Heterogeneity within Antigen-Specific T Cell Responses Revealed by Differential Dynamics of TCR Downregulation.
Authors: Kohrt Holbrook E. , Shu Chen-Tsen , Holmes Susan P. , Weber Jeffrey , Lee Peter P. 
Published: October 2019

Abstract
Cytomegalovirus (CMV) is a known cause of significant morbidity, including CMV pneumonitis, among bone marrow transplant patients. Studies investigating the immune response to CMV pneumonitis within bronchoalveolar lavage samples illustrate a predominance of CD8 T cells. However, CD8 T cell infiltration does not correlate with clinical response. Likewise, cancer vaccine trials have failed to show a correlation between induction of antigen-specific CD8 T cell populations and clinical tumor response. Based on the recent finding that T cells downregulate the T cell receptor (TCR) upon activation, we demonstrate by stimulating antigen-specific T cells with graded amounts of cognate peptides that individual cells lose tetramer reactivity with different dynamics which reflects previously uncharacterized heterogeneity within the population. We analyzed response among virus-, CMV PP65 and EBV BMLF1, and tumor associated antigen- (TAA), MART and gp100, specific CD8 T cell clones and patient samples. Critical T cell response characteristics including sensitivity to antigen, recognition efficiency (RE, also referred to as functional avidity), and relative functional state were calculated based on TCR downregulation. We show TCR downregulation represents an accurate assessment of average T cell function with direct correlation to killing capacity by chromium release (r2 of.907, P<0.01) and degranulation by CD107 mobilization (r2 of.99, P=0.016). However, despite correlation of average T cell function, we found heterogeneity in T cell sensitivity and relative functional status not evident by chromium release or CD107 mobilization alone. TCR downregulation among viral clones with homogeneous tetramer+ populations appeared similar, however one CMV-specific clone was unique in lower RE (7.18, group mean 7.32) and poor functional status (29.6% of tetramer+ CD8 T cells failed to downregulate at supraphysiologic concentrations of stimulating peptide, group mean 9.1%) despite the highest sensitivity (11.6, group mean 8.9). Among CMV-specific patient samples, size of tetramer+ population was not correlated with CD8 T cell sensitivity or RE. Patient 10539 with the largest tetramer+ population (1.8%, group range 0.5–1.8%), demonstrated the lowest sensitivity (10.2, group range 10.2–12.3) and only average RE (9.2, group range 7.8–10.2). TAA-specific CD8 T cell clones and patient samples post-vaccination with heteroclitic peptide contained significant heterogeneity in response to native and heteroclitic peptides. Despite equivalent percent tetramer+ populations, gp100-specific patient samples were significantly less sensitive (8.9 vs 10.8) with lower RE (7.3 vs 9.2) and greater percent relatively nonfunctional (38% vs 2%) to native versus heteroclitic peptide. These findings suggest failure of CD8 T cells specific for CMV or tumor antigens to produce a clinically significant response may be due to heterogeneity in the T cell population. Specifically, in states of low antigen expression, such as chronic viral infection and cancer, CD8 T cells of high sensitivity and RE with adequate functional status may be required for competent in vivo immune response. It is thus important to understand and appreciate the heterogeneity which may exist within antigen-specific T cell populations, and its clinical implications, to better predict efficacy of the immune response to cancer vaccination or following bone marrow transplant.