Application of cyclophosphamide-induced tolerance in α1,3-galactosyltransferase knockout mice presensitized with Galα1-3Galβ-4-GlcNAc antigens

PURPOSE: Hyperacute rejection (HAR) mediated by the natural antibody (nAb) against Galalpha1-3Galbeta-4-GlcNAc (alphaGal) is the major obstacle in xenogeneic organ transplantation. Previously, we reported the acceptance of donor heart grafts in anti-alphaGal nAb-producing galactosyltransferase knockout (GalT KO) mice after cyclophosphamide (CP)-induced tolerance conditioning. In the present study, we applied our tolerance induction conditioning in presensitized recipient mice. METHODS: GalT KO (alphaGal(-/-), H-2(b/d)) recipient mice were presensitized with alphaGal(+) rabbit red blood cells (RRBCs). Presensitized or nonsensitized recipient mice were treated with CP-induced tolerance conditioning, consisting of AKR (alphaGal(+/+), H-2(k)) spleen cells (SC), CP, busulfan (BU), and AKR bone marrow cells (BMC). We assessed the survival of donor hearts and skin grafts and analyzed the production of anti-alphaGal Abs by flow cytometry. RESULTS: Donor mixed chimerism was achieved in the presensitized GalT KO mice treated with CP-induced tolerance conditioning. In parallel with the disappearance of anti-alphaGal Abs, permanent acceptance of donor heart grafts and skin grafts was observed in presensitized and GalT KO mice treated with CP-induced tolerance conditioning. CONCLUSIONS: Both B-cell and T-cell tolerance was achieved in the presence of a higher titer of anti-alphaGal Abs after treatment with CP-induced tolerance conditioning.     

Donor T cells are not required for induction of allograft tolerance in mice treated with antilymphocyte serum, rapamycin, and donor bone marrow cells

BACKGROUND: Postgraft infusion of donor bone marrow cells (BMC) effectively induces tolerance to skin allografts in antilymphocyte serum- and rapamycin-treated recipients in fully major histocompatibility complex-mismatched mouse strain combinations. We used various gene knockout mice to examine the role of donor T cells and B cells in BMC-induced allograft tolerance. METHODS: All recipient mice received ALS on days -1 and 2 and rapamycin (6 mg/kg) on day 7 relative to fully major histocompatibility complex-mismatched skin grafting on day 0. Donor BMC prepared either from mice lacking CD4- and/or CD8a-, or CD3epsilon-expressing cells or B cells, or from corresponding wildtype mice, were given on day 7. The level and phenotypes of chimerism was determined by flow cytometry. RESULTS: All T cell- and B cell-deficient BMC were as effective as wild-type BMC in inducing prolongation of skin graft survival. A low degree of chimerism without donor type T cells was detected in tolerant mice given T cell-deficient BMC or wild-type BMC 60 days after transplantation. Chimeric cells were composed of B cells and macrophages/monocytes. Low level chimerism without donor T or B cells was also present in tolerant mice given B cell-deficient BMC. CONCLUSION: Donor type T cells and T cell chimerism are not required for induction of allograft tolerance by the antilymphocyte serum/rapamycin/donor BMC-infusion protocol. Donor B cells also do not participate in tolerance induction. Thus, infusion of T cell-depleted BMC in conjunction with conventional immunosuppressive regimens will be a simple, safe, and effective way to induce allograft tolerance in clinical organ transplantation.     

Efficacy and Limitations of Natural Killer Cell Depletion in Cyclophosphamide-Induced Tolerance

Purpose We previously developed a cyclophosphamide (CP)-induced tolerance protocol, consisting of an intravenous injection of 1 × 10⁸ donor spleen cells (SC) given on day 0 and an intraperitoneal injection of 200 mg/kg CP given on day 2. In the present study, we modified this protocol with natural killer cell (NK) depletion in recipient mice, and evaluated the efficacy of tolerance induction. Methods We used B10.D2 (H-2^d; IE⁺) and B10 (H-2^b; IE⁻) mice as both donors and recipients. The recipient mice were treated with donor SC, CP, and donor bone marrow cells (BMCs) with or without NK depletion. Results A higher level of mixed chimerism was achieved in the NK-depleted recipients. Survival of both the skin and heart donor grafts was significantly prolonged in the NK-depleted recipients. Donor reactive Vβ11⁺ T cells were found at the same level as in untreated control mice. Pretreatment with recipient NK cell depletion was effective in inducing higher levels of donor mixed chimerism; however, permanent engraftment of donor bone marrow was not achieved. Conclusion Survival of donor grafts was remarkably prolonged in the NK cell-depleted group, but transplantation tolerance could not be induced. Our results suggest that NK cell depletion in CP-induced tolerance conditioning has some effect on the induction of donor-specific tolerance.     

A more persistent tolerance to islet allografts through bone marrow transplantation in minimal nonmyeloablative conditioning therapy

INTRODUCTION: Islet transplantation is a therapeutic approach to prevent diabetes complications. However, the side effects of the required lifelong immunosuppressive regimens to prevent graft rejection restrict the impact of type 1 diabetes. One strategy to overcome these limitations is tolerance induction and graft acceptance through hematopoietic chimerism. In this study we investigated whether tolerance to major histocompatibility complex (MHC) and minor-disparate islet allografts could be induced by minimal nonmyeloablative conditioning and whether more persistent donor-specific islet allografts were accepted if the grafts were implanted with simultaneous bone marrow cells. METHODS: The donor and recipient mice were BALB/c(H-2(b)) and C57BL/6(H-2(d)), respectively. In group 1 streptozotocin-induced diabetic C57BL/6(H-2(d)) mice received only 500 islets of BALB/c(H-2(b)). Group 2 recipients conditioned with antilymphocyte serum, 100 cGy total body irradiation and cyclophosphamide were given islet cells of BALB/c(H-2(b)), but group 3 were simultaneously given 30 x 10(6) BALB/c(H-2(b)) mice BMCs and islet cells similar to group 2. RESULTS: We obtained 5% to 6% allogeneic donor chimerism and 60% graft survival at 80 days after islet transplantation in group 3. We observed lymphocyte infiltration around the islet without destruction of endocrine cells and the presence of strong insulin/glucagon-stained cells in group 3. CONCLUSION: This minimal nonmyeloablative conditioning therapy induced donor chimerism and immune tolerance between MHC- and minor-disparate (BALB/c-->C57BL/6) mice and long-term islet graft survival was obtained through cotransplantation of bone marrow cells.     

Tolerance induction through megadose bone marrow transplantation with two-signal blockade

BACKGROUND: Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. MATERIALS AND METHODS: Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. RESULTS: High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. CONCLUSIONS: The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.     

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection

BACKGROUND: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. METHODS: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. RESULTS: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection. CONCLUSION: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.     

Ability of donor splenocytes with costimulation blockade to induce mixed hematopoietic chimerism and transplantation tolerance

We reported stable mixed chimerism and specific tolerance to a fully allogeneic graft after a minimally myelosuppressive regimen including costimulation blockade (CB), donor bone marrow cells (BMC), and busulfan (Bu), a chemotherapeutic conditioning agent that makes niches for engraftment of BMC. For clinical application, the strategy may have the limitation of the number of donor BMC when a deceased donor offers transplants to multiple recipients. Herein, we examined whether donor splenocytes can serve as an alternative source to induce mixed chimerism and tolerance. When a C57BL/6 (H-2b) recipient was treated with CB (CTLA4-Ig and anti-CD154 mAb, on days 0, 2, 4, 6) and donor BALB/c (H-2d) BMC (2 x 10(7) cells on day 0) in the absence of Bu, survival of BALB/c skin graft was remarkably prolonged but not indefinite (median survival time [MST]: 138 days). The recipients never showed durable chimerism. When the recipient was treated with CB and donor splenocytes ([DST] 2 x 10(7) cells on day 0), survival was not indefinite either (MST: 114 days). When the dose of DST was increased to 2 x 10(8) cells, survival was further prolonged; two of six recipients had indefinite survival (MST: 132 days). Moreover, one recipient showed a low level of chimerism. When treated with CB, donor DST (2 x 10(7) cells on day 0) and Bu (20 mg/kg, day -1), six of seven recipients showed a stable, high level of chimerism and enjoyed tolerance of skin allografts. DST combined with CB and Bu may be an alternative source of hematopoietic stem cells to induce mixed chimerism and transplantation tolerance in our model.     

Allogeneic chimerism established with a mixture of low dose bone marrow cells and splenocytes in sublethally irradiated mice

BACKGROUND: Allogeneic chimerism has been established in graft-accepting recipients and the donor cells in the host may act in a major way to facilitate the induction of tolerance. In this study, we examined the effects of allogeneic chimerism after injecting donor bone marrow cells (BMCs) mixed with splenocytes (SPLCs) to the sublethally conditioned recipients. METHODS: In BALB/c(H-2(d)) to B6(H-2(b)) combination, B6 recipients were irradiated at 7.5 Gy and were injected a mixture of donor BMCs and SPLCs intravenously. On day 90 after injection, the degree of chimerism in peripheral blood lymphocytes (PBL) and in the splenocytes was checked by flow cytometry. RESULTS: In groups which were injected varying BMCs, when > 45 x 10(6) BMCs were injected into B6, a large percentage of donor cells were detected in PBL and in the spleen. In contrast, when < 30 x 10(6) BMCs were injected into B6, only a small percentage of donor cells were detected. In the groups which were injected 3 x 10(6) BMCs with varying SPLCs, when > 10 x 10(6) SPLCs were added, a large percentage of donor cells were detected in PBL and SPLCs, but a small percentage of donor cells were detected with the addition of < 3 x 10(6) SPLCs. A high percentage of chimeric mice showed donor specific tolerance in vitro, mixed lymphocyte responses, and in vivo, skin grafting. In contrast, only a small percentage of chimeric mice showed no donor specific tolerance by skin grafting. CONCLUSION: These findings suggested that even a low dose of BMCs can establish a state of allogeneic chimerism and donor specific tolerance if combined with SPLCs.     

Requirement of a higher degree of chimerism for skin allograft tolerance in cyclophosphamide-induced tolerance

By using a cyclophosphamide (CP)-induced tolerance system, we previously raised the possibility that the degree of chimerism might determine the induction of heart and skin allograft tolerance. When C3H (H-2^k; Thy1.2, Mls-1^b) mice were intravenously primed with 1×10⁸ spleen cells (SCs) from H-2 matched AKR (H-2^k; Thy1.1, Mls-1^a) mice and then treated intraperitoneally with 200 mg/kg CP, the survival of AKR skin grafts was permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism and the clonal destruction of Mls-1^a-reactive CD4⁺V6⁺ T cells in the periphery were observed. When AKR SCs and 100 mg/kg CP were used for conditioning, the survival of the AKR skin grafts was mildly prolonged. The clonal destruction of CD4⁺V6⁺ T cells in the periphery was induced and a minimal degree of mixed chimerism was detectable. The degree of mixed chimerism induced with AKR SCs and 200 mg/kg CP was significantly higher than that with AKR SCs and 100 mg/kg CP during the observation. On the other hand, neither skin allograft prolongation nor permanent mixed chimerism could be induced when C3H mice were treated with AKR SCs and 50 mg/kg CP. In order to increase the degree of mixed chimerism, we injected 1×10⁸ tolerant AKR SCs on day 3 into the recipient C3H mice that had been treated with AKR SCs on day 0 and with 100 mg/kg CP on day 2. The reason that we used tolerant SCs was that untreated AKR SCs caused graft-versus-host disease in most of the recipients. Tolerant AKR SCs were harvested from AKR mice that had been treated with C3H SCs and 200 mg/kg CP 2 weeks earlier, and did not contain regulatory cells. By adoptive transfer, the degree of chimerism was stably and significantly increased in all recipients, and AKR skin graft tolerance was induced in half of the recipients. T-cell-depleted bone marrow cells (BMCs) from untreated AKR mice induced skin allograft tolerance in 83% of recipients. Thus, the present study strongly confirmed the hypothesis that a higher degree of chimerism is required for the induction of skin allograft tolerance in CP-induced tolerance.     

Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene

INTRODUCTION: Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford 'prope' tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. Objectives: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain. MATERIALS AND METHODS: To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice. RESULTS: Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9+/-1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6+/-1.2 days). CONCLUSIONS: IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant (P<0.001) prolongation of the survival of donor skin allografts was observed.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

Active role of chimerism in transplantation tolerance induced by antilymphocyte serum, sirolimus, and bone-marrow-cell infusion

BACKGROUND: A regimen consisting of antilymphocyte serum (ALS), sirolimus, and donor bone-marrow-cell (BMC) infusion induces indefinite skin allograft survival across fully mismatched mouse strain combinations. We investigated the role of chimerism in this transplantation tolerance model. MATERIALS: B10.A (H-2a) mice were treated with ALS on day -1 and 2, sirolimus, and infusion of (C57BL/6xDBA/2)F1 (B6D2F1, H-2(b/d)) BMCs on day 7 relative to DBA/2 (D2) skin grafting on day 0. At postgraft days 30, 50 and 120, the recipient mice were injected intravenously with splenocytes prepared from either naive or D2 mixed chimeric B10.A mice that had been sensitized in vivo to B6. Changes in chimerism and graft survival were monitored. RESULTS: Although D2 skin grafts were rejected with a median survival time of 63.8 days in B10.A mice given ALS and sirolimus alone, they survived more than 200 days in all B10.A mice given ALS, sirolimus, and B6D2F1 BMCs. Chimerism became evident 21 days postgrafting and progressively increased thereafter to 20% at postgraft day 200. Infusion of anti-B6 presensitized cells resulted in depletion of chimeric donor cells and subsequent graft rejection regardless of the timing of injection. Injection of presensitized cells in mice given ALS and sirolimus alone had no effect on graft survival. Injection of presensitized cells that were cytotoxic to alloantigen expressed by BMCs but tolerant to skin reduced, but did not deplete, established chimerism and allowed continued allograft survival. CONCLUSIONS: Chimeric donor cells play a major role in both the early and late phases of transplantation tolerance induced by the ALS, sirolimus, and BMC regimen.     

Antigen-specific T-regulatory cells can extend skin graft survival time in mice

This study investigated the effects of donor antigen-specific CD4(+)CD25(+) T-regulatory cells (Tregs) on skin allografts in mice. An allogeneic skin transplant model was established using donor C57BL/6 or DBA and recipient BALB/c mice. Recipients were divided into 4 groups: control group without intervention (CON; C57BL/6 to BALB/c), rapamycin gavage group (RAP; C57BL/6 to BALB/c), CD4(+)CD25(+) Tregs-treated group (TRE; C57BL/6 to BALB/c), in which recipients received transfusions of CD4(+)CD25(+) Tregs stimulated with C57BL/6-derived immature dendritic cells, and the third-party donor group (DBA; DBA to BALB/c) in which recipients received transfusions of BALB/c CD4(+)CD25(+) Tregs stimulated with C57BL/6-derived immature dendritic cells. Mean (SD) survival time of the skin allografts in the TRE group was 17.0 (3.4) days, significantly longer than in the other groups: CON, 6.9 (1.9) days; RAP, 10.3 (3.0) days; and DBA, 10.8 (3.6) days. The TRE group demonstrated a significantly greater expression of transforming growth factor-β and interleukin (IL)-10. Donor antigen-specific CD4(+)CD25(+) Tregs effectively extend skin allograft survival in mice.     

Requirement of a higher degree of chimerism for skin allograft tolerance in cyclophosphamide-induced tolerance

Abstract By using a cyclophosphamide (CP)‐induced tolerance system, we previously raised the possibility that the degree of chimerism might determine the induction of heart and skin allograft tolerance. When C3H (H‐2k; Thy 1.2, Mls‐lb) mice were intravenously primed with 1x108 spleen cells (SCs) from H‐2 matched AKR (H‐2k; Thy 1.1, Mls‐la) mice and then treated intraperitoneally with 200 mg/kg CP, the survival of AKR skin grafts was permanently prolonged in a tolerogen‐specific fashion. After this treatment, a minimal degree of mixed chimerism and the clonal destruction of Mls‐la‐reactive CD4+Vβ6+ T cells in the periphery were observed. When AKR SCs and 100 mg/kg CP were used for conditioning, the survival of the AKR skin grafts was mildly prolonged. The clonal destruction of CD4 + Vβ6+ T cells in the periphery was induced and a minimal degree of mixed chimerism was detectable. The degree of mixed chimerism induced with AKR SCs and 200 mg/ kg CP was significantly higher than that with AKR SCs and 100 mg/kg CP during the observation. On the other hand, neither skin allograft prolongation nor permanent mixed chimerism could be induced when C3H mice were treated with AKR SCs and 50 mg/kg CP. In order to increase the degree of mixed chimerism, we injected 1x108 tolerant AKR SCs on day 3 into the recipient C3H mice that had been treated with AKR SCs on day 0 and with 100 mg/kg CP on day 2. The reason that we used tolerant SCs was that untreated AKR SCs caused graft‐versus‐host disease in most of the recipients. Tolerant AKR SCs were harvested from AKR mice that had been treated with C3H SCs and 200 mg/kg CP 2 weeks earlier, and did not contain regulatory cells. By adoptive transfer, the degree of chimerism was stably and significantly increased in all recipients, and AKR skin graft tolerance was induced in half of the recipients. T‐cell‐depleted bone marrow cells (BMCs) from untreated AKR mice induced skin allograft tolerance in 83% of recipients. Thus, the present study strongly confirmed the hypothesis that a higher degree of chimerism is required for the induction of skin allograft tolerance in CP‐induced tolerance.     

Allogeneic versus semiallogeneic F1 bone marrow transplantation into sublethally irradiated MHC-disparate hosts. Effects on mixed lymphoid chimerism, skin graft tolerance, host survival, and alloreactivity

In models of tolerance associated with mixed lymphoid chimerism, depletion of Thy 1+ cells from the allogeneic donor inoculum may decrease the level of chimerism achieved and the capacity of donor cells to induce tolerance. To determine whether the apparent role of Thy 1+ cells in the facilitation of bone marrow engraftment and induction of skin graft tolerance is related to alloaggression, the capacity of fully allogeneic C57BL/6J, H-2b BM cells to establish mixed lymphoid chimerism and skin graft tolerance in sublethally irradiated (2.5 Gy x 3) BALB/c, H-2d hosts was compared with that of semi-allogeneic BALB/c x C57BL/6J F1 H-2d/b BM cells which genetically lack the potential for graft-versus-host reactivity against parental recipients. The levels of mixed chimerism observed with allogeneic and semi-allogeneic F1 BM cells were nearly identical: 21.0 +/- 9.7% of spleen cells in H-2b BM-injected and 18.6 +/- 8.8% of spleen cells in H-2d/b BM-injected H-2d hosts were of donor allotype. There was no difference in the fraction of hosts rendered tolerant to C57BL/6J, H-2b skin grafts by H-2b vs. H-2d/b BM at either excess (94% vs. 92% tolerant) or threshold (37% vs. 40% tolerant) numbers of donor cells. Spleen cells from both types of mixed chimeras failed to respond to donor antigens in MLR. Both H-2b and H-2d/b BM-injected H-2d hosts rejected third party C3H/HeJ, H-2k skin grafts and responded to third party stimulators in MLR. Although these nonspecific allo-immune responses were not as strong as the responses of normal animals, they were suppressed to an equivalent degree in both types of chimeras. Graft-versus-host disease, if present in irradiated H-2b BM-injected hosts, did not significantly affect survival compared with survival of irradiated H-2d/b BM-injected animals. These results suggest that the tolerizing capacity of allogeneic BM does not depend upon GVHD and that allogeneic and semi-allogeneic BM establish mixed lymphoid chimerism and induce skin graft tolerance by similar mechanisms across a complete MHC disparity in sublethally irradiated adult hosts.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

CD8 T cell of donor splenocyte mixed with bone marrow cells is more effective than CD4 T cell for induction of donor-specific tolerance in sublethally irradiated mice

BACKGROUND: We previously demonstrated that even a low dose of bone marrow cells (BMCs) established donor-specific tolerance if mixed with splenocytes (SPLCs). In this study, T-cell subsets CD4 (CD4SP) and CD8 (CD8SP) of donor SPLCs were investigated for their contribution to the enhancement of BMC engraftment leading to donor-specific tolerance in sublethally irradiated mice. METHODS: Sublethally irradiated C57BL/6 recipient mice were intravenously injected BMCs mixing with CD4SP or CD8SP harvested from BALB/c donor mice. The degree of chimerism in the peripheral blood lymphocytes (PBLs) and in the SPLCs was analyzed using FACS, mixed lymphocyte reaction, and skin graft transplantation 3 months after injection. RESULTS: Recipients injected with 3 x 10(6) donor BMCs admixed with 10 x 10(6) donor CD8SP established chimerism. However, recipients injected with the same dose of BMCs admixed with 5 x 10(6) CD4SP, 10 x 10(6) CD4SP, and 5 x 10(6) CD8SP did not established chimerism. CD8SP contained 44% of Ly6A/E (Stem Cell Antigen-1 (Sca-1))-positive cells based on FACS analysis, whereas only 6% of CD4SP were positive for Ly6A/E. MLR supernates of donor SPLCs chimeric mice using admixture with CD8SP dominated by Th2 cytokines. In contrast, mixting with MLR supernates from failed chimera showed dominant Th1 cytokines. CONCLUSIONS: CD8SP seems to make a major contribution to enhance BMC engraftment and induce donor-specific tolerance. Ly6A/E (Sca-1)-positive cells need to be further investigated for their contribution to the establishment of chimerism.     

Long-Term Survival of Intestinal Allografts Induced by Costimulation Blockade, Busulfan and Donor Bone Marrow Infusion

Tolerance-inducing strategies that infuse donor bone marrow cells in conjunction with costimulation blockade have not been applied to intestinal transplantation. Intestines from BALB/c mice were transplanted into C57BL/6 recipients treated with anti-CD40L mAb, CTLA4-Ig, donor bone marrow, and busulfan. The majority of mice transplanted after completion of this regimen developed hematopoietic macrochimerism, although the degree of chimerism varied widely between recipients, and experienced long-term allograft survival. T cells from these mice demonstrated donor-specific hyporesponsiveness in vitro. However, T cells from chimeric mice proliferated to donor alloantigen in vivo. Furthermore, chimeric mice bearing intestinal allografts were capable of rejecting subsequently placed donor-strain skin grafts. These data suggest that although long-term allograft survival occurs in the absence of acute or chronic rejection, recipient mice are not completely unresponsive to donor alloantigens. When intestinal transplantation was performed at the time of initial bone marrow infusion (initiation of the chimerism protocol), most recipients failed to develop chimerism and promptly rejected the intestinal allograft. Although this is the most effective protocol that we have tested using this stringent model of transplantation, our observations suggest that modifications will be necessary before it can be reliably applied to the transplantation of highly immunogeneic organs like the intestine.     

Treg‐Therapy Allows Mixed Chimerism and Transplantation Tolerance Without Cytoreductive Conditioning

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short‐course costimulation blockade and rapamycin. This combination treatment led to long‐term multilineage chimerism and donor‐specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3‐transduced Tregs, natural Tregs and TGF‐β induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.     

Mixed chimerism, heart, and skin allograft tolerance in cyclophosphamide-induced tolerance

We elucidated the possible role of chimerism in skin and heart allograft tolerance using cyclophosphamide (CP)-induced tolerance. When C3H (H-2k; Thy1.2, Mls-1b) mice were i.v. primed with 1x10(8) spleen cells (SC) from H-2 matched AKR (H-2k; Thy1.1, Mls-1a) mice and then treated i.p. with 200 mg/kg of CP, the survivals of both AKR skin grafts and heart grafts (HG) were permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism, the clonal destruction of Mls-1a-reactive CD4+Vbeta6+ T cells in the periphery, and the clonal deletion of Vbeta6+ thymocytes were all observed. When AKR SC and 100 mg/kg CP were used for conditioning, the AKR HG were permanently accepted, but the survival of the AKR skin grafts was only mildly prolonged. The clonal destruction of CD4+Vbeta6+ T cells in the periphery and the intrathymic clonal deletion of Vbeta6+ thymocytes were induced in both the SC and the 100 mg/kg CP-treated C3H mice. A minimal degree of mixed chimerism was detectable at 4 and 12 weeks after AKR SC and 100 mg/kg CP treatment, and still did not disappear at 40 weeks. The degree of mixed chimerism induced with SC and 100 mg/kg CP was significantly lower than that with SC and 200 mg/kg CP during the observation. No posttransplant cardiac allograft vasculopathy (CAV) was observed to develop, while both the Th1 type (interferon-gamma) and Th2 type (interleukin-4 and -10) cytokine expressions decreased in the AKR HG of the tolerant C3H mice treated with both AKR SC plus 200 mg/kg CP, and AKR SC plus 100 mg/kg CP. A second set of skin grafts from donor AKR mice survived for more than 100 days in a tolerogen-specific fashion in all C3H mice treated with AKR SC and 200 mg/kg CP and also accepted the AKR HG for over 200 days, while 80% of the C3H mice treated with AKR SC and 100 mg/kg CP and accepted the AKR HG for more than 200 days. These results strongly suggested the following conclusions: 1) the degree of chimerism can strongly influence the induction of skin and heart allograft tolerance, 2) posttransplant CAV does not develop in the donor HG maintained by chimerism-based CP-induced tolerance, 3) the mRNA expression of both Th1 and Th2 type cytokine decreased in the donor HG maintained by chimerism-based CP-induced tolerance, and 4) the induction of skin allograft tolerance is more difficult than the prevention of posttransplant CAV.