Neonatal Tolerance: Donor B Cells Home to Host Lymphoid Organs, Interact With Apoptotic Cells, Produce IL-10 and Prolong Heart Allograft Survival.

Abstract

Introduction: Newborn mice injected with a mixture of semi-allogeneic (F1) bone marrow (BMC) and spleen cells (SC) accept donor-type transplants as adults. B cells are abundant in both BMC and SC and have been reported to tolerize CD8+ T cells. To determine what role, if any, B cells in the tolerizing inoculum play in this classic tolerance model, we studied the effect of purified B cells injected into newborn mice. Methods: F1-B cells and allogeneic-B cells (allo-B) were purified from spleen using StemCell Technologies EasySep® CD19 positive selection kits. C3H/He (C3H; H-2k) neonates were injected within 24 hours of birth with 106 purified B cells, GFP-expressing (C57BL/6 [B6; H-2b] or C3H X B6 [H-2k/b]) or CFSE-labelled (BALB/c [BALB; H-2d] or C3H X BALB [H-2k/d]). Injected cells were tracked by whole body/organ imaging; their interactions and fates were studied with high-resolution optical-sectioning microscopy. Impact on heart allograft survival was also determined. Results: By whole organ imaging (day 3), purified F1-B cells were detected in primary and secondary lymphoid organs (spleen, lymph node and bone). At day 6, injected B cells in spleen were positioned in follicular regions. F1- and allo-B cells both interacted with host CD8+ T cells; F1-B cells remained intact while allo-B cells fragmented and were taken up by host DC. Survival of F1-B cells suggested they induced tolerance in host CD8+ T cells. Consistent with that, F1-B cells also interacted with host apoptotic cells in spleen. Apoptotic cells have been reported to induce B cells to produce IL-10 and in turn convert T cells into Tr1-type regulatory cells. IL-10-producing F1-B cells were observed interacting with host T cells in day 3 spleens. To determine if other mechanisms played a role in prolonging F1-B cell survival we examined host Foxp3 regulatory and apoptotic T cells; in both cases events were few. Purified F1-B cells prolonged survival of donor-type hearts to 100 days in 6 of 7 transplanted mice. Beat scores were low, however, and one day-105 beating heart showed infiltrating host macrophages and T cells including CD8+ T cells. Conclusion: Neonatally-injected purified F1-B cells actively regulate the neonatal immune system and prolong survival of donor-type heart grafts transplanted as adults. By themselves, however, F1-B cells do not induce a robust tolerance suggesting additional F1 cell types are required for the process.