Donor-Specific Regulatory T Cells Acquired from Tolerant Mice Bearing Cardiac Allograft Promote Mixed Chimerism and Prolong Intestinal Allograft Survival

Abstract

The induction of donor-specific transplant tolerance has always been a central problem for small bowel transplantation (SBT), which is thought to be the best therapy for end-stage bowel failure. With the development of new tolerance-inducing strategies, mixed chimerism induced by co-stimulation blockade has become most potent for tolerance of allografts, such as skin, kidney, and heart. However, a lack of clinically available co-stimulation blockers has hindered efficient application in humans. Furthermore, unlike those for other types of solid organ transplantation, strategies to induce robust mixed chimerism for intestinal allografts have not been fully developed. To improve current mixed chimerism induction protocols for future clinical application, we developed a new protocol using donor-specific regulatory T (Treg) cells from mice with heart allograft tolerance, immunosuppressive drugs which could be used clinically and low doses of irradiation. Our results demonstrated that donor-specific Treg cells acquired from tolerant mice after in vitro expansion generate stable chimerism and lead to acceptance of intestinal allograft. Increased intragraft Treg cells and clonal deletion contribute to the development of SBT tolerance.

Keywords: bone marrow transplantation; donor-specific regulatory T cells; mixed chimerism; small bowel transplantation; transplantation tolerance.

Figure 1

In vitro expansion of CD4⁺CD25⁺ Treg cells and comparison with immunosuppressive function of expanded Treg cells. (A) Schematic drawing of the protocol to acquire DSTreg cells from tolerant mice bearing heart allografts. (B) Fresh CD4⁺CD25⁺ T cells and DSTreg cells (5 × 10⁴) were separated by magnetic bead sorting and cultivated in the presence or absence of IL-2 (1000 U/ml), splenic donor APCs (10⁵ cells), and rapamycin (100 nM), or in combination for 7 days at 37°C in 5% CO₂. DSTreg cells proliferated more efficiently in response to the indicated stimulation compared with fresh CD4⁺CD25⁺ T cells as determined by CFSE-labeled cell proliferation assay in vitro. (C) Representative FACS blot depicting Foxp3 expression among CD4⁺ T cells from naïve or tolerant mice after in vitro cultivation. (D) Fresh CD4⁺CD25⁺ T cells, ex vivo-expanded fresh CD4⁺CD25⁺ T cells, DSTreg cells from tolerant mice, and ex vivo-expanded DSTreg cells were assayed for suppressive activity in response to BALB/c spleen cells and third-party spleen cells (C3H) (E). All curves are expressed as percentage of control MLR and represent the combined average values of five individual experiments. Data are shown as mean ± SD (n = 3–5 mice/group). One representative of at least two independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.005 compared among the indicated groups.

Figure 2

DSTreg cell treatment together with low-dose irradiation leads to multilineage mixed chimerism. (A) Groups of C57BL/6J mice were transplantated with fully mismatched BALB/c BM cells (2 × 10⁷) under low doses of irradiation (3 Gy total body irradiation at day −1), co-stimulation blockade with abatacept (CTLA-4-Ig) (0.05 mg at day 2), and three doses of rapamycin (0.1 mg at days 1, 0, and 2) were additionally treated with or without different numbers of expanded DSTreg cells, or expanded fresh Treg cells at day 0. Percentages of successfully induced chimeras are shown. Chimerism was considered to be established if donor cells were detectable by flow cytometry within both the myeloid lineage and at least one lymphoid lineage for the duration of follow-up. (B) Hematopoietic reconstitution was assessed at 3 weeks after BMT. Values for individual mice are shown; bars indicated means. (C) Typical FACS plots of H-2K^b (recipient) versus H-2K^d (donor) staining were carried out 3 weeks after BMT. (D) Donor (H-2D^d) chimerism among leukocytes was assessed by flow cytometry of peripheral blood at multiple time points (2, 4, 8, 12, 16, 20, and 24 weeks post-BMT) and is shown as mean percentage. *P < 0.05, **P < 0.01, ***P < 0.005 compared among the indicated groups.

Figure 3

Chimeras induced by DSTreg cells developed full donor-specific intestine allograft tolerance, and tolerant chimeras displayed hypo-inflammatory responses in the intestinal allografts. (A) Graft survival of intestinal allografts. (B) Representative hematoxylin-and-eosin-stained (original magnification: ×100) sections of intestinal allografts from recipients treated with low-dose irradiation (3 Gy) and BM cells, with or without expanded DSTreg cells, and expanded fresh Treg cells (14 days post-SBT). (C) Allografts were assigned an acute rejection score by a blinded pathologist. (D,E) Cytokine concentrations in the recipient intestinal allografts were measured by ELISA on days 1, 5, 7, and 11 after SBT. Data are shown as mean ± SD (n = 4–7). One representative of at least two independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.005 compared among the indicated groups.

Figure 4

Regulatory mechanism and clonal deletion contribute to intestinal allograft acceptance in chimeras treated with expanded DSTreg cells. (A) Donor reactivity was assessed in MLRs at 8 weeks post-BMT. Donor-specific responses and third-party reactivity were measured in each group. Simulation indices were calculated by dividing the mean cpm from responses against recipient (C57BL/6J), donor (BALB/c), or third-party (C3H) stimulator cells by mean background cpm (i.e., cpm with no stimulator population). The percentage of CD4⁺Foxp3⁺ cells in recipient allografts (B,C), spleen (D), and peripheral blood (E) was measured by multicolor flow cytometry on day 14 after SBT. (F) Deletion of donor-reactive T cells in chimeras was shown by assessing percentages of Vβ11, Vβ5, and Vβ8. Multicolor flow cytometry was used for measurement in selected mice at 8 weeks post-BMT. Chimeras treated with expanded DSTreg cells showed significant peripheral and central clonal deletion among donor-reactive T cells, as measured by percentage of Vβ11 and Vβ5 (but not Vβ8). Data are shown as mean ± SD (n = 4–6). One representative of at least three independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.005 compared among the indicated groups.

Figure 5

Mixed chimeras of DSTreg cell infusion group received donor-specific skin grafts and retained normal immune response to third-party grafts. Skin transplantation was performed 100 days after SBT in recipients (C57BL/6) treated with DSTreg cells, and survival curve of skin grafts from donor-specific Balb/c mice and C3H mice was shown. Donor-specific Balb/c skin grafts survived in most chimeras, whereas C3H skin was rejected. *P < 0.05, **P < 0.01, ***P < 0.005 compared among the indicated groups.