In vitro culture of FL LN specimens over time restores IL-4–induced p-STAT6 in PD-1^hiCD4⁺ T cells but does not require neutralization of PD-1. FL LN specimens were cultured over time as specified in “Methods” with anti–PD-1 neutralizing Ab or with an isotype control Ab, washed twice with PBS, and then restimulated with or without IL-4 for 15 minutes. IL-4–induced p-STAT6 and the expression of PD-1 were measured by phospho-flow cytometry. (A) Representative histograms of IL-4–induced p-STAT6 in FL LN TILs precultured with control Ab or anti-PD-1 Ab for 30 minutes or 48 hours before the cells were washed and restimulated with IL-4 for 15 minutes. (B). Time course of IL-4–induced p-STAT6 is shown in CD4⁺ or CD8⁺ T-cell subsets. Results are shown as FC of IL-4–induced p-STAT6, relative to unstimulated cells for 2 different FL donors. (C) FL LN were precultured in the presence of an isotype control Ab for 30 minutes or 48 hours before washing and restimulated with or without IL-4. IL-4–induced p-STAT6 was then determined in PD-1^−, PD-1^int, or PD-1^hi CD4⁺ TILs. Shown is mean ± SEM; FL, n = 5. (D) Same experiment as described in panel C, using tonsils. Shown is mean ± SEM; tonsil, n = 3. P < .021 as determined by paired 2-tailed t test.