Infiltrating T lymphocytes in the LCH tumor microenvironment are dysfunctional. (A-D) Relative expression of the indicated cytokines in CD8⁺ T cells isolated from LCH lesions (n = 10) and control lymph nodes from a tonsillectomy (n = 7) before and after activation with PMA and ionomycin (PMA/Iono). Data are means ± SD. (E) CD8⁺ T cells isolated from LCH lesions, but not PBMCs from healthy donors (control), were dysfunctional, as determined by mixed lymphocyte reaction (MLR) assay. Data points represent averaged triplicate measures from cells isolated from 4 patients with LCH or controls. (F) The inhibitory activity of Tregs isolated from LCH lesions or freshly isolated “natural” Tregs (nTregs; CD3⁺CD4⁺CD25^Bright), which develop in the thymus during the course of positive and negative selection, Tregs isolated from tonsils obtained from a tonsillectomy, and CD25^Dep (CD3⁺CD4⁺CD25^Low) cells (n = 4 each) were assessed in a CFSE-based suppression assay. Data are the mean ± SD. **P < .01; ***P < .001; ****P < .0001, ns, not significant (P > .05), by 1-way ANOVA with Tukey’s post hoc test.