Rapid assessment of recognition efficiency and functional capacity of antigen-specific T-cell responses.

It is increasingly recognized that cells within an antigen-specific CD8 T-cell population may be diverse in recognition efficiency for target, which may significantly affect the overall efficacy of the response in clinical settings such as viral infections and cancer. CD8 T cells with seemingly identical antigen specificity, particularly those elicited by cancer vaccines, may be heterogeneous for sensitivity and recognition efficiency for the cognate peptide and functional state in vivo. Analysis of individual T-cell clones derived from an antigen-specific T-cell population would provide an accurate assessment of the overall response; however, this is time- and labor-intensive, preventing rapid and routine assessment of patient samples from clinical trials. By stimulating antigen-specific T cells that otherwise appear homogeneous on tetramer staining with graded amounts of cognate peptides, the authors show that individual cells downmodulate surface T-cell receptors (TCR) and thus lose tetramer reactivity with variable dynamics within the T-cell population. The dynamics of TCR downregulation represent an accurate assessment of an individual cell's antigen sensitivity, recognition efficiency, and relative functional state within an antigen-specific population and have direct correlation to killing capacity by chromium release as well as degranulation by CD107 mobilization. Furthermore, despite correlation of average T-cell function by all three techniques, TCR downregulation uncovered heterogeneity in T-cell responses after vaccination among patient samples directly ex vivo. When examined using this novel technique, antigen-specific T cells elicited by vaccination with heteroclitic peptides exhibited significantly different recognition efficiencies for the heteroclitic versus native peptides, translating into differences in functional responses. With advancing cancer vaccine trials, the capacity to detect and functionally characterize antigen-specific T-cell responses in detail is critical. Techniques, as presented here, that rapidly assess the overall antigen sensitivity, recognition efficiency, and functional status of patients' T-cell responses will guide future vaccine trials and immunotherapies.

Microarray analysis reveals differences in gene expression of circulating CD8(+) T cells in melanoma patients and healthy donors.

Circulating T cells from many cancer patients are known to be dysfunctional and undergo spontaneous apoptosis. We used microarray technology to determine whether gene expression differences exist in T cells from melanoma patients versus healthy subjects, which may underlie these abnormalities. To maximize the resolution of our data, we sort purified CD8(+) subsets and amplified the extracted RNA for microarray analysis. These analyses show subtle but statistically significant expression differences for 10 genes in T cells from melanoma patients versus healthy controls, which were additionally confirmed by quantitative real-time PCR analysis. Whereas none of these genes are members of the classical apoptosis pathways, several may be linked to apoptosis. To additionally investigate the significance of these 10 genes, we combined them into a classifier and found that they provide a much better discrimination between melanoma and healthy T cells as compared with a classifier built uniquely with classical apoptosis-related genes. These results suggest the possible engagement of an alternative apoptosis pathway in circulating T cells from cancer patients.