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. 2019 May 29;216(7):1682–1699. doi: 10.1084/jem.20181778

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Figure 5. — Tc17 cells occur in healthy humans and express MAF and RORC. (A) Representative dot plot analysis from four individual donors of IL-17 and IFN-γ production by T cells from human spleen. Cells are gated on CD45RO⁺CD4⁺ T cells (CD3⁺CD4⁺CD8⁻Vα7.2⁻TCRγδ⁻CD45RO⁺) or CD45RO⁺CD8⁺ T cells (CD3⁺CD4⁻CD8⁺Vα7.2⁻TCRγδ⁻CD45RO⁺). (B) Total cell numbers of IL-17 or IFN-γ–producing CD45RO⁺CD4⁺ T cells (upper panel) or CD45RO⁺CD8⁺ T cells (lower panel) from peripheral blood mononuclear cells (n = 5 donors) and spleen cells (n = 4 donors) from healthy donors determined by intracellular flow cytometry staining following stimulation with PMA and ionomycin for 4 h. Cells are gated as in A. Results are pooled from four independent experiments. Individual responses are shown together with the mean ± SEM; exact P values were calculated using Student’s t test. (C) Heat map displaying DE genes. Yellow color represents high expression, and blue represents low expression in effector IL-17⁻CD8⁺ versus IL-17⁺CD8⁺ T cells and IL-17⁻CD4⁺ versus IL-17⁺CD4⁺ T cells. Cells were sorted from spleens of three healthy donors for RNA-seq using a human IL-17 cytokine capture kit following restimulation with PMA and ionomycin. IL-17⁺CD8⁺ T cells (CD3⁺CD4⁻CD8⁺Vα7.2⁻TCRγδ⁻IL-17⁺), IL-17⁻CD8⁺ effector T cells (CD3⁺CD4⁻CD8⁺Vα7.2⁻TCRγδ⁻IL-17⁻), IL-17⁺CD4⁺ (CD3⁺CD4⁺CD8⁻Vα7.2⁻TCRγδ⁻IL-17⁺), and IL-17⁻CD4⁺ cells (CD3⁺CD4⁺CD8⁻Vα7.2⁻TCRγδ⁻IL-17⁻). (D) Mean difference plot highlighting genes significantly increased in human spleen IL-17⁺CD8⁺ T cells (yellow) versus genes increased in human spleen IL-17⁻CD8⁺ T cells (green). Data show log2-fold change compared with mean expression. (E) Protein expression of TCF-1, MAF and RORγt as assessed by flow cytometry from spleen cells of healthy donors. Cells are gated on CD3⁺CD4⁻CD8⁺Vα7.2⁻TCRγδ⁻IFN-γ⁺ (blue) or CD3⁺CD4⁻CD8⁺Vα7.2⁻TCRγδ⁻IL-17⁺ (red). Data show representative histograms from two independent experiments and MFI. (F) Comparison of RNA-seq analyses of human IL-17⁻CD4⁺ versus IL-17⁺CD4⁺ with IL-17⁻CD8⁺ versus IL-17⁺CD8⁺ T cells. (G) Enrichment of up-regulated (Up signature) and down-regulated (Down signature) genes in human splenic IL-17⁺CD8⁺ cells in the colonic cancers identified from the TCGA database (Up signature P value = 0.001; Down signature P value = 0.001).