Identification of regulatory T cells in tolerated allografts

Abstract

Induction of transplantation tolerance with certain therapeutic nondepleting monoclonal antibodies can lead to a robust state of peripheral "dominant" tolerance. Regulatory CD4+ T cells, which mediate this form of "dominant" tolerance, can be isolated from spleens of tolerant animals. To determine whether there were any extra-lymphoid sites that might harbor regulatory T cells we sought their presence in tolerated skin allografts and in normal skin. When tolerated skin grafts are retransplanted onto T cell-depleted hosts, graft-infiltrating T cells exit the graft and recolonize the new host. These colonizing T cells can be shown to contain members with regulatory function, as they can prevent nontolerant lymphocytes from rejecting fresh skin allografts, without hindrance of rejection of third party skin. Our results suggest that T cell suppression of graft rejection is an active process that operates beyond secondary lymphoid tissue, and involves the persistent presence of regulatory T cells at the site of the tolerated transplant.

Figure 1.

The experimental system. CBA/Ca or CP1-CBA were made tolerant to B10.BR skin grafts by treatment with nondepleting CD4 and CD8 mAbs. 100 d after tolerance induction the tolerated skin grafts, or autologous control skin, were removed and transplanted onto “empty” mice (either adult thymectomized and T cell–depleted CBA-CP1 mice, or RAG1^−/−-CBA mice). After 30 d the mice were transfused with 10⁷ splenocytes from naive CBA/Ca mice, together with a fresh B10.BR skin graft. The possible outcomes are: rejection, when a nontolerant preexisting state permits the transfused cells to mediate graft rejection; or acceptance of the skin grafts, when tolerated grafts lead to a tolerance state that is nonpermissive for graft rejection by the transfused splenocytes.

Figure 2.

Tolerated skin grafts can transfer the tolerant state upon regraft. CP1-CBA mice were thymectomized at 4 wk of age, and depleted of T cells with 0.25 mg CAMPATH-1H. (A) At day −30, these mice were transplanted with tolerated B10.BR skin grafts from tolerant CBA/Ca (▪), CBA/Ca skin from the CBA/Ca tolerant to B10.BR skin grafts (▴), or CBA/Ca skin from naive donors (▾). A control group of mice did not receive any initial skin graft (♦). All mice were transfused with 10⁷ spleen cells from naive CBA/Ca at day −1, and transplanted with a fresh B10.BR skin on the following day. Only mice with tolerated skin grafts resisted the challenge transfusion of nontolerant splenocytes and accepted the B10.BR skin grafts indefinitely (▪, n = 5, MST > 100 d, P < 0.002 to other groups). In all other groups the B10.BR skin grafts were rejected at a similar rate. (B) Tolerant mice were grafted with both BALB/c (▪) and B10.BR (▴) skin grafts in the same graft bed, 60 d after challenge with naive CBA/Ca splenocytes and a fresh B10.BR skin. Only BALB/c skin grafts were rejected (P < 0.007).

Figure 3.

Tolerance is not due to microchimerism. Empty CP1-CBA mice were transplanted at day −30 with tolerated B10.BR skin grafts from tolerant CBA/Ca (▪), (B10.BR × CBA/Ca)F₁ skin grafts (▾), or (B10.BR × CBA/Ca)F₁ skin grafts transplanted 30 d before into syngeneic hosts (▴). A control group of mice did not receive any initial skin graft (♦). All mice were transfused with 10⁷ spleen cells from naive CBA/Ca at day −1, and transplanted with a fresh B10.BR skin on the following day. Only recipients of tolerated skin grafts resisted the transfusion of nontolerant splenocytes and accepted the B10.BR skin grafts indefinitely (▪, n = 5, MST > 100 d, P < 0.002). In all other groups the B10.BR skin grafts were rejected at a similar rate.

Figure 4.

Tolerance is due to regulatory T cells present in the skin graft. Empty CP1-CBA mice were transplanted at day −30 with tolerated B10.BR skin grafts from tolerant CBA/Ca (▪), tolerated B10.BR skin grafts from tolerant CP1-CBA (▴), or (B10.BR × CBA/Ca)F₁ skin (▾). A control group of mice did not receive any initial skin graft (♦). All mice were transfused with 10⁷ spleen cells from naive CBA/Ca at day −1, and transplanted with a fresh B10.BR skin on the following day. Mice transplanted with tolerated B10.BR skin grafts from tolerant CP1-CBA (▴) were depleted of infiltrating T cells by treatment with 0.25 mg CAMPATH-1H at days −30 and −1. Recipients of tolerated B10.BR skin grafts from tolerant CBA/Ca were also treated with CAMPATH-1H as described. Only recipients of tolerated skin grafts whose T cells had not been ablated resisted the transfusion of nontolerant splenocytes and accepted B10.BR skin grafts indefinitely (▪, n = 5, MST > 100 d, P < 0.002). In all other groups the B10.BR skin grafts were rejected at a similar rate. Note that one animal in the tolerated skin, T cell–depleted group (▴) rejected the initial B10.BR graft before transfusion with CBA/Ca splenocytes.

Figure 5.

T cells expand from the tolerated B10.BR skin grafts. RAG1^−/−-CBA mice were grafted with tolerated B10.BR skin from tolerant CBA/Ca mice (▪), CBA/Ca skin from CBA/Ca mice tolerant to B10.BR skin grafts (▴), and tolerated B10.BR skin grafts from tolerant CP1-CBA mice (▾ and ♦). Tolerated B10.BR skin grafts in group ♦ were depleted of putative infiltrating T cells with 0.25 mg CAMPATH-1H at day −1. (A) Blood samples were collected 30 d after skin grafting and analyzed by FACS^®. The graph represents the percentage of CD4⁺ T cells within blood mononuclear cells. The percentage of CD4⁺ T cells that expanded from tolerated skin grafts is significantly higher than in the animals grafted with CBA/Ca skin from tolerant syngeneic donors (P < 0.05, unpaired t test). (B) FACS^® staining from a mouse of the tolerated skin group (▾), showing that expanded T cells are mainly CD4⁺CD25⁻. (C) All mice were transfused with 10⁷ spleen cells from naive CBA/Ca 1 wk after blood tests, and transplanted with a fresh B10.BR skin on the following day (day 0). Recipients of tolerated B10.BR skin grafts whose putative regulatory T cells had not been depleted resisted the challenge with transfused CBA/Ca splenocytes and accepted the B10.BR skin grafts indefinitely (▪ and ▾, MST > 100 d, P < 0.05). Mice that were recipients of tolerated B10.BR skin grafts depleted of T cells rejected the grafts shortly after transfusion of CBA/Ca splenocytes (▴, MST = 22 d). Recipients of CBA/Ca skin from CBA/Ca mice tolerated to B10.BR skin grafts also rejected B10.BR skin grafts (♦, MST = 20.5 d).