Id2 expression level determines the development of effector vs. exhausted tissue-resident memory CD8+ T cells during CNS chronic infection

Tissue-resident memory T cells (Trm) play a central role in regional immunity within non lymphoid tissues to efficiently protect against reinfection as well as to control persistent pathogen infection. Although single-cell transcriptomics analysis of Trm in various diseases have uncovered a phenotypic and functional heterogeneity of Trm, the underlying molecular mechanisms directing such diversity are poorly defined. To better understand these mechanisms, we used the model of infection with the parasite Toxoplasma gondii (T. gondii), which establishes a persistent infection in the central nervous system (CNS) controlled by brain CD8+ Trm. Through single-cell transcriptomic analysis of brain CD8+ T cells from T.gondii-infected mice, we uncovered that brain Trm subsets have an heterogenous expression of the transcriptional regulator Id2, and that these differences in Id2 levels correlated with different functional states. Using mixed bone marrow chimeras, our results revealed that T-cell specific Id2-deficiency caused parasite-specific Trm to develop an aberrant phenotype in a cell-intrinsic manner, characterized by diminished effector functions and reduced expression of the tissue retention molecules CD49a and CXCR6. This phenotype was similarly observed upon overexpression of E2A, a transcription factor negatively regulated by Id2, in parasite-specific CD8+ T cells. Conversely, the loss of Id2 in brain-infiltrating CD8+ T cells led to the accumulation of exhausted PD1+ Tox+ CD8+ Trm cells, whereas Id2 overexpression repressed the acquisition of the exhaustion phenotype and promoted the accumulation of pathogen-specific CD8+ T cells in the brain. Overall, our study indicates that Id2 expression level dictates the acquisition of an effector vs. exhausted phenotype by CD8+ Trm during chronic CNS pathogen infection. Overall design: Single-cell RNA-seq dataset (CITE-seq) was obtained from one experiment in which C57BL/6 mice were chronically infected with T.gondii.GRA6-OVA and analyzed at day 154pi. Spleens or brains were pooled from 4 infected mice. Cells from pooled spleens or brains were labeled with TotalSeqC CD3 and CD8 antibodies (Biolegend), and with PE-coupled dCODE Kb-OVA (SIINFEKL) and Kb-SIYRYYGL control dextramer, to enable retrospective identification of OVA-specific cells among total CD8+ T cells. CD8+ T cells were purified using MACS Miltenyi mouse CD8a+ T Cell Isolation kit (Miltenyi Biotec 130-104-075) before proceeding to single-cell emulsion with 15946 splenic CD8+ T cells and 11856 brain CD8+ T cells, respectively. Single-cell libraries were generated with Chromium Next GEM Single Cell 5' Kit v2 and 5' Feature Barcode Kit according to the manufacturer's protocol (10X Genomics). Library size and quality were verified on a Fragment Analyzer (Agilent). Libraries were sequenced on 1 lane of SP flowcell with Illumina NovaSeq6000 sequencer, using paired-end sequencing (2 x 150) and a single 8 bp-long index.