Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes

Abstract

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.

Figure 1

In vitro pre-activated Foxp3⁺ Treg induce durable tolerance to folly allogeneic bone marrow grafts, (a) BALB/c hosts were grafted with B6 bone marrow and injected with BALB/c Treg pre-activated in vitro with B6 APC. Hematopoietic chimerism was assessed by FACS analysis of peripheral blood mononuclear cells (PBMC) three weeks after bone marrow transplantation. Typical FACS plots of H-2K^b vs. H-2K^d staining are shown in the upper panels. In the lower panels, the percentage of donor (H-2K^b+) cells among PBMC from individual mice is shown, (b) Phenotype of Treg before (upper panels) and after in vitro culture with donor-type APC (lower panels). In right panels, black lines indicate staining with antibody to Foxp3, gray curves staining with isotype-matched control antibodies, (c, d) B6 hosts were grafted with allogeneic donor bone marrow from indicated (c) or BALB/c (d) donors with or without Treg. Hematopoietic reconstitution was assessed at 3 weeks (c) or at indicated time points (d). Values for individual mice are shown, bars indicate means, (e) B6 hosts were grafted with a mixture of BALB/c and SJL bone marrow cells with or without Treg of indicated specificity. Hematopoietic reconstitution by cells of BALB/c (■) and SJL (□) origin was assessed at 3 weeks, (f) B6 hosts were grafted with B6 (□) or (■) DBA/2 bone marrow and injected with B6.Thy1.1 Treg cultured in vitro in presence of DBA/2 APC. At indicated time points, splenocytes were analyzed by FACS. Indicated is the percentage of Thy1.1 Treg among CD4⁺ splenocytes. The FACS plots indicate Foxp3-staining on Thy1.1⁺CD4⁺ splenocytes. Horizontal bars indicate mean values. ***P<0.001, **P<0.01 (Student’s t test).

Figure 2

Treg prevent acute and chronic skin allograft rejection, (a) Left panel: B6 recipient mice were preconditioned with sublethal irradiation only (○, n=4) or combined with injection of Treg pre-activated in vitro with donor-type (DBA/2) APC immediately after irradiation (“D 0”, ●, n=8) or three weeks later, just prior to DBA/2 (or control B6) skin transplantation (“D 21”, ▴, n=10). Control mice were irradiated and received a syngeneic skin graft (□, n=4). Skin allograft survival was monitored daily by assessment of macroscopic signs of rejection. Right panel: B6 hosts were irradiated, injected with donor DBA/2 bone marrow with (●, n=12, █, n=8) or without (▵, n=6) Treg. Three weeks later, DBA/2 (or control SJL) skins were transplanted and their survival monitored, (b) SJL hosts were irradiated, injected with B6 bone marrow and Treg cultured with B6 APC, and grafted, three weeks later, with B6 and SJL skins on opposing flanks (n=4). Survival of “target” B6 (●) and third party DBA/2 (○) skins was monitored, (c) As in (a, right panel), but Treg were pre-cultured with (B6 x DBA/2)F1 APC (▵,n=3; ●, n=8,█, n=8). (d) Scoring of infiltrates of DBA/2 skins transplanted on mice that had received DBA/2 bone marrow and Treg cultured with DBA/2 (n=12) or (B6 x DBA/2)F1 (n=8) APC. (e-h) Representative features of skin histopathology 100 days after transplantation (HE, hematoxylin and eosin; F4/80, immunohistochemistry with an antibody to F4/80; Luna, Luna’s eosinophil stain). Scale bars represent 200 μm (HE), 400 μm (F4/80), or 40 μm (Luna).

Figure 3

Treg prevent acute and chronic cardiac allograft rejection (a) B6 recipient mice were preconditioned with sublethal irradiation only (○, n=2) or with irradiation and injection of Treg pre-activated in vitro with donor-type (DBA/2) APC immediately after irradiation (“D 0”,▴, n=5) or three weeks later (“D 21”, █, n=5). Three to eight weeks later, recipient mice were transplanted with donor DBA/2 hearts. Control mice were irradiated and received a syngeneic B6 heart graft (□, n=4). Cardiac allograft survival was monitored daily (for 100 days) by abdominal palpation, (b) B6 hosts were irradiated, injected with donor DBA/2 bone marrow with (●, n=12, □, n=9) or without (▵n=2) Treg cultured in vitro with DBA/2 (●) or (B6 x DBA/2)F1 (D) APC. Three to eight weeks later, recipient mice were transplanted with donor DBA/2 hearts. Cardiac allograft survival was monitored daily, (c) Clinical score of DBA/2 cardiac allograft rejection 100 days after transplantation into sublethally irradiated hosts grafted with DBA/2 bone marrow and injected with Treg pre-activated in vitro with DBA/2 (n=12) or (B6xDBA/2)F1 (n=9) APC, as indicated. *** P< 0.001 (Student’s t test). (d,e,f) Representative features of cardiac histopathology 100 days after transplantation of B6 (d) or DBA/2 (e,f) hearts in B6 hosts. Specificity of injected Treg is indicated in the figure. Scale bar represents 200 μm in left panels and 50 μ in right panels.