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Prior studies have demonstrated that neonatal B cells possess unique immunoregulatory properties. Specifically, neonatal B-1 cells produce interleukin (IL)-10 in response to toll-like receptor stimulation; this modulates innate and adaptive alloimmune responses. Here, we examine whether this process plays a critical role in neonatal transplant tolerance induction.We used a murine model of neonatal transplant tolerance induction, whereby female C57BL/6 mice injected with male spleen cells 3 days after birth acquire the ability to accept a male skin transplant in adulthood.We investigated the role of B cells in this model by using mice with targeted genetic B-cell deficiencies (including muMT, JH−/−, and XID mice). In addition, we examined the role of IL-10 in this model using IL-10−/− mice. Transplant tolerance induction was neither dependent on B cells nor on IL-10.Despite the role of neonatal B cells in suppressing innate and adaptive alloimmune responses, B cells are dispensable for neonatal transplant tolerance induction in this experimental model.