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Background In response to pathogens, CD8 + T cells reprogram their metabolism to fuel a proliferative burst of antigen-specific T cells. Engineering their metabolic programming functionally augments CD8+ T cell responses, yet mechanistic studies understanding the direct effects of T cells are limited due to the complex cellular systems used to study T cell responses. Thus, the direct impact of metabolic programming on T cell phenotype and TCR receptor (TCR) repertoire selection remains unknown. Here, we synthesized nanoparticle-based artificial antigen presentation cells (aAPCs) and used the system as a model of endogenous expansion to stimulate primary CD8+ T cells. We introduced the broad glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), and probed antigen-specific cell phenotype and TCR repertoire as a readouts of metabolic intervention.Methods aAPCs were synthesized by conjugation of PEG-biotin modified H-2K b-Ig fusion proteins loaded with SIYYRGL, SVYDFFVWL, SIINFEKL, or SIIVFEKL and anti-mouse CD28 to 200 nm streptavidin-coated superparamagnetic iron oxide nanoparticles. Endogenous expansion of antigen-specific T cells was performed via negative selection of CD8+ T cells from healthy C57BL/6J mice co-cultured with aAPCs and 10 ng/mL of hIL-2 in the presence of 1 uM of DON or vehicle. Characterization of antigen-specific cells was performed on day 7 post-stimulation. Repertoire analysis was conducted from sorted antigen-specific cells sequenced with Adaptive Biotechnologies’ immunoSEQ platform. In vivo killing assays were assessed by adoptive transfer of antigen-specific T cells into lymphodepleted mice, followed with peptide-pulsed splenocytes as target cells.Results We found that transient glutamine antagonism with DON has a direct effect on epitope-specific T cell phenotype by upregulating memory markers and in a novel fashion function as a ‘clonal filter,’ enriching high-affinity CD8 + T-cell clones without sacrificing expansion or effector differentiation. This was apparent at a clonal level, where DON-treated antigen-specific cells showed distinct repertoire usage compared to vehicle-treated controls. Moreover, repertoire usage was narrowed in DON groups. Finally, the effect of glutamine inhibition skews towards cells with high-affinity TCRs and enhances their long-term ability to kill in vivo.Conclusions Collectively, these findings introduce metabolic blockade as a rapid, non-genetic strategy to pre-select durable, high-affinity T cells. Our research provides insight into how modulating the glutamine axis focuses the resultant CD8 + response using artificial platforms as in vitro models to reveal T cell intrinsic factors. Adoption of artificial antigen presenting platforms could provide a well-controlled method to further study the interplay between T cell metabolism and TCR repertoire to inform strategies for developing new, more effective cellular immunotherapies (figure 1).Ethics Approval All procedures for animal experiments were approved by the Johns Hopkins University’s Animal Care and Use Committee and compliant with the National Institutes of Health guidelines.Abstract 819 Figure 1Graphical abstract