Introduction: Solid organ transplantation is the gold standard therapy for many end-stage organ diseases. Although short-term graft survival is excellent due to the use of potent immunosuppressive regimens, incidence of chronic allograft dysfunction and graft failure remains unchanged. Global immunosuppressants are also associated with adverse side effects including severe infection, malignancy and target end-organ damage. Novel, targeted approaches are required to more effectively modulate the immune system and to improve long-term graft outcomes. One such strategy is that of cellular therapy. An enriched population of human IL-10+ B cells with regulatory function (Breg) has been identified in tolerant, human kidney transplant recipients; mouse IL-10+ Breg confer protection of allograft in mouse models of transplantation. However, human Breg are poorly characterised, are rare in peripheral blood and there is currently no method by which to obtain such cells in large numbers; thus limiting the potential for human Breg as a clinical therapy. Here we describe, for the first time, ex vivo generation and characterisation of human IL-10+ B cells with both in vitro and in vivo regulatory function (EXPB10 cells). Methods: Human CD19+ B cells from healthy individuals were negatively selected and stimulated for 7 days with CD154+ CHO cells and the cytokine combination IL-2, IL-4 and IL-10 to generate IL-10+ B cells. Phenotype and suppressive function of EXPB10 cells were examined in vitro. The ability of EXPB10 cells to regulate in vivo immune responses was tested using a clinically relevant humanised mouse model of skin transplantation. Human CD19+ B cells in peripheral blood from both healthy human subjects and patients suffering from squamous cell carcinoma of the skin were examined for human Breg subsets by flow cytometry. Results: EXPB10 cells produced IL-10, up-regulated expression of the putative Breg marker TIM-1, and suppressed CD4+ T cell proliferation and Th1 cell differentiation in vitro. Suppression was partially dependent on IL-10 and CD154. EXPB10 cells were highly activated, CD19+TIM-1+CD25+CD71+CD73- B cells which demonstrated dependency on autocrine IL-10. TIM-1 may have modulated intracellular pathways downstream of IL-10, thereby driving EXPB10-mediated suppression of CD4+ T cell responses. A second, CD154-independent suppressive mechanism was identified which may rely on purinergic signaling. EXPB10 cells significantly prolonged human graft survival in a humanised mouse model of skin transplantation and were able to generate and expand functionally suppressive human T regulatory cells (Treg). Examination of CD19+ B cells in human peripheral blood identified an endogenous CD19+CD25+CD71+CD73- B cell subset which was enriched for TIM-1, similar to EXPB10 cells. This endogenous B cell subset was also significantly enriched in patients with squamous cell carcinoma of the skin compared to a control cohort of healthy human subjects. Discussion: Human CD19+IL-10+ B cells with regulatory function can be generated ex vivo and can prolong human allograft survival in a complex, biological environment. Mechanistic data demonstrated that this human Breg subset was dependent on the cognate interaction between CD154 and CD40, as well as the autocrine action of IL-10. TIM-1 may act as a gatekeeper of regulatory function by modulating the EXPB10 cell's response to IL-10. Such subtleties in cellular behaviour offer the potential for further manipulation to better control an immune response. The identification of CD19+ B cells in human peripheral blood with a similar phenotype as EXPB10 cells, and moreover the finding that this subset was enriched in patients diagnosed with cancer, suggests that endogenous Breg may exist with similar cellular properties to that of EXPB10 cells. This potent suppressor population may represent a novel cellular therapy which could be used alone or as an adjunct to other immunosuppressive regimens in the context of transplantation, autoimmunity and inflammation.