e22055 Background: Immune checkpoint inhibitors (ICI) targeting checkpoints has achieved cures in half of stage IV melanoma patients yet there remains a critical need to identify why other half remain either ICI resistant or relapse. Recent studies show that multiple subpopulations of tumor-infiltrating CD8⁺PD-1⁺ T cells, previously known to be exhausted, are in fact metabolically active. We have identified a sub population of CD8⁺PD-1⁺ T cells resistant melanoma that are metabolically active, highly proliferative; and has high glutamine metabolic pathway. Rapidly proliferating cells utilize the glucose-lactic acid pathway for faster energy production and can metabolize glutamine for energy. A recent study showed that glutamine metabolism blockade improves immunotherapy response by enhancing cytotoxic T cell function. However, the role of glutamine in dysfunctional CD8⁺T cells has not be studied. Methods: Using patient-derived ICI resistant tumors (n = 8), tumor infiltrating lymphocytes (TILs) were isolated and sorted for total CD8⁺ T cells for single cell RNA sequencing, and then into CD8⁺PD-1⁺TIM-3⁺ T cells for bulk RNA analysis. FACS analysis was performed to assess the protein expression of SLC1A5 (a major glutamine transporter) and ki67. Seahorse Fuel Flex Test was employed to examine oxidative phosphorylation and mass spectrometry analysis was performed on sorted CD8⁺PD-1⁺TIM-3 TILs to assess the glutamine and lactic acid content. Results: A volcanic plot from bulk RNAseq data demonstrated an increase in glutamine pathway activity as well as the expression of SLC1A5 in CD8⁺PD-1⁺TIM3⁺ T cells compared to the peripheral CD8⁺ T cells. Similarly, scRNAseq analysis showed an increased expression of ki67 and SLC1A5 in MAD CD8⁺ T cells compared to the effector and naïve CD8⁺ T cells. FACS analysis of TILs gated for CD3⁺CD8⁺,showed an increased protein expression of SLC1A5 and ki67 in the PD-1⁺TIM-3⁺ population. A seahorse assay using sorted CD8⁺PD-1⁺ TIM3⁺ TILs from human samples confirmed an increased oxidative consumption rate (OCR) and increased dependency of glutamine metabolism in MAD CD8⁺ T cells compared to healthy CD8⁺ T cells. Importantly, mass spectrometry of CD8⁺PD-1⁺ TIM3⁺ T cells showed a significant increase in glutamine and lactate supporting our hypothesis that MAD CD8⁺ T cells have increased glutamine metabolism due to anaerobic glycolysis. Conclusions: Glutamine is a major metabolic source for rapidly proliferating CD8⁺PD-1⁺TIM-3⁺ dysfunctional T cells and inhibition of glutamine metabolism can be leveraged as immunotherapy in ICI resistant melanoma.