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.

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 TCR affinity has 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 flow and CITE-Seq counts of 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 (TILs) 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. Additionally, we found in a progressing TME 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.

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.

<div>Abstract<p>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.</p></div>

<p>Intra cluster differential gene expression between Tet-High and Tet-Low cells</p>