Interconnected lineage trajectories link conventional and NK-like exhausted CD8+ T cells beneficial in T1D [P452_3]

While distinct NK-like CD57+ and PD-1+ CD8+ exhausted T cell populations (Tex) were both linked to beneficial immunotherapy response in autoimmune Type 1 Diabetes (T1D) patients, relationships between these cell types are poorly understood. We show that PD-1+ and CD57+ Tex populations in this context were epigenetically similar, but CD57+ Tex cells displayed unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also showed reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex showed unappreciated gene expression heterogeneity and shared clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment. Overall design: We performed bulk Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) on PBMC samples selected from 4 donors with new onset T1D that were responders to alefacept (T1DAL Trial, ITN). Donors were sampled at baseline (Week 0/Visit 0) and 104 wk (Visit 30) post-treatment with alefacept. PBMC samples were stained with a flow cytometry panel (Table S4) and sorted with the FACS Aria II (Becton Dickinson) cell sorter into 3 subsets of non-naïve CD8+ T cells (singlet, live, CD14- CD19- CD56- CD3+ CD8+ CD4-, not CD45RA+ CCR7+): (1) Double Negative non-Tex (DN, KLRG1- TIGIT-), (2) CD57+ Tex (KLRG1+TIGIT+, CD57+), and (3) CD57- Tex (KLRG1+TIGIT+, CD57-). These subsets were compared for downstream differential accesibility analysis.