Toshiro Iwai

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.

Renal Cancer Treatment with Low Levels of Mixed Chimerism Induced by Nonmyeloablative Regimen Using Cyclophosphamide in Mice

Recently, much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of metastatic renal cancer. Mature donor T cells cause graft-versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor activity associated with this treatment. Hence, the segregation of the graft-versus-tumor activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Here, we show a modified cyclophosphamide-induced tolerance system for the treatment of murine renal cell carcinoma, RENCA, by shifting the equal balance between graft-versus-host and host-versus-graft reactions toward graft-versus-host reaction with donor lymphocyte infusion. Our results clearly show the antitumor activity against RENCA with only low levels of mixed chimerism in the periphery and the in vivo and in vitro acquired immunity against RENCA even when mixed chimerism is almost undetectable. Because the withdrawal of mixed chimerism reduces the risk of GVHD, the antitumor activity is thus sequentially segregated from the initial GVHD in our model. We believe that this is the first unique model system of nonmyeloablative allogeneic hemopoietic cell transplantation to ever be reported for the treatment of renal cancer.

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.

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.

Surgical application for a prolapse of the anterior mitral leaflet by replacing artificial chordae with polytetrafluoroethylene grafts.

PurposeThere are an increasing number of reports concerning mitral valve repair by a reconstruction of the chordae tendinae using expanded polytetrafluoro-ethylene (PTFE) sutures. However, little information is available about extended application or results of this technique for an extended prolapse of the anterior mitral leaflets.MethodsBetween July 1991 and August 2003, 28 patients with moderate to severe mitral regurgitation as a result of a prolapse of anterior leaflets (age range, 15–73 years) underwent mitral valve repair by reconstruction of the artificial chordae with 4-CV expanded polytetrafluoroethylene sutures without a leaflet resection. Either Kay’s suture technique or ring annuloplasty was also performed to correct annular dilatation in all patients.ResultsNo operative death or late mortality was observed. The prolapsed segment, which was successfully repaired, was within 33% of the anterior mitral leaflet (AML) in 6 patients, from 33% to 50% in 5, from 50% to 99% in 11, and 100% in 6 patients. Before discharge, immediate postoperative echocardiography showed less than moderate mitral regurgitation in 28 of 28 patients. The follow-up, consisting of a clinical examination and serial echocardiograms, was complete in all cases and the mean follow-up period was 80.6 months (range, 12–146). There were two failures that required a reoperation because of a worsening mitral regurgitation and hemolytic anemia (elongation of anchored side of papillary muscle). The other two patients required mitral valve replacement due to a progressive regression of the left ventricular function, although the regurgitation worsened from a mild level to a moderate one. When the reoperated patients were excluded from the following data, the degree of mitral regurgitation, estimated by echocardiography performed at recent follow-up period, was none in 10 patients, trivial in 13 patients, and mild in 1 patient. In addition, the systolic and diastolic dimensions of the left ventricle decreased significantly (P < 0.01).ConclusionsThe replacement of artificial chordae was not complicated and it seemed to help to preserve a good relationship among leaflet tissues, chordae, and papillary muscles. We therefore suggest that the extensive use of PTFE artificial chordae appears to be a promising procedure for the repair of all kinds of mitral lesions causing mitral regurgitation.

Effects of cyclosporin A on the activation of natural killer T cells induced by alpha-galactosylceramide.

Background. Natural killer T (NKT) cells play crucial roles in preventing autoimmune diseases and inducing transplantation tolerance. We investigated whether cyclosporin A (CsA), which is generally used in clinical transplantation and autoimmune disease therapy, could modulate the NKT cell activation induced by &agr;-galactosylceramide (&agr;-GalCer) treatment. Methods. C57BL/6 (B6) mice were given daily intraperitoneal injections of CsA (30 or 50 mg/kg) from day −1 and injected intravenously with &agr;-GalCer (2 &mgr;g/mouse) on day 0. The kinetics of NK1.1+CD3+ or NK1.1+Thy1.2+ cells in the liver and spleen were analyzed by flow cytometry. Apoptosis of NK1.1+CD3+ cells, cytokine levels (interleukin [IL]-2, IL-4, IL-10 and interferon [IFN]-&ggr;) in the recipient serum and changes in dendritic cell activation in the spleen were analyzed. Results. In B6 mice treated with &agr;-GalCer, NK1.1+CD3+ cells rapidly decreased in both the liver and spleen, and repopulated to their normal levels by day four, while NK1.1+Thy1.2+ cells rapidly decreased, expanded by day four and reduced to their normal level by day 15. When B6 mice were treated with &agr;-GalCer plus 30 or 50 mg/kg CsA, NK1.1+CD3+ or NK1.1+ Thy1.2+cells were similarly decreased and then expanded via extensive proliferation by day seven or four, respectively. When B6 mice were treated with &agr;-GalCer, substantial amounts of IL-2, IL-4 and IFN-&ggr; were produced, and the surface markers of dendritic cells were upregulated. However, these cytokine productions and maturation of dendritic cells were profoundly suppressed after treatment with &agr;-GalCer and CsA. Apoptosis of NK1.1+CD3+ cells was not affected in mice treated with &agr;-GalCer or &agr;-GalCer and CsA. Conclusions. CsA suppresses &agr;-GalCer-induced cytokine productions and dendritic cell maturation of mouse NKT cells but does not decrease NK1.1+CD3+ cells on day one. The modulation of NKT-mediated immunoregulatory functions by CsA requires careful consideration in clinical transplantation and autoimmune disease therapy.

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.