ICPi treatment promotes AIHA development in HOD⁺OTII⁺ mice. Young (aged 2-3 months) male and female HOD⁺OTII⁺ mice were treated 14 days with antibodies against PD1, Lag-3, CTLA-4, and IL-10R (ie, ICPi, represented in red). Control mice were treated with phosphate-buffered saline (PBS; blue). (A) Whole blood was collected at baseline, after 14 days of treatment, and 14 days after treatment cessation, and sera were analyzed for RBC autoantibodies by flow crossmatch. (B) The frequency of autoantibodies after 14 days of ICPi treatment was stratified by sex: 28 of 36 females and 18 of 28 males produced autoantibodies. (C) Hematocrit and bilirubin concentration demonstrates that ICPi treated (red) but not control mice (blue) exhibited RBC hemolysis and became anemic. (D) Linear regression analysis showing a significant negative correlation between RBC autoantibody levels and hematocrit (R² = 0.47; P < .0001). (E) Serum cytokine levels 2 weeks after ICPi or PBS control treatment. (F) Levels of reactive oxygen species (ROS) in peripheral RBCs. Data in panels A, B, C, and F are combined from 6 independent experiments with 4 mice per group. Data for bilirubin are combined from 3 to 5 independent experiment with 4 mice/group. Data in panel E are combined from 2 independent experiment with 10 mice per group. Statistical significance was determined by Pearson correlation, Mann-Whitney test, or repeated measures 2-way analysis of variance (ANOVA) with Sidak multiple comparisons test: ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. Igs, total immunoglobulins; MFI, mean fluorescence intensity.