Donor HLA-DR Drives the Development of De Novo Autoimmunity Following Lung and Heart Transplantation

Abstract

Individuals harbor preexisting HLA-DR/DQ-restricted responses to collagen type V (ColV) mediated by Th17 cells under Treg control, both specific to peptides that bind to inherited HLA class II antigens. Yet after transplant, the donor-DR type somehow influences graft outcome. We hypothesized that, long after a lung or heart allograft, the particular HLA-DR type of the mismatched transplant donor transforms the specificity of the "anti-self" response. This could explain why, over long term, certain donor DRs could be more immunogenic than others.

Methods: We analyzed 7 HLA-DR15^neg patients who had received a lung allograft from a DR15⁺ donor. To determine the mechanism of acquired specificity in self-reactivity, we analyzed the kinetics of DR1 (host) and DR15 (donor) peptide restriction in a heart transplant model using DR-transgenic mice.

Results: Beyond 1.5 years post-lung transplant, all patients tested had acquired DR15-restricted immune responses to ColV peptides. These responses were either unrestrained Th17 type (n = 4) or Th17 controlled by Treg arising early (<5 y) or late (>7 y) after transplant (n = 4). Treg suppression via conventional (transforming growth factor-β [TGF-β]) and extracellular vesicle-associated (IL-35) cytokines correlated with superior outcomes. Naïve DR1 and DR15 transgenic mice had preexisting DR-restricted responses, exclusively to ColV fragments containing DR1- or DR15-binding peptides. When HLA-DR1 transgenic recipients of a HLA-DR15 heart developed ColV reactivity post-transplant, mice that acutely rejected (20-25 d) responded only to the DR1-restricted ColV peptide epitope. In animals whose grafts survived long term, we could detect acquisition of DR from the transplant donor onto the surface of recipient dendritic cells, and immune responses against a donor DR15-restricted ColV peptide.

Conclusions: These results might explain how certain donor HLA-DR types redirect host immune responses to novel peptides of critical self-antigens. Unless regulated, such responses may predispose the allograft to chronic rejection.

FIGURE 1.

Donor HLA−DR15 results in 2 types of DR15−restricted responses to ColV post-lung transplantation. A, Development of unrestrained donor HLA−DR15−restricted autoimmune responses to ColV in DR15^neg patients ≥1.5 y after lung transplant. Three patients who had received a graft from a DR15⁺ donor were analyzed by Tv−DTH. PPG26 developed responses to the DR15−restricted peptide p1049 as well as to the DR1/15 cross-reactive peptides p799 and p1439. No response to the DR1−restricted ColV peptide p629 was noted. In DR15^neg patients PPG96 and PPG99, we also found strong responses both to ColV and to the DR15−restricted peptide p1049. PPG99 also had a strong response to the donor DR1–restricted ColV peptide p629. PPG96 also responded to p629; whether this is due to peptide presentation by the donor DR is uncertain. B, Development of regulated donor DR15–restricted responses to ColV in DR15^neg patients ≥1.5 y after lung transplantation. PPG95, PPG97, and PPG98 also received a lung allograft from a DR15⁺ donor. While their PBMC did not demonstrate any ColV or ColV peptide reactivity, post-transplant neutralization of TGF−β (PPG98), IL−35 (PPG97), or both (PPG95) revealed a regulated response to ColV as well as the DR15−restricted peptide p1049 post-transplant. PBMC, peripheral blood mononuclear cells; TGF−β, transforming growth factor−β; Tv−DTH, trans-vivo delayed type hypersensitivity.

FIGURE 2.

Longitudinal analysis of the development of donor DR15–restricted ColV autoimmunity and its regulation. Patient L86 (HLA−DR1,11) received a lung transplant from a HLA 1,15 donor. Note the strong anti−ColV response at year 7, including a strong donor DR15–restricted response to p1049, as well as a DR1−restricted response to p629 as published previously (7). Tv−DTH responses in the same patient were reanalyzed at 14 y post-transplant. The recipient now had low responses to ColV and p1439 and no response to p1049. But neutralization of TGF−β and IL−35 led to an increase in responses to ColV and p1439 and uncovered a response to the DR15−restricted p1049 but not to the self (DR1)−restricted p629. PBMC, peripheral blood mononuclear cells; TGF−β, transforming growth factor−β.

FIGURE 3.

HLA−DR15 (donor)−restricted immune responses develop in transgenic mice with chronically rejecting, but not acutely rejecting, DR−transgenic murine cardiac grafts. A, Naïve HLA A2/DR1 transgenic mice received heterotopic cardiac transplants from HLA−DR15 transgenic donors. Two mice whose grafts were still beating, at a very low score, were sacrificed at day 19 and day 36 post-transplant, and 1 graft survived to day 89. All others survived long term, with evidence of chronic rejection. All 4 syngeneic grafts were accepted and survived long term. B(i), A2/DR1 recipients of the DR15 heart grafts were tested for reactivity to various antigens of interest. During both acute (5/13) and chronic rejection (6/13), all recipients developed Tv−DTH responses to ColV, with low responses seen toward vimentin and donor antigen. (ii) Mice that rejected the grafts acutely (<25 d) developed immune responses to the DR1−binding peptide p629 but not to the DR15 epitope p1049. However those rejecting chronically responded to the donor DR15–restricted peptide p1049, as well as the DR1/15 cross-reactive peptides p799 and p1439 (*Unpaired t−tests P < 0.05). Tv−DTH, trans-vivo delayed type hypersensitivity.

FIGURE 4.

Acquisition of donor HLA−DR by recipient APCs. Naïve WT B6 mice were used as recipients of cardiac grafts from HLA−DR15 transgenic mice. HLA−DR expression in the recipients was analyzed using a pan DR antibody. A, Acquisition of donor HLA−DR on graft−infiltrating dendritic cells at day 14 post-transplant: B6 recipient of a DR15 graft was sacrificed at day 14 post-transplant. Live CD45⁺ graft−infiltrating cells were analyzed for donor HLA−DR expression. HLA−DR expression was noted on the graft−infiltrating dendritic cells, as seen by the small shift in the histogram (red) as compared with the negative control (black). Red line, Test DR15 to B6; Blue, WT DR15⁺ cells; Black, WT B6 cells. B, Acquisition of donor HLA−DR on lymph node DC at day 35 post-transplant: B6 recipient rejected the DR Tg heart at day 35 post-transplant. Lymph node cells were analyzed for HLA−DR expression. HLA−DR was expressed on both mDCs and pDCs, but not on monocytes (data not shown). Red line, Test DR15 to B6; blue, WT B6 cells; black, HLA−DR PE FMO. DC, dendritic cells.