Major cytokines driving polyfunctional product CD4⁺and CD8⁺T cells by CD19 stimulation that distinguish responders to the therapy from nonresponders. (A-B) Single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules was performed on product T cells from 20 patients treated with CAR T cells. The analysis was performed on all product cells or select CD4⁺ and CD8⁺ T cells. The product T cells were first stimulated with CD19-expressing target cells or control NGFR cells before the analysis. The graphs show PSI (mean ± standard error) with or without CD19 stimulation for all cells and for CD4⁺ and CD8⁺ subsets separately. The main cytokine drivers for each product T-cell subpopulation are also shown. (C-D) Product CD4⁺ and CD8⁺ T-cell PSI profiles were broken down per cytokine, between patient groups with no response and OR to CAR T-cell therapy. Only CD4 and CD8 cytokines that were upregulated relative to mock stimulation are shown. Each cytokine PSI level reflects its average secretion intensity in polyfunctional single cells. The diagram shows the cytokines that contribute to the polyfunctionality index in the CD8⁺ and CD4⁺ T-cell populations. (E-F) Polyfunctional strength of major cytokines and chemokines in CD4⁺ and CD8⁺ CAR T cells from nonresponders and responders. FD, fold difference; GM-CSF, granulocyte-macrophage colony-stimulating factor.