Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation in vivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes.

T-cell receptor (TCR) binding strength to peptide-MHC antigen complex influences numerous T-cell functions. However, the vast diversity of a polyclonal T-cell repertoire for even a single antigen greatly increases the complexity of studying the impact of TCR affinity on T-cell function. Here, we determined how TCR binding strength affected the protein and transcriptional profile of an endogenous, polyclonal T-cell response to a known tumor-associated antigen (TAA) within the tumor microenvironment (TME). We confirmed that the staining intensity by flow cytometry and the counts by sequencing from MHC-tetramer labeling were reliable surrogates for the TCR-peptide-MHC steady-state binding affinity. We further demonstrated by single-cell RNA sequencing that tumor-infiltrating lymphocytes (TIL) with high and low binding affinity for a TAA can differentiate into cells with many antigen-specific transcriptional profiles within an established TME. However, more progenitor-like phenotypes were significantly biased towards lower affinity T cells, and proliferating phenotypes showed significant bias towards high-affinity TILs. In addition, we found that higher affinity T cells advanced more rapidly to terminal phases of T-cell exhaustion and exhibited better tumor control. We confirmed the polyclonal TIL results using a TCR transgenic mouse possessing a single low-affinity TCR targeting the same TAA. These T cells maintained a progenitor-exhausted phenotype and exhibited impaired tumor control. We propose that high-affinity TCR interactions drive T-cell fate decisions more rapidly than low-affinity interactions and that these cells differentiate faster. These findings illustrate divergent forms of T-cell dysfunction based on TCR affinity which may impact TIL therapies and antitumor responses.

Antigens Expressed by Breast Cancer Cells Undergoing EMT Stimulate Cytotoxic CD8+ T Cell Immunity.

Antigenic differences formed by alterations in gene expression and alternative splicing are predicted in breast cancer cells undergoing epithelial to mesenchymal transition (EMT) and the reverse plasticity known as MET. How these antigenic differences impact immune interactions and the degree to which they can be exploited to enhance immune responses against mesenchymal cells is not fully understood. We utilized a master microRNA regulator of EMT to alter mesenchymal-like EO771 mammary carcinoma cells to a more epithelial phenotype. A computational approach was used to identify neoantigens derived from the resultant differentially expressed somatic variants (SNV) and alternative splicing events (neojunctions). Using whole cell vaccines and peptide-based vaccines, we find superior cytotoxicity against the more-epithelial cells and explore the potential of neojunction-derived antigens to elicit T cell responses through experiments designed to validate the computationally predicted neoantigens. Overall, results identify EMT-associated splicing factors common to both mouse and human breast cancer cells as well as immunogenic SNV- and neojunction-derived neoantigens in mammary carcinoma cells.

Granzymes: The Molecular Executors of Immune-Mediated Cytotoxicity.

Cytotoxic T lymphocytes, differentiated CD8+ T cells, use multiple mechanisms to mediate their function, including release of granules containing perforin and granzymes at target cells. Granzymes are a family of cytotoxic proteases that each act on unique sets of biological substrates within target cells, usually to induce cell death. Granzymes are differentially expressed within T cells, depending on their environment and activation state, making the granzyme cytotoxic pathway dynamic and responsive to individual circumstances. In this review, we describe what is currently known about granzyme structure, processing, and granzyme-induced cell death in the context of cancer and in some other inflammatory diseases.