CD4+CD25+ Regulatory T Cells Control Induction of Autoimmune Hemolytic Anemia.
Authors: Yazdanbakhsh Karina , Mqadmi Amina , Zheng Xiaoying 
Published: October 2019

Abstract
Autoimmune Hemolytic anemia (AIHA) is the result of increased destruction of red blood cells (RBCs) due to the production of antibodies against self antigens. Anemia can be severe and life-threatening. The underlying mechanism of autoimmunity is the result of breakdown of immune tolerance, but the molecular and cellular basis for the induction of AIHA remains to be fully defined. To further our understanding of mechanisms that trigger AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA. Anemia is induced by repeated injection of rat RBCs resulting in development of erythrocyte autoantibodies as well as rat-specific immunoglobulins. The severity of the autoimmune disease is strain dependent. We found that in about 20–30% of C57/Bl6 mice repeatedly immunized with washed rat RBCs, there is breakdown of tolerance and development of pathogenic autoantibodies resulting in decreased hematocrit, reticulocytosis and increased destruction of transfused syngeneic mouse RBCs. To identify the immunological factors contributing to the incidence of AIHA, we analyzed the role of specific T regulatory subsets in controlling AIHA in C57/Bl6 mice. Previous studies documented that depletion of selected regulatory CD4+ T cell subsets (CD25+, CD62L+ and CD45RBlow) can induce different degrees of autoimmune disorders. However, the nature of the regulatory T cell subset in the induction of AIHA has not yet been studied. To test the role of CD25+ T regulatory cells in the induction of AIHA, 10 week old C57/Bl6 mice (n=10) were treated with 500 μg of anti-CD25 antibody six hours prior to immunization with rat RBCs on a weekly basis for four weeks. Following this repeated challenge the incidence of AIHA increased from 20 to 90%. Treatment with isotype control antibody prior to weekly injections of rat RBCs for four weeks resulted in the expected 20% incidence of AIHA. Furthermore, weekly treatment with anti-CD25 alone for four weeks did not result in development of AIHA, indicating that the depletion of CD25 cells in combination with rat RBC stimulus was important for the development AIHA. To test whether anti-CD25 treatment also increased the levels of autoantibodies directed against other non-erythroid antigens, we measured the levels of antibodies to double stranded DNA (anti-ds DNA) characteristic of systemic autoimmune disease and found significantly elevated levels in anti-CD25/rat RBC immunized mice, as compared to control mice treated with rat RBCs alone. Interestingly, treatment with anti-CD25 alone did not result in increased levels of anti-ds DNA, indicating that selective depletion of CD25+ does not result in the development of autoimmunity and that an additional signal is required to activate autoreactivity. In addition, the levels of alloantibodies against rat RBCs in anti-CD25/rat RBC immunized mice were elevated as compared to mice treated with rat RBCs alone, consistent with a heightened immune hypersensitive state. Importantly, adoptive transfer of purified splenic population of CD4+CD25+ from mice that had undergone weekly injections of rat erythrocytes for 12 weeks into naïve C57/Bl6 mice (n=5) prevented the induction of autoantibody production whereas transfer of CD4+CD25-T cells into naïve mice (n=6) significantly elevated the autoantibody levels following weekly immunization with rat RBCs. These findings emphasize an important suppressive role for CD4+CD25+ in prevention of AIHA. Altogether, our data provide new insight regarding the mechanism for breakdown of tolerance in antibody-mediated autoimmunes disease which may help to establish therapeutic strategies for treatment of AIHA.