Fig. 9.

Suppressive function of iT_reg cells declines with disease severity in COPD subjects. (A, Left) Flow cytometry histograms show proliferation of CFSE-labeled CD4⁺ T cells (from healthy donors) stimulated for 72 h in vitro with α-CD3/CD28 and cultured alone (gray curves) or in the presence of various numbers of flow-sorted iT_reg cells (ratio from 1:1 to 1:8) from NS healthy subjects, S healthy subjects, and COPD patients at different disease stages. Numbers in plots indicate the percentage of CFSE dilution in CD4⁺ T cells cultured alone (Upper) or in the presence of iT_reg cells from NS, S, and COPD subjects at different disease stages. The iT_reg cells were flow-sorted as reported in the experimental procedure (SI Appendix, Fig. S13). (A, Right) Percentage of suppression exerted by iT_reg cells in the above conditions. Data are from n = 3 independent experiments (at least n = 2 subjects in 2 technical replicates), and are expressed as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001 statistical significance between COPD at GOLD stage II and healthy (NS and S) controls. ^#P < 0.05 statistical significance between COPD patients at GOLD stage II and NS subjects by 2-way ANOVA corrected for multiple comparisons Tukey test. ^§P < 0.05 statistical significance between COPD patients at GOLD stage IV and S subjects by 2-way ANOVA corrected for multiple comparisons Tukey test. (B) Scatter plots with bar histograms show comparison of suppression ability exerted by iT_reg cells (at the indicated ratio) isolated from COPD subjects on proliferation of CFSE-labeled CD4⁺ T cells stimulated for 72 h in vitro with α-CD3/CD28. Data are from n = 3 independent experiments (at least n = 2 COPD subjects in 2 technical replicates), and are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-tailed Mann–Whitney test.