Anti-CD3ε induces splenic B220^lo b-cell expansion following anti-CD20 treatment in a mouse model of allosensitization

Antibodies targeting T cells and B cells are increasingly used for immunosuppression in clinical transplantation. However, the impact of T-cell depletion by antibodies on B-cell homeostasis is poorly understood. Using a mouse model of allosensitization with skin allograft, we investigated whether targeting T cells by anti-CD3ε alters peripheral B-cell homeostasis and alloantibody responses following B-cell depletion by anti-CD20. We found that anti-CD3ε induced a discrete B220. ^lo, but not a conventional B220. ^hi subset, in the spleens of the allosensitized mice 14 days after anti-CD20 treatment. The splenic B220. ^lo cells were refractory to anti-CD20 depletion. Flow cytometry revealed that the splenic B220. ^lo cells were phenotypically similar to the B220. ^lo AA4.1. ⁺ CD23. ^- sIgM. ^lo sIgD. ^- developing B cells (pre-B to immature B) normally presented in the bone marrow. Despite the presence of the splenic B220. ^lo cells, mice treated with combined anti-CD3ε/CD20 produced limited alloantibodies in response to the primary skin allografts. Alloantibody production increased significantly in the mice following re-immunization by donor-specific splenocytes. We conclude that anti-CD3ε can induce an expansion of B220. ^lo B cells in the spleens after B-cell depletion by anti-CD20. These B cells are not producing alloantibodies, but re-immunization of the mice with alloantigen leads to risk of alloantibody response.