ICPi treatment promotes expansion of proinflammatory CD4⁺T cells. Spleens were collected from ICPi- (red) and PBS-treated (blue) young (aged 2-3 months) HOD⁺OTII⁺ mice after 14 days of treatment. Splenocytes were processed into single-cell suspensions and stained with antibodies to delineate T-cell subsets. (A) The frequency of Tregs identified as FoxP3⁺ Tregs (CD4⁺CD25⁺FoxP3⁺) and Tr1 Tregs (CD4⁺FoxP3^–CD25^–CD49b⁺Lag3⁺IL10⁺) of total CD4⁺ T cells was calculated. To determine Tr1 Tregs, enriched CD4⁺ T cells were stimulated for 72 hours at 37°C with PMA (50 ng/mL) and ionomycin (500 ng/mL), incubated 1 hour with brefeldin A, and stained to identify the Treg subset. (B) CD4⁺ T cells were stained with antibodies against CD62L and CD44 to quantify the percentage of CD62L⁺CD44^– naïve and CD62L^–CD44⁺ effector cells. To determine THs, enriched CD4⁺ T cells were stimulated for 24 hours at 37°C with PMA (50 ng/mL) and ionomycin (500 ng/mL), incubated 1 hour with brefeldin A, and stained intracellularly for IFN-γ and IL-17A. (C-E) Representative flow plots (C) and frequency of TH1 (D) and TH17 cells (E). (F) Ratio of Tregs-to-TH17 cells was calculated for both groups of mice. (G) Frequency of CD4⁺ T cells expressing CD200, ICOS, PD1, Lag-3, and CTLA-4. Different clones than those contained within the ICPi cocktail were used for PD1, Lag-3, and CTLA-4 staining. Data shown are cumulative of 4 to 5 independent experiments with 5 mice per group. Statistical significance was determined by Mann-Whitney test; ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.