In nonautoimmune recipients, induction of mixed and complete chimerism with hematopoietic progenitor cells from MHC (HLA)-matched or -mismatched donors are effective approaches for induction of organ transplantation immune tolerance in both animal models and patients. But it is still unclear whether this is the case in autoimmune recipients. With the autoimmune diabetic NOD mouse model, we report that, although mixed and complete MHC-mismatched chimerism provide immune tolerance to donor-type islet and skin transplants, neither mixed nor complete MHC-matched chimerism does. The MHC-mismatched chimerism not only tolerizes the de novo developed, but also the residual pre-existing host-type T cells in a mismatched MHC class II–dependent manner. In the MHC-mismatched chimeras, the residual host-type peripheral T cells appear to be anergic with upregulation of PD-1 and downregulation of IL-7Rα. Conversely, in the MHC-matched chimeras, the residual host-type peripheral T cells manifest both alloreactivity and autoreactivity; they not only mediate insulitis and sialitis in the recipient, but also reject allogeneic donor-type islet and skin grafts. Interestingly, transgenic autoreactive BDC2.5 T cells from Rag1+/+, but not from Rag1−/−, NOD mice show alloreactivity and mediate both insulitis and rejection of allografts. Taken together, MHC-mismatched, but not MHC-matched, chimerism can effectively provide transplantation immune tolerance in autoimmune recipients.

Induction of mixed and complete chimerism with hematopoietic progenitor cells from HLA-matched or -mismatched donors effectively provides immune tolerance to donor-type organs such as kidney in nonautoimmune patients (18). However, it is still unknown whether this approach can be applied to autoimmune patients such as patients with type 1 diabetes (T1D) who need islet and/or kidney transplantation. Allografts can be rejected by both alloimmunity and autoimmunity in autoimmune recipients (912), pathogenic autoreactive T cells can be cross-reactive with alloantigens (13), and pathogenic autoreactive T cells in NOD mice and T1D patients possess promiscuous TCRs that have cross-reactivity (1418); but it remains unclear whether the T cells that mediate alloimmunity and autoimmunity can be the same T cells.

We recently reported that, in prediabetic autoimmune NOD mice, induction of mixed chimerism with MHC-mismatched, but not -matched, donor bone marrow (BM) transplants was able to re-establish thymic deletion of de novo developed host-type autoreactive T cells, as well as reverse autoimmunity, and prevent T1D development (19). However, whether mismatched mixed chimerism can tolerize pre-existing memory autoreactive T cells remains unclear; the impact of MHC-matched or -mismatched chimerism on organ transplantation immune tolerance in late-stage diabetic NOD mice also remains unclear. Reversal of autoimmunity in overt diabetic NOD mice is much more difficult than preventing autoimmunity in prediabetic NOD mice, and the mechanisms remain unclear. It might be because of the presence of a high percentage of memory pathogenic T cells (2024).

In the current studies, late-stage diabetic NOD mice were conditioned with radiation-free anti-CD3/CD8 conditioning and transplanted with BM and CD4+ T-depleted spleen cells from donors, as previously described (19, 25, 26), or the mice were conditioned with myeloablative total body irradiation (TBI) and transplanted with T cell–depleted (TCD) BM only. We found that, under the anti-CD3/CD8 conditioning, MHC-mismatched BM and spleen cells induced mixed or complete chimerism in a donor cell dose-dependent manner, and both provide immune tolerance to donor-type islet and skin grafts. Interestingly, MHC-matched donor BM and spleen cells could only induce mixed but not complete chimerism in diabetic NOD mice, and the mixed chimerism did not provide immune tolerance to donor-type islet or skin grafts.

In contrast, both MHC-matched and -mismatched TCD donor BM cells induced complete chimerism in diabetic NOD mice conditioned with myeloablative TBI, as judged by lack of de novo developed host-type thymocytes in the thymus or B cells/granulocytes/macrophages in the BM. Surprisingly, MHC-mismatched, but not -matched, complete chimerism was able to provide immune tolerance to donor islet and skin grafts, although both had similar percentage of residual host-type T cells that accounted for ∼5% of total T cells, and these cells were mainly CD4+ T memory cells. The residual host-type T cells in the MHC-mismatched complete chimeras appeared to be anergic with upregulation of PD-1 and downregulation of IL-7Rα. Anergic T cells were reported to upregulate PD-1 and downregulate IL-7Rα (2729).

The residual host-type T cells in the MHC-matched chimeras appeared to have both autoreactivity and alloreactivity, and mediated the graft rejection. Consistently, transgenic autoreactive T cells that possess alloreactivity mediated rejection of donor islet and skin grafts. Finally, MHC-mismatched mixed chimerism tolerized the cross-reactive transgenic autoreactive T cells and prevented rejection of donor-type skin and islet grafts. Therefore, MHC-mismatched chimerism is required for tolerizing the autoreactive T cells that possess alloreactivity and for induction of transplantation tolerance in autoimmune recipients.

Female NOD/LtJ, BDC2.5 NOD, C57BL/6, H-2g7 C57BL/6, BALB/c, and DBA/2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were maintained in a pathogen-free room at City of Hope Research Animal Facilities (Duarte, CA). The animal use procedures were approved by the institutional committee of City of Hope. Induction of chimerism, chimerism typing, skin transplantation, flow cytometry analysis, and histopathology were described in our previous publications (19, 25, 26, 30) and the Supplemental Materials and Methods. BDC2.5-Tetramer SRLGLWVRME and control tetramer AMKRHGLDNYRGYSL were obtained from the National Institutes of Health Tetramer Facility.

NOD mice >12 wk were routinely checked twice a week for urine and/or blood glucose. Mice having blood glucose levels >300 mg/dl for 3 consecutive days were diagnosed as diabetic. The diabetes of >3 wk after onset is defined as late-stage diabetes. One week after onset of diabetes, NOD mice were implanted with insulin pellets (Linshin, Toronto, ON) for control of hyperglycemia.

The late-stage diabetic NOD mice were conditioned with sequential injection of FcR- nonbinding anti–CD3-IgG3 (145-2C11-γ3) and FcR-binding (FB) anti-CD3 (145-2C11) in addition to anti-CD8 (116-13.1) Abs as previously described (25). In brief, late-stage diabetic NOD mice were conditioned on day −8 with sequential injection of FcR- nonbinding anti-CD3 and FB anti-CD3/anti-CD8 with a 12 h interval. On day −3, mice were given an injection of FB anti-CD3/anti-CD8 again. On day 0, BM and CD4+ T-depleted spleen cells from MHC-mismatched C57BL/6 (H-2b) or MHC-matched congenic C57BL/6 (H-2g7) mice were injected.

Depletion of CD4+ cell was performed with anti-CD4 micromagnetic beads and AutoMACS sorter (Miltenyi Biotec, Auburn, CA). The purity was >95%. Production of anti-CD3 and anti-CD8 Abs were previously described (30). After BM transplantation, insulin NPH (1 U every other day; Novo Nordisk) was given to keep mice alive when blood glucose level was >500 mg/dl. Chimerism was initially determined via blood screen of CD45.2, TCRβ, B220, and Mac1/Gr1, and confirmed by checking donor- and host-type B220+ cells in BM and donor- and host-type CD4+CD8+ thymocytes in the thymus, as described in our previous publications (19, 25).

Pancreatic islets were isolated from wild-type (WT) C57BL/6, congenic C57BL/6, or NOD-SCID mice after digestion with collagenase XI (Sigma) and isolated with Histopaque-1077 (Sigma). Islets sizing >50 μm were handpicked and packed into a small tubing as previously described (26). Donor islets were implanted under kidney capsule as described by others (31). Usage of low-dose islets (200 islets/mouse) was based on our previous work indicating that induction of MHC-mismatched chimerism allows for low dose of islets to reverse diabetes (26).

CD11c+ cells were enriched using anti-CD11c microbeads and passing cells over magnetic columns. Sorted Thy1.2+CD4+ T cells together with irradiated (3000 cGy) allogeneic or syngeneic CD11c+ DCs (0.1 × 106 each) from spleens were cultured in a U-bottom 96-well plate for 3 d, and [3H]thymidine deoxyribose (1 μCi/well) was added 18 h before harvest. The stimulation index was calculated using the following formula: (cpm of responder with stimulator − cpm of responder alone)/(cpm of responder alone).

Comparison of survival of skin grafts was evaluated by the log-rank test (Prism version 4.0; GraphPad, San Diego, CA). Comparison of means among multiple groups was evaluated with one-way ANOVA. Comparison of two means was analyzed using unpaired two-tailed Student t test.

Mixed chimerism was induced in late-stage diabetic NOD (H-2Kd, I-Ag7, CD45.1) mice, using MHC-mismatched WT C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC (both MHC class I [MHC I] and MHC II)-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donor BM transplants under a radiation-free anti-CD3/CD8 conditioning regimen as previously described (19, 25).

Three weeks after BM transplantation, mixed chimeric recipients were selected by checking chimerism in blood (data not shown) and then donor-type islet and skin grafts were transplanted. The mixed chimerism status was confirmed by checking cells in the spleen, thymus, and BM at the end of experiments, ∼100 d after induction of chimerism (see later). Implantation of 200 donor-type islets under the kidney capsule of MHC-mismatched mixed chimeras reversed diabetes in all (6/6) recipients, and the islet grafts showed no signs of infiltration by ∼100 d after islet or skin graft transplantation (Fig. 1A, left panels). In contrast, implantation of the same numbers of donor-type islets in the MHC-matched mixed chimeras reversed diabetes in only ∼30% (2/6) of recipients, and their islet grafts were severely infiltrated (p < 0.01; Fig. 1A, right panels). Similarly, all (6/6) donor-type skin grafts were accepted long term without signs of rejection in MHC-mismatched mixed chimeras, but none (0/6) of the skin grafts was accepted in the MHC-matched mixed chimeras, and they were rejected with severe infiltration 30–80 d after transplantation (p < 0.01; Fig. 1B).

FIGURE 1.

MHC-mismatched, but not MHC-matched, mixed chimerism provides immune tolerance to tissue transplants in autoimmune recipients. Mixed chimerism was induced in late-stage diabetic NOD (H-2Kd, I-Ag7, CD45.1) mice, using MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Three weeks after BM transplantation, 200 donor islets were implanted under kidney capsule, and skin grafts (1 × 1 cm2) were implanted on flank. Blood glucose levels and skin graft survival were monitored every other day. Insulin (1 U every other day) was given to keep mice alive when blood glucose was >500 mg/dl. Islet and skin grafts were harvested 100 d after transplantation or upon skin graft rejection. (A) Curves of blood glucose levels (top panels) and representative H&E staining pattern of islet grafts (bottom panels), n = 6. (B) Survival curves of skin grafts (top panels) and representative H&E staining pattern of skin grafts (bottom panels), n = 6. (C) Representative flow-cytometry patterns of mixed chimerism 100 d after induction of HCT (n = 6). (Left panel) CD45.2 (donor) TCRβ+ cells and B220+ cells in the spleen. (Middle panel) Gated CD45.1+ host-type and CD45.2+ donor-type thymocytes were shown in CD4 versus CD8. (Right panel) Gated BM B220+ cells were shown in CD45.2+ versus forward scatter.

FIGURE 1.

MHC-mismatched, but not MHC-matched, mixed chimerism provides immune tolerance to tissue transplants in autoimmune recipients. Mixed chimerism was induced in late-stage diabetic NOD (H-2Kd, I-Ag7, CD45.1) mice, using MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Three weeks after BM transplantation, 200 donor islets were implanted under kidney capsule, and skin grafts (1 × 1 cm2) were implanted on flank. Blood glucose levels and skin graft survival were monitored every other day. Insulin (1 U every other day) was given to keep mice alive when blood glucose was >500 mg/dl. Islet and skin grafts were harvested 100 d after transplantation or upon skin graft rejection. (A) Curves of blood glucose levels (top panels) and representative H&E staining pattern of islet grafts (bottom panels), n = 6. (B) Survival curves of skin grafts (top panels) and representative H&E staining pattern of skin grafts (bottom panels), n = 6. (C) Representative flow-cytometry patterns of mixed chimerism 100 d after induction of HCT (n = 6). (Left panel) CD45.2 (donor) TCRβ+ cells and B220+ cells in the spleen. (Middle panel) Gated CD45.1+ host-type and CD45.2+ donor-type thymocytes were shown in CD4 versus CD8. (Right panel) Gated BM B220+ cells were shown in CD45.2+ versus forward scatter.

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The mixed chimerism status was confirmed at the end of experiments, ∼100 d after induction of chimerism. In both MHC-mismatched and -matched mixed chimeras, approximately one in three of T and B cells in spleen were host type (Fig. 1C, left panels); the de novo developed CD4+CD8+ thymocytes, as well as B220+ BM cells, contained both donor- and host-type cells (Fig. 1C, middle and right panels), indicating true mixed chimeric status, as we previously defined (19). In addition, consistent with previous reports (32), donor-type islet and skin grafts were accepted in nonautoimmune BALB/c recipients after induction of mixed chimerism with MHC-matched DBA/2 BM transplants (Supplemental Fig. 1). These results demonstrate that MHC-mismatched, but not MHC-matched, mixed chimerism is able to provide immune tolerance to donor-type islet and skin transplants in autoimmune recipients, although MHC-matched mixed chimerism is only able to provide transplantation immune tolerance in nonautoimmune recipients.

T cells in NOD mice are resistant to radiation-induced apoptosis and often persist after induction of chimerism with radiation-based conditioning (31, 33). Therefore, induction of complete chimerism using TBI provides an opportunity to study T cell tolerance focused only on peripheral mechanisms. First, we tested whether MHC-matched complete chimerism provided transplantation immune tolerance to residual host-type T cells in autoimmune NOD mice, although MHC-matched mixed chimerism did not (Fig. 1). We induced complete chimerism in late-stage diabetic NOD mice with TCD donor BM cells (10 × 106) from MHC-matched or -mismatched donors after myeloablative TBI (850 cGy) conditioning, as previously described (19). To check whether radiation-resistant T cells did persist after lethal TBI conditioning and induction of chimerism, we compared the percentage of residual host-type T cells in the chimeras 3 wk after BM transplantation. This was the time when islet and skin transplantation were performed in the present studies, as described in Fig. 1. We found that there was a small and similar percentage of residual host-type T cells among blood mononuclear cells or splenocytes of MHC-mismatched or matched recipients, although the B220+ B and Mac1+/Gr1+ macrophages/granulocytes were almost all donor type (Fig. 2A, 2B). The residual host-type T cells in both recipients were predominantly (∼90%) CD4+ T cells, and most of them were CD44hiCD62Llo effector memory T cells (Fig. 2C). Consistent with complete chimerism status, there was no de novo developed host-type CD4+CD8+ T cells in the thymus or de novo developed host-type B cells in the BM of either type of recipient (Fig. 2D, 2E). These results confirm that residual T memory cells exist in the complete chimeras after myeloablative TBI conditioning and transplantation with TCD-BM from MHC-mismatched or -matched donors.

FIGURE 2.

Residual host-type memory T cells persist in complete chimeras after myeloablative TBI conditioning. Late-stage diabetic NOD mice were irradiated (850 cGy, TBI) and reconstituted with TCD-BM from MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Three weeks after HCT, chimerism levels and status of residual host-type T cells in blood, spleen, thymus, and BM were measured (n = 5–6). (A and B) Representative staining pattern showing CD45.2 (donor) versus TCRβ, B220, and Mac1/Gr1 in the blood and spleen. (C) Gated CD45.1+TCRβ+ host-type cells are shown in CD4 versus CD8; gated CD45.1+TCRβ+CD4+ cells are shown in CD62L versus CD44. (D) Gated CD45.2+ and CD45.1+ thymocytes are shown in CD4 versus CD8. (E) Gated B220+ cells in BM are shown in CD45.1 versus CD45.2.

FIGURE 2.

Residual host-type memory T cells persist in complete chimeras after myeloablative TBI conditioning. Late-stage diabetic NOD mice were irradiated (850 cGy, TBI) and reconstituted with TCD-BM from MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Three weeks after HCT, chimerism levels and status of residual host-type T cells in blood, spleen, thymus, and BM were measured (n = 5–6). (A and B) Representative staining pattern showing CD45.2 (donor) versus TCRβ, B220, and Mac1/Gr1 in the blood and spleen. (C) Gated CD45.1+TCRβ+ host-type cells are shown in CD4 versus CD8; gated CD45.1+TCRβ+CD4+ cells are shown in CD62L versus CD44. (D) Gated CD45.2+ and CD45.1+ thymocytes are shown in CD4 versus CD8. (E) Gated B220+ cells in BM are shown in CD45.1 versus CD45.2.

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Second, we tested whether the residual host-type T cells mediated islet and skin graft rejection, when the grafts were implanted 3 wk after induction of complete chimerism. We observed that islet transplantation reversed diabetes in all (6/6) MHC-mismatched recipients, and the islet grafts showed no signs of infiltration (Fig. 3A, left panels). In contrast, although islet transplantation reversed diabetes in most (5/6) of the MHC-matched recipients, all islet grafts had severe infiltration (Fig. 3B, middle panels). Similarly, whereas all (6/6) MHC-mismatched recipients accepted skin grafts for >100 d, and the grafts showed no signs of infiltration, none (0/6) of the MHC-matched recipients accepted the skin grafts, and all of the skin grafts were rejected with severe infiltration by 70 d after transplantation (p < 0.01; Fig. 3B). The complete chimerism status was confirmed 100 d after skin and islet transplantation by observing that >99% of CD4+CD8+ thymocytes in the thymus and B220+ cells in BM were donor type (Supplemental Fig. 2A). The mismatched and matched complete chimeras were also found to have similar percentage (3–6%) of residual host-type T cells in the spleen (Fig. 3C), which were predominantly (>85%) CD4+ T cells (Supplemental Fig. 2B). This confirms that the radiation-resistant memory T cells persist throughout the experimental period regardless despite the otherwise complete chimerism status in the myeloablative TBI-conditioned recipients given donor TCD-BM cells.

FIGURE 3.

MHC-mismatched, but not MHC-matched, complete chimerism provides immune tolerance to tissue transplants in autoimmune recipients. Late-stage diabetic NOD mice were irradiated (850 cGy, TBI) and reconstituted with TCD-BM from MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Additional MHC-matched groups were coinjected with purified donor CD8+ T cells (8 × 106, purity >98%). Donor-type islet and skin grafts were implanted and harvested as described previously. (A) Curves of blood glucose levels (top panels) and representative H&E staining pattern of islet grafts (bottom panels), n = 4–6. (B) Skin graft survival curves (top panel) and representative H&E staining pattern of skin grafts, n = 4–6. (C) Representative flow cytometry patterns of gated splenic TCRβ+ cells that are shown in CD45.2 (donor marker) versus forward scatter, n = 4. (D) Total thymocytes are shown in CD4 versus CD8. (Left panel) MHC-mismatched chimeras with donor-type skin graft acceptance, showing normal percentage of CD4+CD8+ thymocytes. (Middle panel) MHC-matched chimeras with donor-type skin graft rejection, showing normal percentage of CD4+CD8+ thymocytes. (Right panel) MHC-matched chimeras with additional donor CD8+ T cell infusion and donor-type skin graft acceptance, showing loss of CD4+CD8+ thymocytes.

FIGURE 3.

MHC-mismatched, but not MHC-matched, complete chimerism provides immune tolerance to tissue transplants in autoimmune recipients. Late-stage diabetic NOD mice were irradiated (850 cGy, TBI) and reconstituted with TCD-BM from MHC-mismatched C57BL/6 (H-2Kb, I-Ab, CD45.2) or MHC-matched congenic C57BL/6 (H-2Kd, I-Ag7, CD45.2) donors, respectively. Additional MHC-matched groups were coinjected with purified donor CD8+ T cells (8 × 106, purity >98%). Donor-type islet and skin grafts were implanted and harvested as described previously. (A) Curves of blood glucose levels (top panels) and representative H&E staining pattern of islet grafts (bottom panels), n = 4–6. (B) Skin graft survival curves (top panel) and representative H&E staining pattern of skin grafts, n = 4–6. (C) Representative flow cytometry patterns of gated splenic TCRβ+ cells that are shown in CD45.2 (donor marker) versus forward scatter, n = 4. (D) Total thymocytes are shown in CD4 versus CD8. (Left panel) MHC-mismatched chimeras with donor-type skin graft acceptance, showing normal percentage of CD4+CD8+ thymocytes. (Middle panel) MHC-matched chimeras with donor-type skin graft rejection, showing normal percentage of CD4+CD8+ thymocytes. (Right panel) MHC-matched chimeras with additional donor CD8+ T cell infusion and donor-type skin graft acceptance, showing loss of CD4+CD8+ thymocytes.

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Third, we tested whether residual host-type memory T cells in the MHC-matched complete chimeras mediated donor graft rejection. Accordingly, we used MHC-matched donor CD8+ T cells to deplete the residual host-type T cells. We found that donor-type CD8+ T cells depleted the residual host T cells in a dose-dependent manner, and addition of 8 × 106 donor CD8+ T cells reduced the residual host-type CD4+ T by >20-fold and reached near-complete depletion by 3 wk after BM transplantation (p < 0.01; Supplemental Fig. 2C). In further experiments, myeloablative TBI-conditioned diabetic NOD mice were transplanted with TCD-BM and CD8+ T cells (8 × 106) from MHC-matched donors. Three weeks after BM transplantation, the recipients were transplanted with donor-type islet and skin grafts. As expected, infusion of donor CD8+ T cells prevented islet and skin graft rejection in the MHC-matched complete chimeras, as compared with the recipients given donor TCD-BM alone (p < 0.01; Fig. 3A, 3B, right panels). And the prevention of graft rejection was associated with loss of residual host-type T cells in the spleen (Fig. 3C). Unfortunately, the injected donor CD8+ T cells also caused graft versus host disease (GVHD), as judged by lack of de novo developed donor-type CD4+CD8+ thymocytes (Fig. 3D), as well as mild hair loss and skin damage on the head and ears of the recipients (Supplemental Fig. 2D). Taken together, induction of complete chimerism in myeloablative conditioned late-stage diabetic NOD mice with TCD-BM from MHC-mismatched, but not MHC-matched, donors is able to tolerize residual host-type pre-existing T memory cells, and the process can take place in the absence of donor T cell–mediated graft-versus-autoimmunity (GVA) effect.

To dissect the mechanisms by which peripheral host-type memory T cells are tolerized in the complete chimeras given donor TCD-BM cells that do not mediate donor T cell–mediated GVA effect, we induced complete chimerism with TCD-BM cells from MHC-mismatched WT or MHC II−/− C57BL/6 donors or MHC-matched H-2g7 donors as described earlier. Three weeks after induction of complete chimerism, we checked the residual host-type T cells regarding surface marker changes related to T cell anergy such as upregulation of PD-1 and downregulation of IL-7Rα (2729), surface marker changes related to apoptosis such as upregulation of FAS (34), and surface marker changes related to T cell exhaustion such as upregulation of TIM-3 (35, 36). We found that, although there was no significant difference between host-type T cells from recipients given TCD-BM from MHC II−/− mismatched or MHC-matched donors, the host-type T cells from recipients given TCD-BM from MHC-mismatched WT donors upregulated PD-1 expression (p < 0.01; Fig. 4A) and downregulated expression of IL-7Rα (p < 0.01; Fig. 4B), as compared with T cells from recipients given MHC II−/− mismatched or MHC-matched donor TCD-BM cells, but no significant difference was observed with T cell expression of FAS or TIM-3 (Fig. 4C, 4D). These results indicate that donor APC expression of mismatched MHC II can tolerize residual host-type T cells.

FIGURE 4.

MHC-mismatched complete chimerism tolerizes residual host-type T cells in a mismatched MHC II–dependent manner. Three weeks after induction of complete chimerism with TCD-BM cells from MHC-mismatched, MHC-matched, or MHC-mismatched MHC II−/− donors, residual splenic host-type T cells were identified with CD45.1, CD4, and TCRβ, and measured for surface expression of PD-1, IL-7Rα, FAS, and TIM-3. One representative flow cytometry pattern and mean fluorescence (± SE) are shown. n = 3–6. (A) PD-1 expression levels. (B) IL-7Rα expression levels. (C) FAS expression levels. (D) TIM3 expression levels.

FIGURE 4.

MHC-mismatched complete chimerism tolerizes residual host-type T cells in a mismatched MHC II–dependent manner. Three weeks after induction of complete chimerism with TCD-BM cells from MHC-mismatched, MHC-matched, or MHC-mismatched MHC II−/− donors, residual splenic host-type T cells were identified with CD45.1, CD4, and TCRβ, and measured for surface expression of PD-1, IL-7Rα, FAS, and TIM-3. One representative flow cytometry pattern and mean fluorescence (± SE) are shown. n = 3–6. (A) PD-1 expression levels. (B) IL-7Rα expression levels. (C) FAS expression levels. (D) TIM3 expression levels.

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Induction of chimerism in nonautoimmune BALB/c recipients with TCD-BM cells from MHC-matched donors provided immune tolerance to donor-type islet and skin grafts (Supplemental Fig. 1A), induction of mixed chimerism (Fig. 1), even complete chimerism (Fig. 3), in autoimmune recipients with TCD-BM cells from MHC-matched donors was not able to induce tolerance. Thus, we tested whether the residual T cells in the chimeric recipients that rejected allogeneic skin and islet grafts still had autoreactivity by implanting syngeneic islet grafts from NOD-SCID donors. Accordingly, islets from NOD-SCID mice were implanted under the kidney capsule of the MHC-matched or -mismatched complete chimeras 100 d after the first donor-type islet and skin transplantation. The same experiments were also performed with mixed chimeras. Two weeks after transplantation, the islet grafts were evaluated with histopathology. We observed that, although syngeneic host-type islet grafts in the MHC-mismatched complete chimeras showed no signs of infiltration, the islet grafts in the MHC-matched complete chimeras showed severe infiltration (p < 0.01; Fig. 5A, upper panels). Similar results were also observed with the mixed chimeras (p < 0.01; Fig. 5A, lower panels). Autoimmune NOD mice usually have sialitis (37). Whereas MHC-mismatched complete or mixed chimeras eliminated sialitis, consistent with our previous report (25), MHC-matched complete or mixed chimeras still had severe sialitis (p < 0.01; Fig. 5B). The results indicate that the T cells in the MHC-matched chimeric NOD recipients that reject donor-type islet and skin grafts appear to have both alloreactivity and autoreactivity. The results also indicate that MHC-mismatched chimerism, either mixed or complete, can tolerize the peripheral residual T cells that possess both autoreactivity and alloreactivity.

FIGURE 5.

MHC-mismatched mixed or complete chimerism is required for tolerizing host-type autoreactive T cells. One hundred days after donor islet and skin transplantation, 200 islets from host-type syngeneic NOD-SCID mice were implanted under the capsule of the other kidney of the mixed or complete chimeras. Two weeks after second islet transplantation, the grafts and the salivary glands of the recipients were harvested for histopathology and representative H&E staining histopathology is shown. n = 6. (A) NOD-SCID islet grafts; (B) salivary glands. The graft sites are circled and arrows indicate areas of infiltration. Original magnification ×200.

FIGURE 5.

MHC-mismatched mixed or complete chimerism is required for tolerizing host-type autoreactive T cells. One hundred days after donor islet and skin transplantation, 200 islets from host-type syngeneic NOD-SCID mice were implanted under the capsule of the other kidney of the mixed or complete chimeras. Two weeks after second islet transplantation, the grafts and the salivary glands of the recipients were harvested for histopathology and representative H&E staining histopathology is shown. n = 6. (A) NOD-SCID islet grafts; (B) salivary glands. The graft sites are circled and arrows indicate areas of infiltration. Original magnification ×200.

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To directly test whether T cells that possess both autoreactivity and alloreactivity were tolerized after induction of chimerism, we turned to transgenic BDC2.5 NOD mice. Autoreactive BDC2.5 CD4+ T cells express Vβ4Vα1+ TCRs that recognize chromogranin A (38, 39). We observed that sorted BDC2.5 CD4+ T cells from Rag1+/+, but not Rag1−/−, NOD mice proliferated vigorously in response to MHC-mismatched C57BL/6 DC stimulation, although none of them proliferated to syngeneic DC stimulation in the absence of exogenous autoantigens (p < 0.01; Fig. 6A). CD4+ T cells from both Rag1+/+ and Rag1−/− NOD mice were BDC2.5-tetramer+, indicating they both retained the autoreactive TCR (Fig. 6B). These results indicate that BDC2.5 CD4+ T cells from Rag1+/+ NOD mice possess both autoreactivity and alloreactivity, but BDC2.5 CD4+ T cells from Rag1−/− NOD mice possess autoreactivity only.

FIGURE 6.

MHC-mismatched chimerism tolerized transgenic autoreactive CD4+ T cells that possess alloreactivity and mediate rejection of donor-type islet and skin transplants. (A) Thy1.2+CD4+ T cells were sorted, respectively, from spleen of prediabetic BDC2.5 Rag1+/+ and BDC2.5 Rag1−/− NOD mice. The sorted T cells were cultured with DCs (0.1 × 106 each per well) from syngeneic WT NOD or MHC-mismatched WT C57BL/6 mice. The proliferation was measured by [3H]thymidine incorporation. Stimulation indexes (mean ± SE) from three replicate experiments are shown. (B) Representative staining pattern of T cells among blood mononuclear cells from BDC2.5 Rag1+/+ and BC2.5 Rag1−/− mice. Gated CD4+ TCRβ+ cells are shown in histogram of BDC2.5-Tetramer versus control tetramer. (C) BDC2.5 Rag1+/+ or BDC2.5 Rag1−/− NOD mice were transplanted with skin grafts from MHC-mismatched C57BL/6 donor. Skin graft survival curves (top panel) and representative H&E staining of skin grafts are shown. Original magnification ×200. n = 4. (D) Mixed chimerism was induced in BDC2.5 NOD mice, using MHC-mismatched C57BL/6 or MHC-matched congenic C57BL/6 donors. Donor skin and islet grafts were implanted and harvested as described earlier. The survival curves of skin grafts and representative H&E staining patterns are shown for each group, and a representative H&E staining pattern of donor-type islet grafts is shown as well (n = 4–6). Original magnification ×200.

FIGURE 6.

MHC-mismatched chimerism tolerized transgenic autoreactive CD4+ T cells that possess alloreactivity and mediate rejection of donor-type islet and skin transplants. (A) Thy1.2+CD4+ T cells were sorted, respectively, from spleen of prediabetic BDC2.5 Rag1+/+ and BDC2.5 Rag1−/− NOD mice. The sorted T cells were cultured with DCs (0.1 × 106 each per well) from syngeneic WT NOD or MHC-mismatched WT C57BL/6 mice. The proliferation was measured by [3H]thymidine incorporation. Stimulation indexes (mean ± SE) from three replicate experiments are shown. (B) Representative staining pattern of T cells among blood mononuclear cells from BDC2.5 Rag1+/+ and BC2.5 Rag1−/− mice. Gated CD4+ TCRβ+ cells are shown in histogram of BDC2.5-Tetramer versus control tetramer. (C) BDC2.5 Rag1+/+ or BDC2.5 Rag1−/− NOD mice were transplanted with skin grafts from MHC-mismatched C57BL/6 donor. Skin graft survival curves (top panel) and representative H&E staining of skin grafts are shown. Original magnification ×200. n = 4. (D) Mixed chimerism was induced in BDC2.5 NOD mice, using MHC-mismatched C57BL/6 or MHC-matched congenic C57BL/6 donors. Donor skin and islet grafts were implanted and harvested as described earlier. The survival curves of skin grafts and representative H&E staining patterns are shown for each group, and a representative H&E staining pattern of donor-type islet grafts is shown as well (n = 4–6). Original magnification ×200.

Close modal

We tested whether BDC2.5 Rag1+/+ and Rag1−/− NOD mice could reject skin grafts from MHC-mismatched C57BL/6 donors. Consistent with a previous publication (38), BDC2.5 Rag1−/− NOD mice rapidly developed diabetes ∼2–3 wk after birth (data not shown). For BDC2.5 Rag1−/− mice to survive long enough to go through the skin graft experiments, 15-d-old pups were injected with 50 μg anti-CD3 and then transplanted with skin grafts 4 wk later. BDC2.5 NOD mice in Rag1+/+ background rejected the MHC-mismatched skin graft within 20 d with severe infiltration (Fig. 6C), and consistent with their autoreactive nature, these mice also had infiltration in native islets (Supplemental Fig. 3A). In contrast, the BDC2.5 Rag1−/− NOD mice did not reject or infiltrate the skin grafts (p < 0.05; Fig. 6C), although all four mice developed diabetes despite the initial anti-CD3 injection (Supplemental Fig. 3B). These results indicate that transgenic T cells that possess both autoreactivity and alloreactivity can mediate rejection of allografts.

Finally, we induced mixed chimerism in BDC2.5 Rag1+/+ NOD mice with BM cells from MHC-mismatched or -matched C57BL/6 donors (Supplemental Fig. 3C, 3D) and tested whether the mixed chimeras rejected or accepted donor-type skin and islet grafts. We observed that induction of MHC-mismatched, but not MHC-matched, mixed chimerism in those BDC2.5 NOD mice prevented rejection of donor-type skin (p < 0.01; Fig. 6D). The donor skin grafts in the MHC-mismatched chimeras showed no infiltration, but those in the MHC-matched chimeras showed severe infiltration (Fig. 6D, upper right panels). Similarly, the donor islet grafts in the MHC-mismatched chimeras showed no infiltration, but those in the MHC-matched chimeras showed severe infiltration (Fig. 6D, lower right panels). These results demonstrate that MHC-mismatched, but not MHC-matched, mixed chimerism can tolerize residual peripheral autoreactive T cells that possess alloreactivity and mediate rejection of allografts.

With the autoimmune NOD mouse models of islet and skin transplantation, we have observed that: 1) T cells that possess both autoreactivity and alloreactivity play an important role in mediating rejection of allografts; 2) MHC-mismatched, but not MHC-matched, chimerism can tolerize the radiation-resistant residual host-type T cells in the periphery of autoimmune NOD recipients conditioned with myeloablative TBI; 3) MHC-mismatched mixed chimerism is sufficient, but MHC-matched mixed or even complete chimerism is not sufficient for mediating transplantation immune tolerance in autoimmune recipients; and 4) MHC-mismatched, but not MHC-matched, chimerism can tolerize the autoreactive T cells that also possess alloreactivity.

It has been shown that MHC-matched mixed chimerism is sufficient to tolerize alloreactive T cells and provides immune tolerance to donor-type organ transplants in nonautoimmune laboratory animals and humans (1, 3, 5, 40). However, we found that MHC-matched mixed or even complete chimerism is not sufficient to tolerize the autoreactive T cells that mediate rejection of donor-type islet or skin transplants. It is of interest that induction of mixed chimerism in a systemic autoimmune lupus patient with HLA-matched donor BM was not able to reverse autoimmunity, and the patient still had a lupus flare (3). Thus, our observation is of potential clinical significance.

We also observed that the radiation-resistant residual host-type T cells are with memory phenotype, namely, autoreactive T memory cells. Those autoreactive T cells appeared to possess alloreactivity and mediate rejection of allogeneic islet and skin grafts, as elimination of the residual host-type CD4+ T cells was able to prevent rejection of donor-type skin graft in the MHC-matched complete chimeras. Consistently, transgenic autoreactive BDC2.5 T cells from WT Rag1+/+, but not from Rag1−/−, NOD mice possessed alloreactivity and mediated allograft rejection. Also, MHC-mismatched, but not MHC-matched, mixed chimerism can tolerize those autoreactive T cells with alloreactivity. These suggest that T cells that possess both autoreactivity and alloreactivity may play an important role in autoimmune pathogenesis and in allograft rejection in autoimmune recipients. Consistently, we also observed that in chronic graft versus host disease (GVHD) recipients, there were alloreactive CD4+ T clones that possess autoreactivity and mediate autoimmune-like chronic GVHD (41). Our observations are also consistent with others’ reports that autoreactive or alloreactive T cells can express promiscuous TCRs that are capable of responding to more than one Ag (13, 4245). In addition, recent reports indicate that some autoreactive T cells can express more than one TCR as a means of surviving thymic negative selection (46). T cells that have dual TCRs play an important role in mediating GVHD (47, 48). Previous reports indicate that autoreactive T cells in T1D recipients of islet transplants play an important role in mediating the rejection of islet transplants (12). Our observations provide some new insights into the potential mechanisms for these observations. Whether the autoreactive T cells that mediate allograft rejection express one promiscuous TCR or express dual TCRs are subject to future studies.

It is difficult to tolerize T memory cells that have alloreactivity and mediate allograft rejection (49). We found that MHC-matched chimerism cannot tolerize the residual radiation-resistant autoreactive T memory cells with alloreactivity in the periphery, but infusion of donor CD8+ T cells could eliminate those T memory cells via GVA effect. Unfortunately, consistent with our recent report (50, 51), the alloreactive donor CD8+ T cells also caused chronic GVHD in TBI-conditioned recipients. In contrast, MHC-mismatched chimerism induced with donor TCD-BM can tolerize the radiation-resistant autoreactive T memory cells in the periphery in a mismatched MHC II–dependent manner. Also, the interaction with mismatched MHC II appeared to induce anergy of the autoreactive CD4+ T memory cells with alloreactivity, as suggested by the residual T cell upregulation of PD-1 and downregulation of IL-7Rα expression. Future functional studies will be required for testing this hypothesis, and for determining the molecular mechanisms by which mismatched MHC triggers this process.

We should point out that it is clear that residual host-type CD4+ T cells in MHC-mismatched complete chimeras cannot, but the residual host-type CD4+ T cells in the MHC-matched complete chimeras can mediate rejection of donor-type skin and islet grafts. However, the mechanisms regarding how MHC-mismatched compete chimerism prevents residual host-type T cell rejection of donor-type skin and islet grafts are less clear. Besides T cell tolerance induced by interaction with mismatched MHC II on donor APCs, there are alternative explanations. First, although we used donor TCD-BM to establish complete chimerism, we still cannot totally exclude the possibility of GVA effect on the residual host-type T cells mediated by residual donor-type T cells or NK cells in the graft. However, GVA effect usually manifests with elimination or marked reduction of the residual host-type T cells. But in the MHC-mismatched complete chimeras, the residual host-type CD4+ T cells existed with relative stable percentage even 100 d after induction of complete chimerism. Thus, the GVA effect is probably minimal in TBI-conditioned recipients given TCD-BM. Second, residual host-type T cells cannot recognize donor-type alloantigen in the MHC-mismatched NOD chimeras in the absence of host-type DCs, because NOD mice were reported to have potent indirect CD4+ T cell response to alloantigens (52). We have observed that in the MHC-mismatched mixed chimeric NOD recipients, the presence of similar percentage of host-type CD4+ T cells did not mediate any rejection either. Thus, prevention of rejection by lack of recognition of alloantigen by residual host-type CD4+ T cells in the MHC-mismatched complete chimeras is also unlikely. Therefore, our results suggest that MHC-mismatched chimerism, that is, MHC-mismatched donor MHC II, can tolerize alloreactive T cells in the chimeric recipients, and future studies are required to figure out the molecular mechanisms that drive this tolerance.

Expression of protective MHC II molecules by thymic DCs can mediate deletion of autoreactive T cells (53, 54), and our previous report showed that MHC-mismatched chimerism mediated thymic deletion of de novo developed host-type autoreactive T cells (19). Based on our previous publications and present results, we conclude that donor APC with mismatched MHC can not only tolerize the radiation-resistant residual memory autoreactive T cells in the periphery, but also the de novo developed autoreactive thymocytes. This provides an explanation on how MHC-mismatched mixed and complete chimerism both can provide immune tolerance to donor-type islet and skin transplants in autoimmune recipients. Whether particular donor and host MHC combination is required for induction of transplantation tolerance via induction of mixed chimerism is a subject of future studies.

In summary, this study has revealed an important basic requirement for induction of mixed chimerism as an approach for establishing organ transplantation immune tolerance in autoimmune recipients. This study suggests that a donor carrying mismatched autoimmune-resistant HLA loci might be required for induction of organ transplantation tolerance in an autoimmune patient via induction of either mixed or complete chimerism.

We are grateful to Dr. Arthur Riggs for continuous encouragement and support of this research. We thank Lucy Brown at the City of Hope (COH) Flow Cytometry Facility and Sofia Loera at the COH Anatomic Pathology Laboratory for excellent technical assistance, and we thank Dr. Richard Ermel and his staff at the COH Research Animal Facility for providing excellent animal care. We thank the National Institutes of Health Tetramer Facility for producing the BDC2.5 Tetramer and Control Tetramer used in this article.

This work was supported by the Excellence Award of the Beckman Research Institute of City of Hope and a private donation from the DePasquale family.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

COH

City of Hope

FB

FcR-binding

GVA

graft-versus-autoimmunity

GVHD

graft-versus-host disease

MHC I

MHC class I

TBI

total body irradiation

TCD

T cell–depleted

T1D

type 1 diabetes

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data