Supplemental figure 1: 38 BPAR 29 ACR 4 AMR 17 SOC rejection therapy 12 mTORi implementation 10 Banff 2A or 2B received rATG followed by Tac 1 Banff 1A resolved with CCS+rATG 1 Banff 1B resolved with CCS+rATG 2 Banff 1B CCS followed by Tac

Treatment of central nervous system (CNS) autoimmune disorders frequently involves the reduction, or depletion of immune-competent cells. Alternatively, immune cells are being sequestered away from the target organ by interfering with their movement from secondary lymphoid organs, or their migration into tissues. These therapeutic strategies have been successful in multiple sclerosis (MS), the most prevalent autoimmune inflammatory disorder of the CNS. However, many of the agents that are currently approved or in clinical development also have severe potential adverse effects that stem from the very mechanisms that mediate their beneficial effects by interfering with CNS immune surveillance. This review will outline the main cellular components of the innate and adaptive immune system that participate in host defense and maintain immune surveillance of the CNS. Their pathogenic role in MS and its animal model experimental autoimmune encephalomyelitis (EAE) is also discussed. Furthermore, an experimental model is introduced that may assist in evaluating the effect of therapeutic interventions on leukocyte homeostasis and function within the CNS. This model or similar models may become a useful tool in the repertoire of pre-clinical tests of pharmacological agents to better explore their potential for adverse events.

Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35–55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8 + T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8 + T cell clonotypes (CD8 EXP ). Gene expression of CD8 EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8 EXP , independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8 EXP into Jurkat 76 cells (TCR –/– ) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8 EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8 EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8 EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8 + T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

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Immune surveillance of the CNS is critical for preventing infections, however there is no accepted experimental model to assess the risk of infection when utilizing disease-modifying agents. We tested two approved agents for patients with multiple sclerosis (MS), glatiramer acetate and fingolimod, in an experimental model of CNS immune surveillance. C57BL/6 mice were infected with the ME49 strain of the neuroinvasive parasite Toxoplasma gondii (T. gondii) and then treated with GA and fingolimod. Neither treatment affected host survival, however differences were observed in parasite load and in leukocyte numbers in the brains of infected animals. Here we demonstrate that this model could be a useful tool for analyzing immune surveillance.

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