Donor lymphocyte infusion (DLI) has been shown to induce donor-specific hyporesponsiveness or even immune-tolerance among alloreactive T cells in different organ transplantation models. However, the optimal cell source or subset for DLI effects is not known. B cells constitute a complex system of uniquely well-equipped antigen-presenting cells (APCs) and exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function, potentially regulating the immune response, and are therefore, ideal candidates for DLI.

. In this study, we investigated the immune-regulatory roles of murine B cell subsets as tolerogenic APCs targeting alloreactive T cells.

Mononuclear cells from the peritoneal cavity, spleen, and liver of B57CL/6 mice were stained with various combinations of mAbs directed against IgM, CD19, CD21, CD11b, CD5, CD80, CD86 and MHC class I and II, PD-L1, and PD-L2, followed by phenotypic analyses by multi-parameter flow cytometry. Naïve B cells were divided into subsets: CD21^–IgM^low follicular B cells, CD21⁺IgM^high marginal zone B, and CD21^–IgM^high B-0 cells in the spleen, and CD11b⁺CD5⁺ B-1a cells, CD11b⁺CD5^– B-1b, and CD11b^–CD5^– B-2 cells in the peritoneal cavity and liver. Among the B cell subsets, CD11b⁺CD5⁺ B-1a cells included MHC class II⁺CD80⁺CD86⁺ PD-L1⁺PD-L2⁺ B cells. These B cells expressed surface molecules necessary for the efficient Ag-presentation to T cells together with apoptosis inducing ligands, potentially imparting their tolerogenic potential upon alloantigen recognition of T cells via the direct pathway. To utilize these tolerogenic B cells for DLI, we developed a method for isolation, and enrichment of these B cells without interfering with PD-L1 and PD-L2 functions. Using fluorescence-activated cell sorting (FACS) of CD19⁺CD11b⁺CD5⁺ B-1a cells, PD-L1⁺PD-L2⁺ B cells were enriched to 54.9%. Additionally, FACS of CD19⁺CD11b⁺CD5⁺CD21^– B-1a cells enriched PD-L1⁺PD-L2⁺ B cells further, to 62.8 %.

The newly identified tolerogenic B cells expressing both, PD-L1 and PD-L2 may be optimal for DLI, demonstrating a novel concept of inhibition or tolerization of alloreactive T cells in organ transplant recipients.

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