Reversing T cell dysfunction in a novel in vitro 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 in vitro system that recapitulates hallmark exhaustion features in human CD8 + and CD4 + T cells, including increased PD-1 + Tim-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-1 + Tim-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 in vitro 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.