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Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse

Fig 8

Generation of anti-mouse autoantibody in erythroid cells of bone marrow and spleen in mice with induced AIHA.

Mice were given intraperitoneal injections of 2x108 rat erythrocytes weekly for 5–6 weeks to induce AIHA. Mice were sacrificed and their bone marrow and spleen cells harvested. Cells isolated were stained with anti-mouse CD71-PE, anti-mouse Ter119-APC and 7AAD, after blocking with anti-mouse CD16/32, to determine the proportion of live erythroid cells as described in S3 Fig. The erythroid cells were co-stained with F(ab’)2 anti-mouse IgG-PE to detect the presence of autoantibodies. Erythroid cells in different stages of maturation (proerythroblasts, erythroblast A, B and C) were gated and autoantibody binding was analyzed in each of them. Presence of membrane-bound autoantibody in the total erythroid populations of bone marrow and spleen after 6 injections is shown in panel A (ANOVA test for bone marrow, p<0.001 and spleen, p = 0.001). Panels B and C show the binding of autoantibody in erythroid cells at various stages of differentiation in bone marrow and spleen (ANOVA test, p<0.001 for both bone marrow and spleen,) respectively. Each bar on the graph represents mean ± SEM of observations. n = 4 control and 6 AIHA-induced mice. *p<0.05, **p<0.01 and ****p<0.001 for comparison of groups (Student t-test).

Fig 8