Allogeneic Murine Plasmacytoid Dendritic Cells Induce Regulatory T Cells In Vitro Which Can Prolong Cardiac Allograft Survival In Vivo.

Abstract

Background. We have found that without immunosuppression, life-sustaining, full MHC-mismatched mouse kidney allografts were spontaneously accepted in the DBA/2 to B6 strain combination. Using B6.Foxp3DTR mice, we have shown that Foxp3+ cells are necessary to maintain spontaneous kidney allograft tolerance. Pathological analyses of accepted kidney allografts demonstrated unique, Tregrich organized lymphoid structures (TOLS), which were rich in Foxp3+ Tregs and plasmacytoid dendritic cells (pDCs), and which were distinct from tertiary lymphoid structures. Methods. To test whether pDCs could promote Treg production, we isolated pDCs from DBA mouse bone marrow and cultured them in vitro with naïve CD4<sup style=“font-family: ‘Times New Roman', serif;”>+[/sup] CD25<sup style=“font-family: ‘Times New Roman', serif;”>- [/sup]T cells from B6 mouse spleen in the presence of IL-2 and TGFβ. We then analyzed these cells for suppressive capacity by MLR utilizing CFSE dilution in responding CD4+CD25- B6 T cells. Results. We observed an increased expression of Foxp3 in the co-cultured T cells. Addition of Foxp3+ T cell/pDC cultures also suppressed proliferation in vitro when CFSE labeled naïve B6 T cells were stimulated by nonspecific anti-CD3/anti-CD28 beads (mean 95.8% vs 6.0% proliferating cells, p<.001) or irradiated allogeneic stimulators in sensitized mice (mean 13.8% vs 3.75% proliferating cells, p<.001). This suppression was greater than that induced by CD4+CD25+ Foxp3+ nTregs isolated from B6 mice and cultured in an equal ratio to responder T cells after bead (mean 67.8% vs 6.0% proliferating cells, p<.001) or allogeneic (mean 7.7% vs 3.75% proliferating cells, p<.02) stimulus. Adoptive transfer of the induced murine Foxp3<sup style=“font-family: ‘Times New Roman', serif;”>+[/sup] T cell and pDC mixture into B6 recipients 2 weeks prior to heterotopic DBA/2 heart transplantation resulted in prolongation of allograft survival (MST =11 days, n=6) compared to vehicle treated controls (MST = 6 days, n=6) or compared to pDCs coinjected with co-cultured B6 naïve T cells that were non-induced (no IL-2/TGFβ) (n=3, MST=7 days) (p<.002). Conclusions. Allogeneic pDCs can promote the generation of highly suppressive Foxp3+ Tregs in vitro which have the ability to prolong vascularized allograft tolerance in vivo without any further treatment.