Resident Physician University of Rochester Medical Center
Introduction: Urothelial carcinoma (UC) is commonly treated with therapeutics that target the immune system, such as checkpoint immunotherapy or Bacillus Calmette-Guerin (BCG). While assessment of T cell function is of utility in predicting response to these therapies, current methods for evaluating systemic T cell immunity or intratumoral immune behavior are limited. As antigenically activated T cells produce extracellular vesicles (EVs) carrying markers of cell subtype and activation state and these are found in the blood, we hypothesized that serum T cell derived EVs could be used to interrogate the systemic antitumor T cell response. The purpose of this study was to evaluate a novel microfluidic strategy ‘catch and display for liquid biopsy’ [CAD-LB] for the rapid assessment of T cell biomarkers on individual EVs from T cell conditioned medium or blood. Methods: T cell derived EVs from cultured cells. We hypothesized that T cell derived EVs have markers of T cell function that mirror their cell of origin. To test this, we assessed markers of activation and exhaustion on EVs secreted by T cells. Activation and exhaustion were induced in vitro by stimulation with anti-CD3/CD28 beads, mimicking the in vivo stimulation of T cells in an antigen-rich tumor environment. T cell subtype and activation state were assessed by CAD-LB and flow cytometry. T cell markers on serum EVs. We developed an affinity purification method for CD3+ EVs and subsequently evaluated the level of T cell biomarkers on these using CAD-LB in UC specimens from patients. Results: 1. T cell marker expression on EVs from conditioned medium from resting, activated and exhausted cells followed the pattern present on the EV producing T cells. 2. T cell derived EVs from UC patient serum identified markers of activated and functionally exhausted cells, indicating that we can capture the full range of T cell functionality with CAD-LB. 3. Detected T cell EVs with markers of non-circulating tissue-resident cells (present in tumor, secondary lymphoid and peripheral tissue) demonstrate that EVs capture the systemic immune responses. Conclusions: T cell derived EVs capture the phenotype and identity of their cell of origin. CAD-LB detects a range of T cell populations in serum. Future work will test if T cell EVs capture tumor and systemic antitumor immunity, which may help identify UC patients more likely to respond to immunotherapy. SOURCE OF Funding: None.
