Multimodal delineation of a layer of effector function among exhausted CD8 T cells in tumors [scRNA-seq]

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. Overall design: Adoptively transferred CD45.1; OT-I; Cd69-TFP CD8 T cells were sorted from B78chOVA tumors d12 post transfer into four populations based on the CD69:TFP quadrants (Q1: TFP+/CD69-, Q2: TFP+/CD69+, Q3: TFP-/CD69-, and Q4: TFP-/CD69+). Cells were sorted from 8 tumors grown in separate mice and pooled by quadrants for barcoding and analysis. Sorted cells were separately labeled with lipid and cholesterol-modified oligonucleotides (LMO's) according to McGinnis et. al. Following 2 washes with PBS + 0.1% BSA, cells were pooled for encapsulation in one lane of a 10X 3' v3 kit with a target cell number of 18,000.