Allogeneic T regulatory cell-mediated transplantation tolerance in adoptive therapy depends on dominant peripheral suppression and central tolerance

Abstract

T regulatory cells (Tregs) represent agents to mediate tolerance to allografts so that the use of immunosuppressive drugs is avoided. In this regard, we previously demonstrated that the adoptive transfer of allogeneic Tregs into IL-2Rbeta(-/-) mice prevented autoimmunity and led to allograft tolerance. Here, we investigated the requirements and mechanisms that favor this long-lasting tolerance. The most potent tolerance required exact matching of all alloantigens between the adoptively transferred allogeneic Tregs and allogeneic skin grafts, but tolerance to such allografts that lacked expression of major histocompatibility complex class I or II molecules also occurred. Thus, Tregs are not required to directly recognize major histocompatibility complex class II alloantigens to suppress skin transplant rejection. Depletion of allogeneic Tregs substantially, but not completely, abrogated this form of tolerance. However, thymocytes from allogeneic Treg adoptively transferred IL-2Rbeta(-/-) mice did not reject the corresponding allogeneic skin graft in secondary Scid recipients. Consistent with a requirement for a deletional mechanism in this IL-2Rbeta(-/-) model, a small number of wild-type T cells readily abrogated the immune tolerant state. Collectively, these findings indicate that full tolerance induction is largely dependent on substantial Treg-mediated suppression and thymic deletion of alloreactive T cells and may represent general conditions for Treg-mediated transplantation tolerance.

Figure 1

Skin graft survival when the donor Treg cells and allograft differ in only a single MHC class I or II alloantigen. Eight- to 12-week-old BALB/c IL-2Rβ^−/− mice that were adoptively treated with the indicated population of Treg cells at birth (denoted as the donor Treg strain→IL-2Rβ^−/− recipient strain) received BALB/c, C3H, and B6^bm3 (B6^bm3) or B6^bm12 (B6^bm12) skin grafts. (A) Tolerance to B6^bm3 skin. (B) Tolerance to B6^bm12 skin. The number of mice/group, combined from 2 independent experiments, and the MST of the allogeneic skin grafts are listed within each panel of the figure.

Figure 2

Skin graft survival when the donor Treg cells and allograft differ in major or minor alloantigens. Eight- to 12-week-old B6 IL-2Rβ^−/− mice that were adoptively treated with the indicated population of Treg cells at birth (denoted as the donor Treg strain→IL-2Rβ^−/− recipient strain) received BALB/c, C3H, B6^bm3, and B6^bm12 (A) or BALB/c, BALB.B, B6, and C3H (B) skin grafts. (A) Tolerance to B6^bm3 skin. (B) Tolerance to BALB.B skin. The number of mice/group from a single experiment and the MST of allogeneic skin grafts are listed within each panel of the figure.

Figure 3

Skin graft survival when the allograft lacks surface expression of MHC class I or II molecules. Eight- to 12-week-old BALB/c IL-2Rβ^−/− mice that were adoptively treated with the indicated population of Treg cells at birth (denoted as the donor Treg strain→IL-2Rβ^−/− recipient strain) received skin grafts from BALB/c, C3H, and B6 MHC-deficient mice. (A) Tolerance to B6 MHC class II–deficient (B6 II^−/−) skin. (B) Tolerance to MHC class I–deficient (B6 β₂m^−/−) skin. The number of mice/group from a single experiment and the MST of allogeneic skin grafts are listed within each panel of the figure.

Figure 4

Skin graft survival in the absence of donor Treg cells. Eight-week-old B6 Rag2^−/−/γc^−/− mice (H-2^b) received 7 × 10⁶ unfractionated (A) or H-2^d donor cell–depleted (B) spleen cells from BALB/c→B6^−/− mice. Rag2^−/−/γc^−/− recipients were then transplanted with BALB/c, C3H, and B6 (B6) skin 2 days after the cell transfer. Mice were monitored up to 90 days after which they were sacrificed to assess the presence of the donor Treg cells. MST of allogeneic skin grafts are listed within each panel of the figure. Data represent 9 mice/group, combined from 2 independent experiments.

Figure 5

MLR by T cells or APCs from IL-2Rβ^−/− mice that were adoptively transferred with allogeneic Treg cells. (A) MLR responses by WT and recipient-derived T cells to allogeneic APCs. The source of the responding T cells is listed below the x-axis. Unfractionated spleen cells (2 × 10⁵) isolated from WT BALB/c and BALB/c→BALB/c^−/− mice or spleen cells depleted of H-2^b+ donor cells (2 × 10⁵) isolated from B6→BALB/c^−/− mice were cultured with BALB/c (left), B6 (center), or C3H (right) APCs (2 × 10⁵) for 96 hours. (B) MLR response of WT B6 T cells to APC derived from the spleen of indicated mice as listed below the x-axis. ³H-thymidine was added in the last 6 hours of culture. Data represent the mean ± SEM of 2 to 3 independent experiments with the use of mice that were either tolerant skin graft recipients (B6→BALB/c^−/−) or mice that rejected both donor skin (BALB/c^+/+ and BALB/c→BALB/c^−/−).

Figure 6

Central tolerance in IL-2Rβ^−/− mice that were adoptively transferred with allogeneic Treg cells. Eight-week-old BALB/c^scid mice (H-2^d) received a 4:1 mixture of FACS-sorted SP CD4⁺ and CD8⁺ cells (0.5 × 10⁶ total) isolated from the thymus of WT BALB/c, BALB/c→BALB/c^−/−, or B6→BALB/c^−/− mice, the latter of which were further sorted to exclude any donor H-2^b+ cells. BALB/c^scid recipients were then transplanted with BALB/c, C3H, and B6 donor skin 2 days later. Mice were monitored up to 90 days, after which they were sacrificed to assess the presence of Treg cells. (A). Skin graft survival in BALB/c^scid recipients that received the indicated donor thymocytes. The number of mice in each group of recipients and the MST of allogeneic skin grafts are listed within each the panel. (B) Rejection score of B6 skin grafts. (C) Percentage of CD4⁺Foxp3⁺ Treg cells in the whole spleen of individual recipient mice. (D) Percentage of Treg cells within the splenic CD4⁺ T-cell subset. Staining for MHC class I^d and I^b confirmed that all CD4⁺ Foxp3⁺ T cells were of H-2^d origin. Data are 4 to 5 mice/group from a single experiment.

Figure 7

Ability of naive T cells to break skin graft tolerance of IL-2Rβ^−/− mice that were adoptively transferred with allogeneic Treg cells. Eight-week-old Rag2^−/−/γc^−/− mice (H-2^b) received 7 × 10⁶ unfractionated spleen (SP) cells isolated from BALB/c→B6^−/− mice along with the indicated numbers of unfractionated (A-B) or CD25-depleted (C) naive T cells isolated from IL-2Rβ^−/− (A) or WT CD45.1 B6 congenic (B-C) mice. The naive T cells were from autoimmune-free IL-2Rβ^−/− mice due to the transfer of syngeneic Treg cells at birth. Rag2^−/−/γc^−/− recipients were then transplanted with BALB/c, C3H, and B6 skin 2 days later. The number of mice in each group of recipients, combined from 2 independent experiments, and the MST of skin grafts are listed within each the panel.