specific for an immunodominant Gag epitope. Recall stimulation using HIV-1 AI9 peptide administered in vivo resulted in microglia displaying elevated levels of major histocompatibility complex class II and programmed death-ligand 1, and demonstrating tissue-wide reactive gliosis. Immunostaining further confirmed this glial activation. Taken together, these results indicate that specific, adaptive recall responses from bTRM can induce reactive gliosis and production of neurotoxic mediators.
Project description:Most tissues are populated by tissue-resident memory T cells (TRM cells), which are adapted to their niche and appear to be indispensable for local protection against pathogens. Here we show that human white matter-derived brain CD8+ T cells can be subsetted into CD103-CD69+ and CD103+CD69+ T cells both with a phenotypic and transcription factor profile consistent with TRM cells. Specifically, CD103 expression in brain CD8+ T cells correlates with reduced expression of differentiation markers, increased expression of tissue-homing chemokine receptors, intermediate and low expression of the transcription factors T-bet and eomes, increased expression of PD-1 and CTLA-4, and low expression of cytolytic enzymes with preserved polyfunctionality upon activation. Brain CD4+ T cells also display TRM cell-associated markers but have low CD103 expression. We conclude that the human brain is surveilled by TRM cells, providing protection against neurotropic virus reactivation, whilst being under tight control of key immune checkpoint molecules.
Project description:Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.
Project description:Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen-specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein-1) or LAG-3 (lymphocyte activation gene-3), and were capable of making IFN-? (interferon-?). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.
Project description:When trying to conceive 1% of couples have recurrent miscarriages, defined as three or more consecutive pregnancy losses. This is not accounted for by the known incidence of chromosomal aneuploidy in miscarriage, and it has been suggested that there is an immunological aetiology. The endometrial mucosa is populated by a variety of immune cells which in addition to providing host pathogen immunity must facilitate pregnancy. Here we characterise the endometrial CD8-T cell population during the embryonic window of implantation and find that the majority of cells are tissue resident memory T cells with high levels of CD69 and CD103 expression, proteins that prevent cells egress. We demonstrate that unexplained recurrent miscarriage is associated with significantly decreased expression of the T-cell co-receptor CD8 and tissue residency marker CD69. These cells differ from those found in control women, with less expression of CD127 indicating a lack of homeostatic cell control through IL-7 signalling. Nevertheless this population is resident in the endometrium of women who have RM, more than three months after the last miscarriage, indicating that the memory CD8-T cell population is altered in RM patients. This is the first evidence of a differing pre-pregnancy phenotype in endometrial immune cells in RM.