Date of Award

January 2016

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Richard L. Edelson

Abstract

Individual cancers, even of the same cell type, express unique arrays of distinctive tumor antigens, requiring accurate laboratory measurement of induced immunity against them problematic. Fluorescently tagged reagents (dextramers) that selectively engage clonal T-cell receptors (TCR) can cytofluorographically quantify both frequency and avidity antigen-specific T-cells, but cannot be synthesized without prior identification of the relevant antigen. Since clinically evident tumors may contain as many as 300 unique point mutations capable of generating a large number of uniquely antigenic proteins, and since procurement of such information for each cancer is currently unrealistic, it is presently only possible to assess responses to anti-cancer immunotherapy by clinical determination of estimation of the tumor burden capacity. Therefore, there is a need to develop methodology that can quantify the collective anti-tumor T-cell response, without prior identification of the full array of expressed tumor antigens. We have developed a practical high-resolution method to measure antigen-specific CD8 T-cell responses, via T-cell proton extrusion, an immediate result of selective TCR engagement by antigen presenting cells. The fluorescent emission characteristics of hydroxypyrene trisulfonate (HPTS) correlate with solution-phase proton concentrations, manifesting as increased emission signals. We exploit this TCR characteristic within the context of T-cell activation and show that stimulation with anti-CD3 immunoglobulin stimulates measureable TCR to release protons to a significantly higher degree than unengaged TCR (p<0.001), both mouse and human systems. Specific mouse CD8 T-cell responses to an exogenous tumor antigen (the eight amino acid derivative SIINFEKL of transfected ovalbumin) and human CD8 T-cell responses to a melanoma-associate tissue antigen (MART-1) differed from control (p<0.001). Human CD8 T-cell responses to MART-1 peptide, presented by dendritic antigen presenting cells (DC) could be similarly distinguished from that to control peptide (gp100), even when the frequency of MART-1 responsive CD8 T-cells was titrated down from 23 percent to 1% (p<0.001), as measured in parallel by dextramers. When implemented in a tumor-responsive animal model, treatment groups showed higher emission intensities compared to control groups at time points. These preliminary results confirm the practicality of real-time assessment of antigen-specific TCR engagement, by proton release, a methodology which may become applicable to T-cell responses to any collective group of antigens.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 12/31/2019

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