Granzymes are a class of cytotoxic proteases and are the primary mechanism utilized by T cells to directly eliminate cancer cells. Each granzyme acts upon a unique set of substrates in target cells to induce cytotoxicity through a range of different mechanisms. Granzymes are some of the most differentially regulated genes in CD8+ tumor infiltrating lymphocytes, relative to T cells outside of the tumor microenvironment (TME). We and others have determined by microarray and qPCR that granzyme F is highly upregulated in the TME. Granzyme F expression is restricted to a small subset of antigen-experienced and exhausted T cells, as determined by flow cytometry-based detection of granzyme F RNA transcripts and may represent a marker of a unique T cell cytotoxic function. Single cell RNA sequencing of CD8 TIL has revealed that granzyme F-high expressing cells are unique from both granzyme A and B expressing cells, and that it is therefore likely these TIL utilize a unique mechanism of cytotoxicity in their elimination of cancer cells. Recombinant granzyme F has previously been shown to induce a unique form of cell death, characterized as being caspase-independent and resulting in rupture of target cell plasma membrane. By over expressing granzyme F we are determining if this mechanism of cell death is leverageable to improve the cytotoxic capacity of TIL, and if induction of different forms of T cell-mediated cytotoxicity can modulate the immunogenicity of the TME. These experiments are designed to provide insight into how to improve adoptive T cell therapies by directly improving cytotoxicity, the terminal step of T cell interaction with tumor cells. This work was supported by the National Institutes of Health NIAID training grant (Training Program in Immunology; T32-AI07405) award to Zachary Hay
Granzymes are a class of proteases produced by CD8 T cells to mediate their cytotoxic activity against target cells. The objective of this project is to determine the mechanism through which CD8 T cells are executing their granzyme-mediated cytotoxic function and how the tumor microenvironment (TME) impacts the specific granzymes produced. By micro-array and qPCR we determined that CD8 T cells in the TME have altered expression of specific granzymes. Granzyme F had one of the most altered expressions of all genes examined in antigen-specific CD8 T cells taken from a spleen relative to those taken from a tumor. We further examined the effects of the TME on granzyme expression by sorting tumor infiltrating CD8 T cells on exhaustion markers and found a relationship between exhaustion and granzyme expression, characterized by a decrease in granzyme A and an increase in granzyme B mRNA expression. We will determine the influence different ex vivo culture conditions have on differential granzyme expression and function of CD8 T cells. The expression of different granzymes may have further implications for modulation of the TME through the form of cancer cell death induced, especially the increase in granzyme F which has been suggested to induce a more immunogenic form of cell death. Additionally, the impact of exhaustion on the cytotoxic activity of CD8 T cells from the TME may represent a means for exhausted CD8 T cells to regulate their cytotoxic activity by altering the granzymes they express.
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