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

Hilal Saraç^1*Rachael Nicholson¹Rebecca N. Graham¹Meera Augustus²Dympna Connolly²Lindsay Lim¹Sofia Lourenco^1*

• ¹Cancer Research Horizons, London, United Kingdom
• ²Cancer Research Horizons, Cambridge, United Kingdom

T cell exhaustion, caused by chronic antigen stimulation within the tumor microenvironment, is increasingly recognized as an underlying factor in tumor progression and immunotherapy resistance. Here we established a robust in vitro model of human CD8+ and CD4+ T cell exhaustion using chronic stimulation with soluble anti-CD3/anti-CD28 and performed RNP CRISPR to investigate the ability of RAS GTPase activating protein 2 (RASA2) to modulate exhausted T cell function. RASA2 depletion prior to exhaustion improved effector cytokine and granzyme secretion in both CD8+ and CD4+ T cell compartments. Uniquely, in addition to blocking the onset of exhaustion, we aimed to reverse this dysfunctional phenotype by performing CRISPR knockout (KO) in exhausted T cells (Tex) following chronic stimulation. RASA2 depletion also improved the effector function of exhausted T cells in this context, albeit to a lesser extent compared to a blocking context. Interestingly, the impact of RASA2 depletion on secreted effector proteins was distinct between CD8+ and CD4+ T cells, suggesting different roles for RASA2 in modulating the differentiation pathways to exhaustion along with reversibility of dysfunctional states between these two compartments. This novel in vitro pipeline enables the interrogation of T cell exhaustion biology with increased throughput and physiological relevance compared to other approaches, ultimately supporting the development of novel strategies to overcome cancer immunotherapy resistance.