Multimodal delineation of a layer of effector function among exhausted CD8 T cells in tumors

The anti-tumor function of CD8 T cells is limited through well-established pathways of T cell exhaustion (TEX). Strategies to capture emergent functional states amongst this dominant trajectory of dysfunction are necessary to find pathways to durable anti-tumor immunity. By leveraging transcriptional reporting (by the fluorescent protein TFP) of the T cell activation marker Cd69, related to upstream AP-1 transcription factors, we define a classifier for potent versus sub-optimal CD69+ activation states arising from T cell stimulation. In tumors, this delineation acts an additional functional readout along the TEX differentiation trajectory, within and across TEX subsets, marked by enhanced effector cytokine and granzyme B production. The more potent state remains differentially prominent in a T cell-mediated tumor clearance model, where they also show increased engagement in the microenvironment and are superior in tumor cell killing. Employing multimodal CITE-Seq in human head and neck tumors enables a similar strategy to identify Cd69RNAhiCD69+ cells that also have enhanced functional features in comparison to Cd69RNAloCD69+ cells, again within and across intratumoral CD8 T cell subsets. Refining the contours of the T cell functional landscape in tumors in this way paves the way for the identification of rare exceptional effectors, with imminent relevance to cancer treatment. To identify patterns of gene expression within six broadly defined cell populations, we also performed cell sorting for bulk RNA-sequencing from freshly resected primary human tumors including compartments denoted: 1. “Live”: All viable cells at the time of sorting, 2. “Tconv”: sorted conventional CD4+ and CD8+ T cells, 3. “Treg”: CD25+ CD4+ (enriched for regulatory) T cells, 4. “Myeloid”: Lymphocyte-negative HLA-DR+ (enriched for myeloid) cells, 5. “Stromal”: CD45- CD44+Thy1+ cells, and 6. “Tumor”: all other CD45- cells. We use the bulk RNASeq data from the 'Tconv' compartment for this study *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************