Figure 2.

(See previous page). Tumor-infiltrating CD4⁺FoxP3⁻IL-10+ T cells are potent suppressors of T cell function and their phenotype corresponds to Tr1 cells. Tumor-infiltrating CD4⁺CD25⁻ T cells were activated with anti-CD3/CD46 or anti-CD3/ICOS antibodies for 24–48 h, then stained for IL-10 and magnetically sorted into IL-10^low and IL-10^high fractions, which were both co-cultured at a 1:10 ratio with CFSE-labeled PBMCs from healthy donors stimulated with phytohemagglutinin (PHA) for 5 d (A) T cell proliferation and TNFα production measured by flow cytometry in PHA-stimulated PBMCs cultured alone or in the presence of IL-10^low or IL-10^high fractions of tumor infiltrating CD4⁺CD25⁻ T cells. (B) Collective analysis of the percentages of suppression of T cell proliferation and TNFα production from eight patients. (C) Effect of blocking IL-10R on the suppressive capacity of CD4⁺IL-10^high cells. Cells were cultured as described above in the presence of 30 μg/mL of neutralizing anti-IL-10R antibody or an irrelevant isotype control antibody. (D) Expression of CD49b and LAG-3 on tumor-infiltrating CD4⁺ T cells activated with antibodies to CD3 and ICOS for 24 h. Cells were gated on viable CD3⁺CD4⁺ T cells and FoxP3⁺CD127⁻ Tregs were excluded from the analysis. Histograms show the expression of IL-10 and ICOS in different populations based on the expression of CD49b and LAG-3. (E) CD49b and LAG-3 expression in blood, TFL and TILs isolated from a representative patient with HCC. Cells were gated on viable CD3⁺CD4⁺FoxP3⁻ T cells. (F) Collective percentages of CD49b⁺LAG-3⁺ cells within CD4⁺Foxp3⁻ T cells in 21 patients analyzed (HCC n = 8 and LM-CRC n = 13). HCC (red dots) and LM-CRC (blue open dots).