Reversing T cell dysfunction in a novel model of T cell exhaustion reveals differential roles of RASA2.

[INTRODUCTION] T cell exhaustion driven by chronic antigen stimulation limits durable responses to cancer immunotherapy. Using repeated soluble anti-CD3/anti-CD28 stimulation, we established an system that recapitulates hallmark exhaustion features in human CD8 and CD4 T cells, including increased PD-1Tim-3 subsets and loss of IL-2, TNF-α and IFN-γ secretion. We used our platform to explore the role of RASA2 in CD4 versus CD8 T cell exhaustion and assess the feasibility of reversing established exhaustion in T cells.

[METHODS] Primary human T cells underwent six rounds of chronic stimulation to generate exhausted T cells (Tex), while single-stimulated controls (Ts) were rested in IL-2 media. Exhaustion states were assessed by flow cytometry, cytokine profiling, spectral flow cytometry, and scRNA-seq with pseudotime analysis, across timepoints, resting and activation along the exhaustion protocol. CRISPR-Cas9 RNP editing targeting RASA2 was performed either before exhaustion ("blocking") or post exhaustion directly in in vitro generated exhausted T cells ("reversal") across both CD8 and CD4 T cells.

[RESULTS] Chronic stimulation induced robust dysfunction marked by elevated PD-1Tim-3 cells and diminished effector cytokines in both compartments. RASA2 depletion before exhaustion enhanced cytokine and granzyme secretion without altering inhibitory receptor expression. Notably, direct editing of exhausted T cells achieved ~65% RASA2 loss and restored cytokine and granzyme secretion, with CD4 Tex exhibiting greater functional plasticity than CD8 Tex.

[DISCUSSION] This work provides the first demonstration of CRISPR editing directly in generated human exhausted T cells, revealing distinct roles for RASA2 across CD4 and CD8 compartments. This platform enables mechanistic dissection of T cell exhaustion biology with increased throughput and physiological relevance, ultimately supporting the development of novel strategies to overcome cancer immunotherapy resistance.