Infiltrating T lymphocytes in the LCH tumor microenvironment exhibit an exhausted phenotype. (A) Expression of different immune checkpoint receptors on LCH lesion-infiltrating CD8⁺ T cells. Analyses of CyTOF data were performed using the FlowSOM algorithm and visualized as pie charts. (B-C) Quantification of relative expression of PD-1 (n = 15, LCH; n = 10, control) (B) and coexpression of immune checkpoint receptors (n = 5 each) (C) in CD8⁺ T cells, as determined by CyTOF. Data are the means ± SD. (D) Relative expression of perforin and granzyme B in LCH lesion–infiltrating CD8⁺ T cells not expressing PD-1, TIM-3, or LAG-3 or expressing 1 or more of the immune checkpoint receptors (n = 5). Data are the mean ± SD. (E) Pie chart representing FlowSOM algorithm–based assessment of PD-L1 and PD-L2 expression on CD207⁺ gated cells. (F) Effector function of CD8⁺ T cells was restored after treatment for 24 hours with anti-PD-1 but not with anti-PD-L1 antibody, as determined by mixed lymphocyte reaction (MLR) assay. Data points represent averaged triplicate measures from cells isolated from 4 patients with LCH. For all panels, LCH lesions were derived from liver, bone, or spleen. *P < .05, **P < .01, ***P < .001, ****P < .0001, ns, not significant (P > .05), by 1-way ANOVA with Tukey’s post hoc test.