Figure 6

CD4⁺LAP⁺ tumor-infiltrating lymphocytes (TILs) potently suppress the proliferation of CD4⁺CD25⁻ T effector cells. (a) CD4⁺CD25^hiCD127^lo T cells were fluorescence-activated cell sorting (FACS) purified from peripheral blood mononuclear cells (PBMCs), together with CD4⁺LAP⁺ and CD4⁺LAP⁻ T cells from the colorectal tumor of the same patient (see Supplementary Figure S4 online), and coincubated with autologous CD4⁺CD25⁻ T effector cells at the indicated ratios (1=4 × 10⁴ cells). Suppression indicates the percent reduction in proliferation of activated autologous effector T cells over a period of 72 h. Data are inclusive of four independent experiments (2 Dukes’ B, 2 Dukes’ C tumors). (b) Addition of an anti-transforming growth factor-β (αTGF-β) blocking antibody or an anti-interleukin-10R (αIL-10R) blocking antibody partially restored proliferation of effector T cells cultured in an E:T ratio of 25:1. Data are inclusive of three independent experiments (1 Dukes’ A, 1 Dukes’ B, 1 Dukes’ C tumor). (c) A transwell assay was established, whereby carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4⁺CD25⁻ effector T cells were stimulated with anti-CD3/CD28 beads, and the indicated cell subset isolated from blood, colon, or tumor was added to the top insert of the transwell. Cells were left for 72 h before analyzing for CFSE dilution among the stimulated effector T cells. Results indicate the reduction in the proportion of cells that have undergone proliferation in comparison with control wells containing stimulated effectors alone (% suppression). Data are inclusive of two independent experiments (1 Dukes’ A, 1 Dukes’ B tumor). Significant differences are indicated: *P<0.05. LAP, latency-associated peptide.

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