Pathogenesis of immune-mediated demyelinating diseases like multiple sclerosis (MS) is thought to be governed by a complex cellular interplay between immunopathogenic and immunoregulatory responses. We have previously shown that central nervous system (CNS)-specific CD8 T cells have an unexpected protective role in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). In this study, we interrogated the suppressive potential of PLP178-191-specific CD8 T cells (PLP-CD8). Here, we show that PLP-CD8, when administered post-disease onset, rapidly ameliorated EAE progression, and suppressed PLP178-191-specific CD4 T cell responses as measured by delayed-type hypersensitivity (DTH). To accomplish DTH suppression, PLP-CD8 required differential production of perforin and IFNγ. Perforin was not required for the rapid suppressive action of these cells, but was critical for maintenance of optimal longer term DTH suppression. Conversely, IFNγ production by PLP-CD8 was necessary for swift DTH suppression, but was less significant for maintenance of longer term suppression. These data indicate that CNS-specific CD8 T cells employ an ordered regulatory mechanism program over a number of days in vivo during demyelinating disease and have mechanistic implications for this immunotherapeutic approach.

CD8 T-cells predominate in CNS lesions of MS patients and display oligoclonal expansion. However, the role of myelin-specific CD8 T-cells in disease remains unclear, with studies showing protective and pathogenic roles in EAE. We demonstrated a disease-suppressive function for CNS-specific CD8 T-cells in a model where the antigen is exogenously administered in vivo and used for in vitro activation. To probe the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo, we developed a novel approach utilizing infection with Listeria monocytogenes (LM) encoding proteolipid protein peptide (PLP) amino acids 178-191 (LM-PLP). LM-PLP infection preferentially induced PLP-specific CD8 T-cell responses. Despite the induction of PLP-specific CD8 T-cells, LM-PLP infection did not result in disease. In fact, LM-PLP infection resulted in significant amelioration of PLP178-191-induced EAE. Disease suppression was not observed in mice deficient in CD8 T-cells, IFN-γ or perforin. DTH responses and CNS infiltration were reduced in protected mice, and their CD4 T-cells had reduced capacity to induce tissue inflammation. Importantly, infection with LM-PLP ameliorated established disease. Our studies indicate that CD8 T-cells induced by endogenous presentation of PLP178-191 attenuate CNS autoimmunity in models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

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Autoimmune diseases are characterized by regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that CD8+ T-cells associated with acute relapse of multiple sclerosis are significantly deficient in their immune suppressive ability. We hypothesized that distinct CD8+ cytotoxic T-cell (Tc) lineages, determined by cytokine milieu during naïve T-cell differentiation, may harbor differential ability to suppress effector CD4+ T-cells. We differentiated purified human naïve CD8+ T-cells in vitro toward Tc0 (media control), Tc1 and Tc17 lineages. Using in vitro flow cytometric suppression assays, we observed that Tc0 and Tc17 cells had similar suppressive ability. In contrast, Tc1 cells showed significant loss of suppressive ability against ex vivo CD4+ T-cells and in vitrodifferentiated Th0, Th1 and Th17 cells. Of note, Tc1 cells were also suboptimal in suppressing CD4-induced acute xenogeneic graft versus host disease (xGVHD) in vivo. Tc subtypes derived under various cytokine combinations revealed that IL-12-containing conditions resulted in less suppressive cells exhibiting dysregulated cytotoxic degranulation. RNA sequencing transcriptome analyses indicated differential regulation of inflammatory genes and enrichment in GM-CSF-associated pathways. These studies provide insights into the role of T-cell differentiation in CD8 suppressive biology and may reveal therapeutically targetable pathways to reverse suppressive deficit during immunemediated disease.

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that CNS myelinspecific CD8 T cells unexpectedly harbor immune regulatory capacity in both mouse and human. In particular, PLP 178-191 -specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis. We have recently shown that this depends on PLP-CD8 elaborating IFN-g and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell-derived IFN-g remains poorly understood. In this study, we show that although wild-type (WT) PLP-CD8 were robustly suppressive in IFN-gR-deficient mice, IFN-gR-deficient PLP-CD8 exhibited suboptimal suppression in WT mice. Compared with WT counterparts, IFN-gR-deficient PLP-CD8 were defective in suppressing disease in IFN-g-deficient recipients, a scenario in which the only IFN-g available to WT PLP-CD8 is that which they produce themselves. Further, we found that IFN-gR-deficient PLP-CD8 exhibited altered granzyme/IFN-g profiles, altered migration in recipients, and deficits in killing capacity in vivo. Collectively, this work suggests that IFN-g responsiveness allows myelin-specific CD8 T cells to optimally perform autoregulatory function in vivo. These insights may help elucidate future adoptive immunotherapeutic approaches for MS patients.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNSspecific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP 178-191 CD8 T cells suppress disease, whereas PLP 139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP 178-191 CD8 T cells versus nonregulatory PLP 139-151 or OVA 323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3 + subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-g and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP 178-191 CD8 T cells but not within nonregulatory OVA 323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.

Factors regulating self-antigen directed immune-responses in autoimmunity are poorly understood. Signal regulatory protein gamma (SIRPγ) is a human T-cell specific protein with genetic variants associated with type 1 diabetes (T1D). SIRPγ's function in the immune system remains unclear. We show that T1D and relapsing remitting multiple sclerosis (RRMS) subjects have significantly greater frequency of rs2281808 T genetic variant, that correlates with reduced SIRPγ-expression in T-cells. Importantly, reduced SIRPγ-expression in RRMS and T1D subjects was not restricted to T variant, suggesting SIRPγ-expression is also regulated by disease specific factors in autoimmunity. Interestingly, increased frequencies of SIRPγ low T-cells in RRMS and T1D positively correlated with proinflammatory molecules from T-cells. Finally, we show that SIRPγ low T-cells have enhanced pathogenecity in vivo in a GVHD model. These findings suggest that decreased-SIRPγ expression, either determined by genetic variants or through peripherally acquired processes, may have a mechanistic link to autoimmunity through induction of hyperactive T-cells.

Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the pathogenic and regulatory roles these cells play in demyelinating disease. Dissecting the function of B cells in the MS mouse model experimental autoimmune encephalomyelitis (EAE) is largely confined to induction with either the myelin oligodendrocyte glycoprotein epitope MOG35-55 or the fulllength recombinant human MOG protein, the latter representing the most-used B cell-dependent EAE model. There is a clear need to investigate B cell function in additional myelin antigen contexts. Unlike MOG 35-55 , where lack of B cells yields more severe disease, we show here that the immunodominant myelin proteolipid protein epitope (PLP 178-191 ) elicited identical EAE in WT and μMT mice, suggesting an absence of B cell engagement by this peptide. We hypothesized that a longer PLP antigen may better engage B cells and designed a peptide encompassing the extracellular domains (ECD) of PLP. We demonstrate here that PLP ECD -immunized B cell-deficient mice failed to exhibit EAE. In contrast, PLP ECD induced EAE not only in WT mice, but in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B celldependent eAe model.