Graft-Infiltrating PD-L1hi Cross-Dressed Dendritic Cells Subvert Donor-Reactive T Cell Responses in Mouse Liver Transplant Tolerance

Abstract

Introduction: The mechanisms underlying “spontaneous” liver allograft tolerance remain unclear. Given recent reports of the key role of host dendritic cells (DCs) in mouse spleens that express intact donor MHC (cross-dressing) in the instigation of graft rejection[1][2] we investigated the role of cross-dressed (CD) DCs in mouse liver transplantation tolerance. Methods: Liver allografts from C57BL/6 (B6; H-2b) or B6 SJL CD45.1 mice to C3H/HeJ (C3H; H-2 k) recipients were performed. In this combination, unlike heart, skin or kidney allografts, > 90% of liver grafts are accepted without immunosuppressive therapy[3]. Graft non-parenchymal cells (NPC) and splenocytes were examined by flow cytometry and imaging cytometry on post-operative days (POD) 1, 3, 7, 14, 30, and 300. Mixed leukocyte reactions (CFSE-MLR) were also performed to assess DC function. Results: Infiltration by recipient DCs (lineage (−), CD11c (+)) in liver allografts peaked on POD 7, while donor DCs in liver grafts gradually disappeared and could not be detected by POD 7. Interestingly, more than half of the graft-infiltrating recipient DCs at that time displayed donor MHC-I, indicating cross-dressing; these DCs persisted in the graft (approx. 20 % of recipient DC) at least until POD 300. In contrast, only a very minor fraction of cross-dressed DCs (CD-DCs) (0-2%) were detected in the spleen at any time point. Control staining for donor MHC-I using naïve C3H mouse liver NPC or splenocytes was negative. Moreover, especially on POD 7, and persisting until POD 300, CD-DCs isolated from liver grafts expressed higher levels of T cell inhibitory programed death ligand 1 (PD-L1) compared to non CD-DCs (nCD-DCs). Importantly, unlike nCD-DCs, CD-DCs from liver grafts did not stimulate proliferation of allo-reactive donor T cells and markedly suppressed donor-reactive host T cell proliferation in CFSE-MLR. Conclusions: A large proportion of CD recipient DCs with capacity to subvert donor-reactive host T cell responses are evident in liver allografts early post-transplant. Moreover, cross-dressing by graft-infiltrating DCs that also express high levels of PD-L1, persists indefinitely. This suggests that graft-infiltrating host CD-DC may play a key role in regulation of alloimmunity and the promotion of donor-specific liver transplant tolerance.Figure 1: Marked reduction in donor dendritic cells (DCs) and emergence of cross-dressed host DCs in the graft after liver allo-transplantaion. (A) (B) Graft and spleen DCs were analyzed using donor recipient MHC class 1 (A) or CD45.1/ CD 45.2 antibodies (B) after B6 WT graft to C3H recipient transplatation. Data shown are representative data from 3-5 mice.Figure 2: Infiltrating host DCs and CD-DCs peak on 7 POD and PD-L1high CD-DCs persist in the graft for at least 300 POD. (A) Chronological changes in (left) the incidence of total DC and (right) the incidence of CD-DC (as % total DC) in the graft. (B) Expression of PD-L1 by CD-DCs and non (n)CD-DCs in the graft. PD-L1 expression by CD-DCs was significally higher than of nCD-DC, especially on 7POD. 3-6 mice per time point. *: P<0.05, ***: P<0.001Figure 3: Liver CD-DCs markedly sppress donor-reactive host T cell proliferation in CFSE-MLR Spleen (Sp) or liver CD-DCs or nCD-DCs isolated from graft recipients on 7 POD were co- cultured with responder (C3H (host) spleen T cells) and stimulators (B6 (donor) spleen DC) in CFSE-MLR (T cells: DC (each) =10:1). Liver CD-DCs, markedly suppressed host T cell proliferation. % T cell proliferation in indicated. Data are representative of 3 experiments that gave similar results.