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Background Immunogenic tumors frequently elicit a polyclonal T cell response containing a spectrum of T cell receptor (TCR) affinities and avidities. 1 This variation in TCR affinity and avidity results in differences in the antigenic response, kinetics, acquisition of effector phenotypes, and anti-tumor immunity.2–13 T cell states are associated with and enforced by different metabolisms.14–23 However, the metabolic machinery induced by distinct TCR signal strengths, the underlying mechanisms and interplay with milieu, and the impact on T cell phenotype and anti-tumor efficacy is not well understood.Methods We utilized defined antigens, modified versions of these antigens to alter their affinity (altered peptide ligands, APLs) and mice with transgenic expression of the corresponding TCR. We analyzed publicly available RNA sequencing datasets of T cells isolated from draining lymph nodes and tumors. We utilized in vitro cultures with activation of T cells with corresponding native peptide, APLs, anti-CD3/anti-CD28 antibodies, or no stimulation, and evaluated their differentiation and corresponding metabolism with flow cytometry and stable isotope labeling over time.Results RNA Sequencing demonstrated a metabolically active response in tumor-infiltrating CD8+ T cells, with differential metabolic usage based on activating peptide and tissue localization. Stimulation with different activation conditions in vitro demonstrated a spectrum of activation phenotypes based on TCR signal strength, with stronger peptides upregulating CD44 and CD25 faster than weaker peptides, with transient CD69 expression, and ultimately becoming CD44+CD62L+CD25+CD69- with multiple rounds of proliferation by 48 hours post-stimulation (figure 1). In contrast, the weakest peptides had minimal CD44 expression and proliferation, and no CD25 expression. Interestingly, intermediate peptides had a decoupling of activation markers: multiple rounds of proliferation and most of the cells being CD44+CD62L+, but little CD25 expression. Most phenotypes were conserved between TCRs, with some differences in acquisition of CD44 and CD62L expression. Stable isotope labeling with13 C glucose revealed multiple patterns of metabolite accumulation (figure 2): some metabolites are similarly abundant regardless of activation state; others follow a clear accumulation pattern going from high to low signal strength; and a third group shows increased accumulation in sub-optimal but functional TCR stimulation.Conclusions Here we show that differential TCR signal strength elicits distinct metabolic profiles corresponding to the acquisition of some, but not all, effector phenotypes. 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OT-I T cells stimulated for 48 hours with plate-bound antibodies or native or APLs of Ova257-264 SIINFEKL were phenotyped by flow cytometryAbstract 1296 Figure 2Principal component analysis of metabolomics data of OT-I T cells under different activating conditions following 8h of 13C glucose stable isotope labeling starting 24h (left) or 48h (right) post-activation