Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture–based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function. [Display omitted] •Quantitative surface proteomics of primary human T cells•Activation, regulatory T cells, and hypoxia induce bidirectional surfaceome changes•Hypoxia dramatically remodels the primary T cell surface proteome•Both regulatory T cells and hypoxia downregulate nutrient transporter expression The tumor microenvironment (TME) features immunosuppressive regulatory T cells (Tregs) and areas of hypoxia. Given the importance of surface proteins in T cell antitumor function, we performed quantitative cell surface proteomics to determine how these factors affect the primary effector T cell surface proteome (surfaceome). We discovered that Treg coculture and hypoxia reduced expression of nutrient transporters, among other proteins implicated in T cell activation. Together, our findings reveal insights into how these immunosuppressive factors modulate the T cell surfaceome.