The tumor microenvironment of benign and malignant salivary gland tumors.

BACKGROUND: Treatment of salivary gland tumors (SGTs) remains challenging. Little is known about the immune landscape of SGTs. We aimed to characterize the tumor microenvironment in benign and malignant SGTs. METHODS: Eleven benign and nine malignant tumors were collected from patients undergoing curative intent surgery. Specimens were analyzed using mass cytometry by time-of-flight. Immune cell populations were manually gated, and T cells were clustered using the FlowSOM algorithm. Population frequencies were compared between high-grade and low-grade malignancies, corrected for multiple hypothesis testing. RESULTS: There were trends towards increased CD4+ and CD8+ T cells among malignant tumors. High-grade malignancies exhibited trends towards higher frequencies of CD8+ PD-1+ CD39+ CD103+ exhausted T cells, CD4+ FoxP3+ TCF-1+ CD127- Tregs, and CD69+ CD25- CD4+ T cells compared to low-grade malignancies. CONCLUSION: SGTs exhibit significant immunologic diversity. High-grade malignancies tended to have greater infiltration of exhausted CD8+ T cells and Tregs, which may guide future studies for immunotherapy strategies.

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis.

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR Vα- and Vß-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific Vß and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vß chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.