Ichiro Shimizu

Induction of Permanent Mixed Chimerism and Skin Allograft Tolerance Across Fully MHC-Mismatched Barriers by the Additional Myelosuppressive Treatments in Mice Primed with Allogeneic Spleen Cells Followed by Cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 × 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 × 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE−) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vβ11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 × 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).

Regulatory Roles of NKT Cells in the Induction and Maintenance of Cyclophosphamide-Induced Tolerance

We have previously reported the sequential mechanisms of cyclophosphamide (CP)-induced tolerance. Permanent acceptance of donor skin graft is readily induced in the MHC-matched and minor Ag-mismatched recipients after treatment with donor spleen cells and CP. In the present study, we have elucidated the roles of NKT cells in CP-induced skin allograft tolerance. BALB/c AnNCrj (H-2d, Lyt-1.2, and Mls-1b) wild-type (WT) mice or Vα14 NKT knockout (KO) (BALB/c) mice were used as recipients, and DBA/2 NCrj (H-2d, Lyt-1.1, and Mls-1a) mice were used as donors. Recipient mice were primed with 1 × 108 donor SC i.v. on day 0, followed by 200 mg/kg CP i.p. on day 2. Donor mixed chimerism and permanent acceptance of donor skin allografts were observed in the WT recipients. However, donor skin allografts were rejected in NKT KO recipient mice. In addition, the donor reactive Vβ6+ T cells were observed in the thymus of a NKT KO recipient. Reconstruction of NKT cells from WT mice restored the acceptance of donor skin allografts. In addition, donor grafts were partially accepted in the thymectomized NKT KO recipient mice. Furthermore, the tolerogen-specific suppressor cell was observed in thymectomized NKT KO recipient mice, suggesting the generation of regulatory T cells in the absence of NTK cells. Our results suggest that NKT cells are essential for CP-induced tolerance and may have a role in the establishment of mixed chimerism, resulting in clonal deletion of donor-reactive T cells in the recipient thymus.

A technique of cervical aortic graft transplantation in mice.

A new method of mouse aortic graft transplantation into carotid artery was developed with cuff technique. By harvesting the descending aorta of the donor using a small Teflon cuff (external diameter 0.6 mm, internal diameter 0.4 mm) and super fine-tip forceps, and modifying the method of mouse heterotopic heart transplantation with cuff technique, donor descending aortic allografts could be interposed in the common carotid artery of recipient mice. Histological analysis demonstrated neither evidence of tissue damage nor intimal thickening in isograft implanted over 100 days. We strongly recommend that this new model of aortic transplantation in mice is a simple and useful technique for vascular transplantation research.

Heart allograft tolerance without development of posttransplant cardiac allograft vasculopathy in chimerism-based, drug-induced tolerance.

BACKGROUND Recently, we have described a drug (cyclophosphamide [CP] plus busulfan [BU])-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong the survival of heart allografts and inhibit the development of posttransplant cardiac allograft vasculopathy (CAV). METHODS The components of the method are intravenous administration of 1 x 108 allogeneic spleen cells on day 0, intraperitoneal injection of 200 mg/kg of CP and 30 mg/kg of BU on day 2, and intravenous injection of T cell-depleted 1 x 107 allogeneic bone marrow cells from the same strain of mice on day 3. Heart grafting was performed on day 28. Chimerism in peripheral blood was followed by flow cytometric analysis, and histological analysis was performed at various times after grafting. RESULTS In a fully major histocompatability complex (MHC)-mismatched combination of B10.D2 (H-2d, IE+)-->B10 (H-2b, IE-), stable, multilineage-mixed chimerism was observed permanently. B10.D2 heart grafts were accepted permanently in a donor-specific manner, and posttransplant CAV did not develop. CONCLUSIONS These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-lasting heart allograft tolerance without development of CAV.

Role of the Cytokine Profiles Produced by Invariant Natural Killer T Cells in the Initial Phase of Cyclophosphamide-Induced Tolerance

Background. Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance induction protocol. Recently, we reported that invariant natural killer T (iNKT) cells were essential for the tolerance induction in this system. In this study, we evaluated the roles of the cytokines produced by iNKT cells. Methods. DBA/2 (H-2d) mice and BALB/c (H-2d) wild-type (WT) or iNKT knock out (KO) mice were used as donors and recipients. WT recipients received three doses (days −7, −4, −1 or 35, 38, 41) or a single dose (day −1 or 0) of &agr;-galactosylceramide (GC) in conjunction with our conditioning regimen that consisted of 108 donor spleen cells on day 0 and 200 mg/kg CP on day 2. To investigate the iNKT cell function, iNKT KO recipients were reconstituted with cytokine (interferon-&ggr;, interleukin [IL]-4, or IL-10) KO iNKT cells and received donor spleen cells and CP. Results. Mixed chimerism was observed in WT recipients, but was reduced in iNKT KO recipients. However, mixed chimerism was absent in WT recipients given GC on days −7, −4, −1, but not in WT recipients given GC on day 35, 38, 41. Donor skin grafts were chronically rejected when mixed chimerism was diminished. Skin grafts were accepted in iNKT KO recipients reconstituted with iNKT cells from interferon-&ggr;, IL-4, or IL-10 KO mice and receiving our conditioning regimen. Conclusions. Invariant NKT cells were required in the initial phase of the induction of chimerism. Our results indicated that known major cytokines produced by iNKT cells were dispensable for the regulatory function of iNKT cells.

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.

The Immunoregulatory Roles of Natural Killer T Cells in Cyclophosphamide-induced Tolerance

Background. Recent studies have indicated that natural killer T (NKT) cells are essential for the establishment of transplantation tolerance. In the present study, we have elucidated the role of recipient and donor NKT cells in cyclophosphamide (CP)-induced tolerance. Method. DBA/2 (DBA; H-2d) mice were used as donors and BALB/c (BALB; H-2d) wild-type (WT) or V&agr;14 NKT-knockout (KO, BALB/c background) mice were used as recipients. Recipients were treated with CP-induced tolerance regimen, which consists of donor spleen cells (SC) on day 0 and CP on day 2. In some experiments, NKT KO mice, which received NKT cells from either WT, inferon-&ggr; KO, or interleukin-4 KO mice, were treated with tolerant regimen. To deplete Ly49 inhibitory receptors on NKT cells in the recipient mice, anti-Ly49 monoclonal antibody cocktails were injected on day −1 when indicated. Results. Donor skin graft was permanently accepted in recipient BALB WT mice with induction of donor mixed chimerism. On the contrary, donor DBA skin allografts were chronically rejected in NKT KO recipient. Lower levels of mixed chimerism were observed in NKT KO recipients comparing to the WT recipients. The production of interferon-&ggr; or interleukin-4 from NKT cells did not affect the induction of tolerance. Depletion of Ly49 positive NKT cells abrogated the induction of skin graft tolerance. Conclusion. Recipient NKT cells, but not donor NKT cells, were dominantly required for the induction of allograft tolerance. Our results indicated that the single cytokine produced by NKT cells did not mediate the regulatory function in the induction of allograft tolerance.

Efficacy and Limitations of Cyclophosphamide‐induced Tolerance against αGal Antigen

In the present study, we have elucidated the efficacy of two cyclophosphamide (CP)‐induced tolerance protocols for the induction of B‐cell tolerance against Galα1‐3Galβ1‐4GlcNAc (αGal) antigens. α1,3‐galactosyltransferase‐deficient (GalT–/–; H‐2b/d) mice received with 1 × 108 AKR (αGal+/+ H‐2k) spleen cells (SC) followed by 200 mg/kg CP, or alternatively followed by 200 mg/kg CP, 30 mg/kg Busulfan (BU) and 1 × 108 T‐cell‐depleted AKR bone marrow cells (BMC). The generation of both anti‐αGal and anti‐donor antibodies were completely suppressed, but normal antibody production against third party antigens was observed after BALB/c skin grafting in both groups of GalT–/– mice. In GalT–/– mice, treated with SC and CP, mixed chimerism was not observed. Cellular rejection was observed in grafted donor AKR hearts with an absence of humoral rejection, whereas humoral rejection was observed in untreated GalT–/– mice. On the other hand, long‐term mixed chimerism and permanent acceptance of donor AKR skin graft and heart graft were achieved in GalT–/– mice treated with SC, CP, BU and BMC. These results demonstrate the efficacy of classical drug‐induced tolerance in the induction of B‐cell tolerance against αGal antigens. However, induction of stable mixed chimerism was required for the suppression of cellular rejection.

Different expressions of Ly-49 receptors on mouse NK and NK T cells.

NK T cells are a unique T cell lineage and are reported to express Ly-49 molecules which are inhibitory receptors specific for class I molecules. In this study, we examined the expression of activation and inhibitory receptors on NK T cells in different organs of beta2-microgloblin knock out (beta2mKO), C57BL/6 (B6; H-2b), C57BL/10 (B10; H-2b) and B10.D2 (H-2d) mice. The low level expression of inhibitory receptors Ly-49A and G2 on NKT cells as well as NK cells, which are specific for Dd antigen, were observed in B10.D2 mice, but not in beta2mKO, B6, or B10 mice. The small percentage of inhibitory receptor Ly-49C positive NK and NKT cells, which is specific for Kb and Dd antigens, was observed in BMC, LMC and SC of B6, B10 and B10.D2 mice compared to beta2mKO mice. On the contrary, the large percentage of Ly-49C positive NK T cells was observed in thymocytes of B6, B10 and B10.D2 mice compared to beta2mKO mice. Interestingly, Ly-49D activation receptor was hardly detectable on NK T cells in any organs of the 4 strains of mice whereas it was clearly detectable on NK cells. These findings suggest that the unique characteristics of NK T cells may mediate regulatory function in MHC class I antigen-restricted immunity.

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

PurposeWe 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.MethodsWe used B10.D2 (H-2d; IE+) and B10 (H-2b; 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.ResultsA 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.ConclusionSurvival 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.