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Effect of regulatory dendritic cells on IL-10-producing regulatory B cells of skin allograft in mice

Keywords: dendritic cells , skin transplantation , B-lymphocytes , regulatory

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Abstract:

Objective  To investigate the effect of recipient immature dendritic cells (imDCs) loaded with PUVA-treated donor apoptotic splenic lymphocytes (PUVA-SP DC) on IL-10+CD19+regulatory B cells (Breg) and the survival duration of skin allograft in mice. Methods  Bone marrow-derived DCs of C57BL/6 mice were obtained from bone marrow cells by co-culturing with recombinant mouse IL-4 and GM-CSF. Spleen lymphocytes (SP) of BALB/c mice were isolated and prepared as PUVA-SP by treating the cells with 8-methoxypsoralen plus ultraviolet A irradiation. The bone marrow-derived imDCs of C57BL/6 mice were co-cultured with PUVA-SP of BALB/c mice to obtain PUVA-SP DCs. The skin allograft model was then established. Animals were randomly grouped according to different pretreatments as follows: the control group was iv. introduction of PBS (0.2ml) alone 7 days before skin transplantation, the PUVA-SP DC group received an iv. injection of PUVA-SP DCs, the maDC (mature DC) group received recipient maDCs, and the imDC group was given recipient imDCs. Mice were monitored daily from day 6 after transplantation for signs of rejection of skin graft. The recipients' peripheral blood serum samples were then collected and the level of cytokines were measured by using ELISA kits. The survival time of skin allograft was evaluated every day. The expression of IL10+CD19+regulatory B cells was analyzed by flow cytometry. Results  After transplantation, the proportion of IL-10+CD19+Breg in the peripheral blood of PUVA-SP DC group was 7.48%, which was obviously higher than that of imDC group (4.12%), maDC group (3.01%) and control group (2.37%). The serum level of cytokine IL-10 in PUVA-SP DC group was 58.2±0.9ng/ml, and it was significantly higher than that in maDC group (20.1±1.6ng/ml), imDC group (26.2±1.3ng/ml) and control group (19.0±0.6ng/ ml, P<0.01). The survival time of allograft in PUVA-SP DC group was 62.3±2.6d, and it was markedly longer than that in maDC group (20.7±1.9d), imDC group (12.1±1.0d) and control group (11.0±1.3d, P<0.01). Conclusions  Administration of PUVASP DCs, in the absence of an immunosuppressant, may significantly delay allograft rejection. This effect is associated with up-regulation of circulating regulatory B cells with preferential IL-10 secretion.

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