Figure 4.
Intratumoral Tregs are phenotypically and functionally heterogeneous. (A) The expression of 18 checkpoint receptors on CD4+FOXP3+CD25+CD127− Tregs from healthy donor PBMCs (n = 3) and tonsils (n = 3), FL (n = 3), and DLBCL (n = 2) were assessed by t-distributed stochastic neighbor embedding (tSNE) analysis and FlowSOM clustering of mass cytometry data (supplemental Table 1). (Top) Population analysis of Tregs colored based on FlowSOM metaclusters. (Bottom) Density plots of checkpoint receptor protein expression on concatenated Tregs. (B) Heat map of checkpoint receptor expression in activated Tregs (metacluster 2) and resting Tregs (metacluster 1) from the same samples as in panel A. The heat map shows arcsinh ratio of median fluorescence intensity (MFI) normalized to column minimum. (C) Spearman correlation of Treg subset distribution assessed by flow cytometry (5-marker tSNE) with the distribution assessed by mass cytometry (18-marker tSNE). (D) Frequency of activated Tregs among CD4+ T cells assessed by flow cytometry in PBMCs (n = 5) and tonsils (n = 7) from healthy donors, and in FL (n = 15), DLBCL (n = 16), and MCL (n = 10). Horizontal line represents median frequency. (E) Frequency of the activated Treg subset among CD4+ T cells in paired samples, PBMCs, and LNs, from patients with FL (n = 9). (F) Representative flow cytometry histogram of CellTrace Violet dilution in effector CD4+ or CD8+ T cells when stimulated with T-cell-expansion beads (stim. Teff) and cocultured in presence of either actTregs or restTregs purified from an FL sample (left), or calculated as percent suppression of stim. Teff proliferation when cocultured in the presence vs absence of Tregs, (n = 9). (G) (Left) A representative experiment showing TGF-β expression for activated and resting Tregs in the presence or absence of different stimuli. (Right) Summary of TGF-β median fluorescence intensity for unstimulated, activated, and resting Tregs from FL (n = 4) and tonsillar (n = 5) samples. Statistical differences were calculated using Kruskal-Wallis with Dunn multiple comparison test for panel D and Wilcoxon for panels E-G. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Intratumoral Tregs are phenotypically and functionally heterogeneous. (A) The expression of 18 checkpoint receptors on CD4+FOXP3+CD25+CD127− Tregs from healthy donor PBMCs (n = 3) and tonsils (n = 3), FL (n = 3), and DLBCL (n = 2) were assessed by t-distributed stochastic neighbor embedding (tSNE) analysis and FlowSOM clustering of mass cytometry data (supplemental Table 1). (Top) Population analysis of Tregs colored based on FlowSOM metaclusters. (Bottom) Density plots of checkpoint receptor protein expression on concatenated Tregs. (B) Heat map of checkpoint receptor expression in activated Tregs (metacluster 2) and resting Tregs (metacluster 1) from the same samples as in panel A. The heat map shows arcsinh ratio of median fluorescence intensity (MFI) normalized to column minimum. (C) Spearman correlation of Treg subset distribution assessed by flow cytometry (5-marker tSNE) with the distribution assessed by mass cytometry (18-marker tSNE). (D) Frequency of activated Tregs among CD4+ T cells assessed by flow cytometry in PBMCs (n = 5) and tonsils (n = 7) from healthy donors, and in FL (n = 15), DLBCL (n = 16), and MCL (n = 10). Horizontal line represents median frequency. (E) Frequency of the activated Treg subset among CD4+ T cells in paired samples, PBMCs, and LNs, from patients with FL (n = 9). (F) Representative flow cytometry histogram of CellTrace Violet dilution in effector CD4+ or CD8+ T cells when stimulated with T-cell-expansion beads (stim. Teff) and cocultured in presence of either actTregs or restTregs purified from an FL sample (left), or calculated as percent suppression of stim. Teff proliferation when cocultured in the presence vs absence of Tregs, (n = 9). (G) (Left) A representative experiment showing TGF-β expression for activated and resting Tregs in the presence or absence of different stimuli. (Right) Summary of TGF-β median fluorescence intensity for unstimulated, activated, and resting Tregs from FL (n = 4) and tonsillar (n = 5) samples. Statistical differences were calculated using Kruskal-Wallis with Dunn multiple comparison test for panel D and Wilcoxon for panels E-G. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

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