In chronic inflammatory diseases of the central nervous system (CNS), immune cells persisting behind the blood-brain barrier are supposed to promulgate local tissue destruction. The drivers of such compartmentalized inflammation remain unclear, but tissue-resident memory T cells (T RM ) represent a potentially important cellular player in this process. Here, we investigated whether resting CD8 + T RM persisting after cleared infection with attenuated lymphocytic choriomeningitis virus (LCMV) can initiate immune responses directed against cognate self-antigen in the CNS. We demonstrated that time-delayed conditional expression of the LCMV glycoprotein as neo-self-antigen by glia cells reactivated CD8 + T RM . Subsequently, CD8 + T RM expanded and initiated CNS inflammation and immunopathology in an organ-autonomous manner independently of circulating CD8 + T cells. However, in the absence of CD4 + T cells, TCF-1 + CD8 + T RM failed to expand and differentiate into terminal effectors. Similarly, in human demyelinating CNS autoimmune lesions, we found CD8 + T cells expressing TCF-1 that predominantly exhibited a T RM -like phenotype. Together, our study provides evidence for CD8 + T RM -driven CNS immunopathology and sheds light on why inflammatory processes may evade current immunomodulatory treatments in chronic autoimmune CNS conditions.

The mechanisms underlying the chronicity of autoimmune diseases of the central nervous system (CNS) are largely unknown. In particular, it is unclear whether tissue-resident memory T cells (T RM ) contribute to lesion pathogenesis during chronic CNS autoimmunity. Here, we observed that a high frequency of brain-infiltrating CD8 + T cells exhibit a T RM -like phenotype in human autoimmune encephalitis. Using mouse models of neuronal autoimmunity and a combination of T single-cell transcriptomics, high-dimensional flow cytometry, and histopathology, we found that pathogenic CD8 + T cells behind the blood-brain barrier adopt a characteristic T RM differentiation program, and we revealed their phenotypic and functional heterogeneity. In the diseased CNS, autoreactive tissue-resident CD8 + T cells sustained focal neuroinflammation and progressive loss of neurons, independently of recirculating CD8 + T cells. Consistently, a large fraction of autoreactive tissue-resident CD8 + T cells exhibited proliferative potential as well as proinflammatory and cytotoxic properties. Persistence of tissue-resident CD8 + T cells in the CNS and their functional output, but not their initial differentiation, were crucially dependent on CD4 + T cells. Collectively, our results point to tissue-resident CD8 + T cells as essential drivers of chronic CNS autoimmunity and suggest that therapies targeting this compartmentalized autoreactive T cell subset might be effective for treating CNS autoimmune diseases.

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy-and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies.

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CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to less expanded clones. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.