PL2‐AM23‐SN‐30 | Apyrase Reduces Severity of Autoimmune Hemolytic Anemia (AIHA) Secondary to Checkpoint Inhibitors in a Mouse Model

Autoimmune hemolytic anemia (AIHA), due to pathogenic autoantibodies that destroy red blood cells (RBCs), is a severe, and sometimes fatal, disease. The incidence of AIHA secondary to immune checkpoint inhibitor immunotherapy (ICPi) is increasing; indeed, it is the most frequent hematological adverse event due to ICPi and is associated with severe hemolysis and high fatality rates. Because current therapies for AIHA have variable success, with high relapse rates, there is an unmet need to develop more efficacious treatments. In a mouse model, it was shown that RBC-specific autoreactive T cells upregulated checkpoint molecules, the immunosuppressive cytokine IL-10 and its receptor (IL-10R), and purinergic signaling molecules (e.g.,CD39), resulting in tolerance. In addition, infusing antibodies against checkpoint molecules and IL-10R reversed tolerance resulting in pathogenic RBC autoantibodies and appearance of pathogenic CD39 single-positive T cells (CD39SP). As CD39 is a key enzyme involved in hydrolyzing proinflammatory extracellular ATP (eATP) to ADP and AMP, we hypothesized that soluble CD39 (i.e., apyrase) treatment would reduce AIHA severity. HOD mice (expressing an RBC-specific HOD transgene) were bred with OTII mice (with CD4 T cells recognizing HOD) to generate autoreactive HOD+OTII+ mice. HOD+OTII+ mice were treated for 3 weeks with a cocktail of four antibodies (denoted “4Aby”) against IL-10R (500 μg i.p. weekly) and the CTLA4, LAG3, and PD1 checkpoint molecules (200 μg i.p. each every other day) to induce AIHA. Groups of mice received PBS, apyrase (3U), or heat-inactivated apyrase 1 h before each 4Aby treatment. Hematocrit, reticulocyte count, RBC-bound antibodies and antigen levels, and reactive oxygen species (ROS) were assessed in whole blood. Sera were tested for autoantibodies by flow crossmatch and splenocytes collected to evaluate in vitro regulatory T-cell (Treg) suppression and to enumerate CD39SP. Compared with PBS controls, apyrase-treated mice had significantly decreased RBC autoantibody levels (p < .001), reduced inflammation (i.e., decreased ROS; p < .05), and decreased anemia (e.g., decreased reticulocytosis [p < .05), increased hematocrit [p < .05), and increased RBC antigen levels [p < .05]). Reduced RBC autoantibodies correlated with enhanced suppression by Tregs (p < .05) and reductions in CD39SP. There were no significant differences between cohorts treated with PBS or heat-inactivated apyrase. These results reflect three experiments with four mice/group. Reducing eATP with apyrase significantly improves clinical signs of AIHA secondary to ICPi in mice, implicating purinergic signaling in AIHA onset. These data suggest that targeting the purinergic signaling may be effective for treating patients with AIHA. As apyrase has been used therapeutically in humans in other settings, these results may lead to new approaches to treat human AIHA.