Cyclophosphamide-induced Tolerance Research Articles

Cyclophosphamide-Induced Tolerance in Allogeneic Transplantation: From Basic Studies to Clinical Application.

Immune tolerance against alloantigens plays an important role in the success of clinical organ and allogeneic hematopoietic stem cell transplantation. The mechanisms of immune tolerance to alloantigens have gradually been elucidated over time. Although there have been numerous reports to date on the induction of tolerance to alloantigens, the establishment of mixed chimerism is well-known to be crucial in the induction and maintenance of immune tolerance for either of the methods. Since the early 1980s, the murine system of cyclophosphamide (Cy)-induced tolerance has also been examined extensively. The present review focuses on studies conducted on Cy-induced immune tolerance. Clinical data of patients with allogeneic transplantation suggest that the posttransplant Cy method to induce immune tolerance has been successfully translated from basic studies into clinical practice.

Cyclophosphamide-induced tolerance in kidney transplantation avoids long-term immunosuppressive therapy.

There has recently been remarkable progress in immunosuppressive agents, such as tacrolimus and cyclosporine. Therefore, the rate of organ establishment has improved in transplantation. However, immunosuppressive agents generally suppress the function of Tcells. Thus, opportunistic infections, such as cytomegalovirus infection, are still a major problem in kidney transplantation. Induction of specific tolerance to avoid immunosuppressive drug therapy after kidney transplantation is considered as the ultimate goal of transplantation. Various factors induce tolerance that involves establishment of hematopoietic chimerism and various cell subsets. In particular, we have carried out various studies regarding the cyclophosphamide-induced tolerance system. Tolerance is induced after establishment of hematopoietic chimerism after donor bone marrow transplantation. At the clinical stage, kidney transplantation before administration of cyclophosphamide after transfusion of bone marrow to create hematopoietic chimera is considered to be one of the most successful protocols. Furthermore, recent studies have shown the involvement of multiple populations of immune cells in preserving immunological tolerance and promoting long-term renal grafts. The present review focuses on how cyclophosphamide and other immune factors induce tolerance in kidney transplantation.

A reduction of recipient regulatory T cells by cyclophosphamide contributes to an anti‐tumor effect of nonmyeloablative allogeneic stem cell transplantation in mice

We have recently established a unique model system of nonmyeloablative allogeneic stem cell transplantation (SCT) for treatment of murine solid tumors, based on cyclophosphamide-induced tolerance. An injection of allogeneic donor spleen cells and bone marrow cells (BMC) followed by cyclophosphamide treatment induced a stable mixed chimerism with long lasting tolerance to the allografts. A donor lymphocyte infusion (DLI) in the cyclophosphamide-induced tolerant mice exerted strong anti-tumor effects on an MBT-2 murine bladder tumor, MBT-2 via their graft versus tumor (GVT) activity. In the present study, we determined whether a cyclophosphamide-induced reduction of naturally occurring regulatory T cells (Tregs) was associated with the anti-tumor activity in our nonmyeloablative SCT system. The number of recipient CD4+ CD25+ Foxp3+ Tregs significantly decreased 3 days after an intraperitoneal injection of cyclophosphamide in C3H/HeN mice that had been injected with spleen cells and BMC of donor AKR/J mice, compared with the number of CD4+ CD25+ Foxp3- T cells. An adoptive transfer of CD4+ CD25+ T cells from naïve C3H/He x AKR/J F1 mice into recipient mice 1 day after DLI significantly suppressed the expansion and IFN-γ production of host-reactive donor CD4+T cells and hampered the MBT-2 anti-tumor activity when compared with the transfer of CD4+ CD25- T cells. These results indicated that cyclophosphamide-induced reduction of recipient Tregs is associated with retardation of tumor progression via the expansion of host-reactive donor T cells and IFN-γ production after DLI in our nonmyeloablative SCT system.

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

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. 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 alpha-galactosylceramide (GC) in conjunction with our conditioning regimen that consisted of 10(8) 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-gamma, interleukin [IL]-4, or IL-10) KO iNKT cells and received donor spleen cells and CP. 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-gamma, IL-4, or IL-10 KO mice and receiving our conditioning regimen. 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.

Comparison of Outcomes of HLA-Matched Related, Unrelated, or HLA-Haploidentical Related Hematopoietic Cell Transplantation following Nonmyeloablative Conditioning for Relapsed or Refractory Hodgkin Lymphoma

We compared the outcome of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) for patients with relapsed or refractory Hodgkin lymphoma (HL) based on donor cell source. Ninety patients with HL were treated with nonmyeloablative conditioning followed by HCT from HLA-matched related, n=38, unrelated, n=24, or HLA-haploidentical related, n=28 donors. Patients were heavily pretreated with a median of 5 regimens and most patients had failed autologous HCT (92%) and local radiation therapy (83%). With a median follow-up of 25 months, 2-year overall survivals, progression-free survivals (OS)/(PFS), and incidences of relapsed/progressive disease were 53%, 23%, and 56% (HLA-matched related), 58%, 29%, and 63% (unrelated), and 58%, 51%, and 40% (HLA-haploidentical related), respectively. Nonrelapse mortality (NRM) was significantly lower for HLA-haploidentical related (P=.02) recipients compared to HLA-matched related recipients. There were also significantly decreased risks of relapse for HLA-haploidentical related recipients compared to HLA-matched related (P=.01) and unrelated (P=.03) recipients. The incidences of acute grades III-IV and extensive chronic graft-versus-host disease (aGVHD, cGVHD) were 16%/50% (HLA-matched related), 8%/63% (unrelated), and 11%/35% (HLA-haploidentical related). These data suggested that salvage allogeneic HCT using nonmyeloablative conditioning provided antitumor activity in patients with advanced HL; however, disease relapse/progression continued to be major problems. Importantly, alternative donor stem cell sources are a viable option.

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

Hyperacute rejection (HAR) mediated by the natural antibody (nAb) against Gal alpha 1-3Gal beta-4-GlcNAc (alpha Gal) is the major obstacle in xenogeneic organ transplantation. Previously, we reported the acceptance of donor heart grafts in anti-alpha Gal 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. GalT KO (alpha Gal(-/-), H-2(b/d)) recipient mice were presensitized with alpha Gal(+) rabbit red blood cells (RRBCs). Presensitized or nonsensitized recipient mice were treated with CP-induced tolerance conditioning, consisting of AKR (alpha Gal(+/+), 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-alpha Gal Abs by flow cytometry. Donor mixed chimerism was achieved in the presensitized GalT KO mice treated with CP-induced tolerance conditioning. In parallel with the disappearance of anti-alpha Gal Abs, permanent acceptance of donor heart grafts and skin grafts was observed in presensitized and GalT KO mice treated with CP-induced tolerance conditioning. Both B-cell and T-cell tolerance was achieved in the presence of a higher titer of anti-alpha Gal Abs after treatment with CP-induced tolerance conditioning.

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

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. DBA/2 (DBA; H-2) mice were used as donors and BALB/c (BALB; H-2) wild-type (WT) or Valpha14 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-gamma 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. 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-gamma 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. 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 Natural Killer Cell Depletion in Cyclophosphamide-Induced Tolerance

We previously developed a cyclophosphamide (CP)-induced tolerance protocol, consisting of an intravenous injection of 1 x 10(8) 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. We 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. 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 Vbeta11+ 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. 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.

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-2(d), Lyt-1.2, and Mls-1(b)) wild-type (WT) mice or Valpha14 NKT knockout (KO) (BALB/c) mice were used as recipients, and DBA/2 NCrj (H-2(d), Lyt-1.1, and Mls-1(a)) mice were used as donors. Recipient mice were primed with 1 x 10(8) 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 Vbeta6(+) 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.

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.

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-2k; Thy1.2, Mls-1b) mice were intravenously primed with 1 x 10(8) spleen cells (SCs) from H-2 matched AKR (H-2k; Thy1.1, Mls-1a) 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-1a-reactive CD4+Vbeta6+ 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+Vbeta6+ 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 x 10(8) 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.

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.

Heart allograft tolerance without development of posttransplant cardiac allograft vasculopathy in cyclophosphamide-induced tolerance

Heart allograft tolerance without development of posttransplant cardiac allograft vasculopathy in cyclophosphamide-induced tolerance

Different role of cyclophosphamide-induced tolerance in heart and skin allograft tolerance

Different role of cyclophosphamide-induced tolerance in heart and skin allograft tolerance

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 x 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 x 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 Vbeta11+ 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 x 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).

Inability of Cyclophosphamide-Induced Tolerance to Permit Engraftment of Pluripotent Stem Cells Contained in a Moderate Number of Syngeneic Bone Marrow Cells

We have previously described cyclophosphamide (CP)-induced tolerance that comprises an intravenous (i.v.) injection of 1 x 10 8 allogeneic spleen cells (SC) followed, 2 days later, by an intraperitoneal (i.p.) administration of 200 mg/kg of CP. By using this method, we were able to readily induce both long-lasting mixed chimerism and skin allograft tolerance in most of H- 2 matched combinations, but not in H-2 mismatched combinations. In the present study, we have investigated whether the treatment with SC followed by 200 mg/kg CP can permit engraftment of syngeneic pluripotent hematopoietic stem cells (HSC), and whether CP itself can permit the HSC engraftment. Recipient B6 (H-2 b; Ly-5.2) mice received 1 x 10 7, 5 x 10 7 or 1 x 10 8 SC from syngeneic B6.Ly-5.1 (H-2 b; Ly-5.1) mice and administered 2 day later 200 mg/kg CP. Using anti-Ly-5.1 and Ly-5.2 mAbs and a flow cytometry, the origins of lymphoid and myeloid cells injecting the recipients were observed over time. Chimerism was at least initially detectable in all groups treated with SC alone (without 200 mg/kg CP), but became undetectable by 32 weeks after the treatment with SC alone. In recipient B6 mice treated with B6.Ly-5.1 SC and 200 mg/kg CP, on the other hand, lymphoid-chimerism was detectable at 32 weeks in a transferred cell dose-dependent manner, but granulocytechimerism indicating pluripotent HSC engraftment disappeared earlier than 32 weeks after the treatment. In order to further evaluate the effect of CP itself on HSC engraftment, recipient B6 mice were given various doses of CP (50-400 mg/kg) and were injected with T celldepleted 1 × 10 7 BMC from B6.Ly-5.1 mice. Multilineage mixed chimerism over 32 weeks was induced in only one of 11 B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC, although lymphoid chimerism was induced temporarily in recipient B6 mice treated with 200 mg/kg CP followed by B6.Ly-5.1 BMC and persistently in most of recipient B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC.

Lack of pluripotent stem cell engraftment in cyclophosphamide-induced tolerance

Lack of pluripotent stem cell engraftment in cyclophosphamide-induced tolerance

Improved technique of heterotopic cervical heart transplantation in mice.

The method for mouse vascularized heart transplantation have been described using suture and cuff techniques. Technical problems have limited its widespread use. Here, we describe our method of modified cervical heterotopic transplantation with the cuff technique. By using a smaller Teflon cuff (external diameter 0.6 mm, internal diameter 0.4 mm) and superfine-tip forceps, it became possible to directly pull the edge of the carotid artery and evert the proximal end of the artery over the cuff. Similarly, the external jugular vein could be easily everted over a 22-gauge cuff with this direct pulling method. By these modifications, the operation time was reduced. It usually takes 20 min for the donor harvest, 15 min for preparation of the cervical vessels, and 15 min for anastomosis. All procedures from the donor harvest through skin closure of the recipient mice can be completed within 1 hr, and ischemic time is within 25-40 min. This method can be used to investigate cyclophosphamide-induced tolerance and mechanisms of reperfusion injury.

The Induction of Skin Xenograft Tolerance in Rat-to Mouse Combination could be Affected by DFR Mediating Cells and Antibodies against Rat Bone Marrow Cells as well as NK Cells in the Cyclophosphamide-Induced Tolerance System

We investigated whether the prolongation of skin xenograft survival was obtained by a tolerance-inducing method using cyclophosphamide (CY), by which long-lasting skin allograft tolerance could be induced. The long-lasting skin allograft survival could be obtained in the recipient C3H/HeN (C3H) mice which were given 100 [μg of anti-CD4 mAb on day -3, 1 x 10 8 spleen cells (SC) plus 3 x 10 7 bone marrow cells (BMC) derived from C57BL/6 (B6) mice on day -2, 200 mg/kg CY on day 0, and which were grafted with allogeneic B6 skin on day 14. When the C3H mice were treated with anti-CD4 mAb, 1 x 10 8 SC plus 5 x 10 7 BMC derived from F344 rat and CY, the F344 skin grafts survived slightlv longer (about 15 days) than those in untreated recipients (about 8.4 days). Such a prolongation of skin xenograft survival was considered donor-specific because rejection of 3rd party skin grafts from BN rats occurred significantly earlier than that of F344 skin grafts. In the recipient C3H mice treated with anti-CD4 mAb, F344 SC plus BMC and CY, mixed chimerism in the periphery was detected for a few days after CY administration, although intrathymic chimerism was not detected throughout this study. In these recipient C3H mice, cytotoxic T lymphocytes (CTL) against F344 antigens were completely abrogated though the delayed footpad reaction (DFR) remained at a low but significant level. Moreover, though antibody (Ab) activity against F344 SC was completely abrogated, neither Ab activity against F344 BMC, which seemed to have a background common to natural Ab activity, nor NK activity were abrogated by this treatment. These results suggested that DFR mediating cells directly mediated skin xenograft rejection in the recipient mice treated with anti-CD4 mAb, F344 cells, and CY. Such cells may interfere with establishment of mixed chimerism and longlasting skin xenograft tolerance, presumably in cooperation with CY-resistant Ab activity and NK cells.

Prolongation of Kidney Graft Survival by Cyclophosphamide-induced Tolerance in Rats

Prolongation of Kidney Graft Survival by Cyclophosphamide-induced Tolerance in Rats