Prophylactic and therapeutic administration of apyrase ameliorates ICPi-induced AIHA. Whole blood was collected from young (aged 2-3 months) HOD⁺OTII⁺ mice treated with apyrase or with an inactivated apyrase before every ICPi injection. (A-B) Sera were analyzed for RBC autoantibodies by flow crossmatch for HOD-specific autoantibodies (A) and hematocrit percentage (B) was calculated. (C-D) Frequencies of FoxP3⁺ Tregs (C) and CD39SP (D) were calculated. (E) Suppressive activity of CD4⁺CD25⁺ T cells collected from apyrase-pretreated (green), inactive apyrase-pretreated (light blue), ICPi-treated (red), and PBS-treated (blue) HOD⁺OTII⁺ mice. Suppression was assessed by adding CD4⁺CD25⁻ T cells (Tconv) in culture with serial dilutions of CD4⁺CD25⁺ enriched T cells. Cells were stimulated with anti-CD3 10 μg/mL and anti-CD28 20 μg/mL for 72 hours at 37°C. Suppression was expressed as percentage of decreased number of Tconv. (F-G) Kaplan-Meier survival curve (F) and CD39SP level (G) in whole blood of mice treated with ICPi for 14 days followed by injection of PBS (red line) or 5 units of apyrase daily for 14 days (green line). (H) Linear regression analysis showing a significant positive correlation between RBC autoantibody levels and frequency of CD39SP (R² = 0.62; P < .0001). (I) Young (aged 2-3 months) HOD⁺OTII⁺ mice were treated with ICPi for 14 days alone (red; n = 13) or with apyrase treatment beginning at 7 days (ie, when CD39SP are detectable; green; n = 30). Sera from B6 animals were collected as controls (blue). Sera were serially diluted and analyzed for RBC autoantibodies then analyzed in a flow crossmatch. (J) Hematocrit percentage for mice treated with ICPi alone or with apyrase was calculated. Data presented in panels A-D are combined from 3 different experiments with 4 mice per group. Data presented in panels E-F are combined from 2 different experiments. Statistical significance was determined by 2-way ANOVA with Sidak posttest, Mann-Whitney test, and by log-rank test: ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.