Integrated FACS/SCBC phenotypic/functional proteomic analysis of tumor-antigen-specific T-cell populations collected from a melanoma cancer patient participating in an ACT trial. (a) Measurement protocol. MART-1 tumor-antigen-specific CD8+ T cells are separated from the blood of the patient using 10-parameter FACS sorting and then loaded onto an SCBC for assaying 19 secreted effector proteins. (b) Analysis of SCBC data. Unsupervised clustering of the single-cell proteomic data (tree, left) reveals coordinated behaviors that reflect specific immune functions. Correlation coefficients, calculated from single cell assays, are provided for proteins within the specified groupings (In group) and outside those groupings (Out group). The scatter plot (right) shows correlations between two anti-tumor effector proteins (IFN-γ and TNF-α) and also shows that the roughly 10% of the cell population that secretes five or more different proteins are also about 100-fold more active for any given protein, and so dominate the immune response for that phenotype. (c) The population kinetics of the TCR-engineered MART-1+ CD8+ T cells, as a percentage of CD3+ T cells (orange solid curve), along with the polyfunctional index (pie chart areas) for tracking population of the MART-1+ CD8+ T cells secreting five or more proteins. The pie chart composition reflects the relative abundances of those proteins. GB refers to the protein Granzyme B. The dynamics of the polyfunctional cells showed much stronger correlations with the observed clinical responses in the patients. Adapted from  with permission.