Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model

Abstract

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti-T-cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti-T-cell receptor (TCR) alphabeta mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low-dose whole body irradiation, AKR bone marrow transfer (BMT), and anti-CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti-TCR alphabeta antibody, low-dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151.2+/-15.3 days). Mice given the combination of anti-TCR alphabeta mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116.7+/-17.6 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR alphabeta mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.

Figure 1

Kinetics of the chimeric state of donor-derived cells after transfer of fully allogeneic bone marrow cells in the (a) group IV (b) group V, and (c) group VI mice. Treatment and conditions of bone marrow transfer (BMT) are noted in Table 1. Peripheral blood leucocytes were examined by two-color flow cytometry. Donor derived H-2K^k positive cells are plotted as a percentage of all peripheral blood leucocytes. In group VI, a stable high chimerism was obtained compared to the other groups. The asterisks (*) in (b) and (c) indicate the time point at which the skin graft was rejected. The asterisks in (a) indicate that the skin graft was rejected on day 140 (15 weeks after BMT) in the group IV mice.

Figure 2

Results of the mixed leucocyte reaction of spleen cells obtained from recipient mice subject to each treatment protocol, 7 weeks after AKR bone marrow transfer (BMT) (day 84). Treatment and conditions of bone marrow transfer (BMT) are noted in Table 1. In Groups IV, V and VI, the mixed leucocyte reaction was significantly suppressed donor-specifically (*; P < 0·01). For each treatment protocol, the spleens of two mice were analysed, and similar results were obtained.

Figure 3

Skin graft survival after various combinations of treatments. Treatment and conditions of bone marrow transfer (BMT) are noted in Table 1. Skin graft survival was significantly prolonged in the group IV mice than in the other treatment groups.

Figure 4

Up-regulation of CD25 (IL-2Rα) surface expression on spleen cells 24 hr after anti-CD3 mAb administration. After withdrawal from tacrolimus, B6 recipients were treated with treatment protocol IV or VI. Twenty-four hours after anti-CD3 mAb treatment (group IV mice were administered vehicle only), the spleens were removed and the peripheral cells were examined by flow cytometry. Each bar represents the mean±SD of the results from four mice. The group VI mice had a significantly larger percentage of IL-2Rα-positive lymphocytes than the group IV mice.

Figure 5

Suppressed MLR in long-lasting tolerant mice. (a) Group IV tolerant mice; (b) group IV rejected mice. Spleen cells were obtained from recipient mice between day 168 and 185, and incubated with irradiated AKR or BALB/c spleen cells for four days. The MLR of the spleen cells of the long-lasting, group IV tolerant mice, was significantly suppressed donor-specifically (*; P < 0·01). The MLR response of spleen cells obtained from group IV rejected mice, had recovered. Similar results were observed in the group V tolerant and rejected mice, and group VI rejected mice.

Figure 6

Suppressed cytotoxic activity in group IV tolerant mice on day 185. Cytotoxic activity was assayed by the standard ⁵¹Cr-release method. Spleen cells were obtained from recipient mice between day 168 and 185 and used as the effector cells. ⁵¹Cr-labelled AKR or BALB/c spleen cells (concanavalin A-blasts) were used as the target cells. Cytotoxic activity was suppressed donor-specifically in the group IV tolerant mice.