Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection. Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr) but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection, and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner, and is associated with effective parasite control during chronic stage. Conditional invalidation of TAP-mediated MHC class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulate the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections. Overall design: For 3' scRNA-seq: Facs-sorted CD103-negative vs. CD103-positive CD8+ Kb-OVA dex+ T cells from the different biological conditions were obtained in two independent experiments. One experiment comprised an encephalitis (Tg.SAG1-OVA-infected C57BL/6) and latency (Tg.GRA6-OVA-infected C57BL/6) group analyzed at d52pi, and one comprised only the latency group analyzed at d160pi. Single-cell libraries were generated immediately after Facs-sorting using Chromium Single Cell 3’ Library & Gel Bead Kit v3 according to the manufacturer’s protocol (10X Genomics). For CITE-seq: CITE-seq dataset is derived from one experiment in which B6.Ld mice were infected with Tg.GRA6-OVA and analyzed at d46pi. Brain-isolated CD8+ T cells were pooled from 4 mice and labeled with TotalSeqC CD3 and CD8 antibodies (Biolegend), and with PE-coupled dCODE Kb-OVA, Ld-GRA6 and Kb-SIYRYYGL control dextramers, to enable retrospective identification of OVA-specific and GRA6-specific cells among total CD8+ T cells. dCODE dextramer-positive CD8+ T cells were enriched by 2 successive magnetic sorting steps. Single-cell libraries were generated immediately with Chromium Next GEM Single Cell 5' Kit v2 and 5' Feature Barcode Kit according to the manufacturer’s protocol (10X Genomics).