CD4 T cells help CD8 T cells differentiate into competent effector and memory cells. We recently showed that CD4 T cells are essential for CD8 T cell differentiation into brain-resident memory (bTRM) during polyomavirus (PyV) infection. Here we identify IL-21 as the CD4 T cell help. Using MHC-II tetramers for two epitopes in mouse PyV, we show brain PyV-specific CD4 T cells express PD-1 and CXCR5. IL21-VFP reporter mice revealed CD4 T cells as the only cellular source of IL-21 in the brain. 2D micropipette adhesion assays, measuring TCR affinity, showed brain IL-21-producing CD4s have higher affinity TCRs than IL-21− cells. RNAseq analysis of IL-21+ vs. IL-21− brain CD4s corroborates that IL-21 production is positively associated with TCR affinity and that IL-21 producers are positively enriched for TFH signature genes. We further found that IL-21 receptor (IL21R) is required for CD8 TRM differentiation. Brain CD8 T cells upregulated IL21R at 15 days post-infection, coincident with expression of the TRM marker CD103. Few CD8 T cells in brains of IL-21R−/− mice expressed CD103, a finding confirmed using donor anti-PyV CD8 T cells lacking IL-21R in infected wild type (WT) recipients. Unlike IL21R-sufficient CD8s, IL21R−/− CD8 T cells are not maintained in the brain upon systemic CD8 antibody depletion. IL21R−/− CD8s also have a diminished response to a rechallenge in the brain than what we observed with WT CD8 T cells. RNAseq analysis revealed IL21R−/− CD8 T cells were negatively enriched for TRM core signature genes and oxidative metabolism genes compared to IL-21R-sufficient CD8 T cells. Thus, we conclude that IL-21 produced by high-affinity, PyV-specific CD4 T cells in the brain is required for differentiation of CD8 bTRM during PyV CNS infection.

The demyelinating brain disease Progressive Multifocal Leukoencephalopathy (PML), caused by JC polyomavirus (JCV), is a life-threatening complication for patients with HIV/AIDS, hematologic malignancies, rheumatologic diseases, and those receiving long-term immunomodulating therapies. Using mouse polyomavirus (MuPyV), our laboratory developed a robust model of polyomavirus-associated demyelinating leukoencephalitis, complete with viral infection and T cell infiltration. Increasing evidence supports the importance of CD4+ T cells in controlling JCV in the brain and thereby preventing PML. Recent studies in our laboratory show that CD4+ T cells are essential for differentiation and maintenance of MuPyV-specific brain-resident memory CD8+ T cells (bTRM). We find that the help CD4+ T cells are provide to virus-specific CD8+ T cells for their appropriate bTRM differentiation is IL-21. PD-1hiCXCR5hi MuPyV-specific CD4+ T cells are the primary sources of brain IL-21 and co-localize with CD8+ T cells in the brain. Brain MuPyV-specific CD8+ T cells have increased expression of the IL-21 receptor (IL21R) 15 days post-infection, which correlates to the time-point when they begin to express CD103, a canonical TRM marker. TRM maintenance is independent of circulating cells. In line with this property, we show that MuPyV-specific CD8+ T cells are not depleted by parentally administered anti-CD8. In addition, brain CD8+ T cells from IL21R−/ − mice have decreased expression of CD103 and are susceptible to depletion from peripheral anti-CD8. These data indicate that during polyomavirus infection, IL-21 by CD4+ T cells drives CD8+ T cells to differentiate into tissue resident memory in the brain.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating brain disease caused by JC polyomavirus (JCPyV). Most individuals are asymptomatically infected with JCPyV, but those with immunological perturbations are at risk of developing PML. PML pathogenesis is closely linked with the emergence of JCPyV mutants bearing mutations in regions of the VP1 major capsid protein that bind host cell receptors; these mutations may mediate immune escape or result in altered cell tropism/neurovirulence. A frequent PML mutation in VP1, S269F, is located in the receptor-binding pocket of the virus capsid. To investigate the effects of the S269F mutation on viral infection and the immune response, we utilized mouse polyomavirus (MuPyV) as a natural pathogen-host model of JCPyV infection. We generated a MuPyV mutant containing a V296F substitution in VP1, which maps to the S269F mutation in JCPyV, to ask whether this mutation on immune evasion and host cell tropism. The MuPyV DNA genome incorporates cellular histones; thus we generated infectious fluorescent MuPyV by infecting a host cell line expressing an H2B histone-GFP protein. Using this fluorescently-labeled virus, we determined that a VP1 monoclonal antibody (mAb), which neutralizes wild type virus, binds the V296F mutant, but is unable to block viral attachment and infection. We are currently generating cryo-EM reconstructions to visualize the interaction of this mAb with the wild type and V296F mutant viruses. This data suggests that this PML-synonymous mutation in MuPyV may allow escape from antiviral humoral immunity.