Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.     

Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).     

Nondepleting anti-CD4 monoclonal antibody enhances the ability of oral alloantigen delivery to induce indefinite survival of cardiac allografts: oral tolerance to alloantigen

BACKGROUND: We examined whether oral administration of alloantigen could induce the prolonged survival of cardiac allografts. METHODS: Hearts from CBK (H2k+Kb) transgenic or (C57BL/10xCBA)F1 (H2bxH2k) mice were transplanted into CBA (H2k) recipients pretreated orally with 1 x 10(7) donor splenocytes in the presence or absence of a nondepleting anti-CD4 (YTS 177, 200 microg/dose). RESULTS: Modest prolongation of CBK cardiac grafts was induced in CBA mice fed with multiple doses of CBK splenocytes (MST 42 days compared with controls fed with syngeneic CBA splenocytes, 12 days). When the CD4 monoclonal antibody, YTS177, was administered for 2 days before the first oral delivery of CBK splenocytes, all mice accepted their grafts indefinitely (MST > 100 days versus mice treated with anti-CD4 alone, 11.5 days). To determine if feeding multiple doses of alloantigen was essential, CBA mice were given CBK splenocytes orally on a single occasion in combination with the anti-CD4. The majority of the grafts survived indefinitely (MST >100 days). This oral treatment regimen also induced indefinite prolongation of (C57BL/10xCBA)F1 cardiac grafts. CONCLUSION: The induction of unresponsiveness by oral administration of alloantigen can be augmented by a nondepleting anti-CD4, YTS177, when given before the first oral delivery of allogeneic cells.     

Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody

BACKGROUND: We previously showed that intratracheal delivery of alloantigen induced prolonged survival of fully allogeneic cardiac grafts in mice. Here, this treatment protocol was combined with nondepleting anti-CD4 monoclonal antibody (mAb) to induce operational tolerance. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of whole splenocytes from C57BL/10 (H-2b) mice or a 15-mer Kb peptide, with or without intraperitoneal administration of nondepleting anti-CD4 mAb (YTS177). Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. In addition, some naive CBA mice underwent adoptive transfer of splenocytes from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time, 12 days). Mice given intratracheal delivery of whole splenocytes or Kb peptide demonstrated prolonged graft survival (median survival time, 84 and 76 days, respectively). Concurrent administration of YTS177 and intratracheal delivery of splenocytes or Kb peptide resulted in indefinite graft survival. Mice with long-surviving C57BL/10 cardiac grafts showed acceptance of skin grafts from C57BL/10 mice but not BALB/c mice, demonstrating that operational tolerance had been induced. Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of splenocytes or Kb peptide plus YTS177 induced indefinite survival of cardiac grafts in secondary recipients, indicating that regulatory cells had been generated. CONCLUSION: In a murine model, intratracheal delivery of donor splenocytes or Kb peptide combined with YTS177 induced operational tolerance and generated regulatory cells.     

The smell of Tokishakuyaku-san (TJ-23) induces generation of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts in mice

Oral administration of Tokishakuyaku-san (TJ-23), a Japanese herbal medicine, induces prolongation of cardiac allograft survival and generates regulatory cells in mice. Because herbal medicines usually have unique odor, and because smell is supposed to modulate the immune system, we examined whether the odor of TJ-23 induced prolonged allograft survival and regulatory cell generation. Naïve CBA mice (H2^k) and olfactory-dysfunctional CBA mice after a stereotaxic operation underwent transplantation of C57BL/6 (B6, H2^b) hearts, receiving fumigated water only or TJ-23 until rejection. Untreated or treated with water fumigation CBA mice rejected B6 cardiac grafts acutely (median survival times [MSTs], 7 and 8.5 days). When CBA mice were treated with fumigation of TJ-23, allograft survival was significantly prolonged (MST, 48 days). Olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 rejected grafts acutely (MST, 7 days). Treatment with fumigation of TJ-23 also suppressed splenocytes proliferation and interferon-γ production. Secondary CBA recipients of whole splenocytes or CD4⁺ cells from primary TJ-23-treated CBA recipients of B6 cardiac allografts at 30 days after grafting showed prolonged survival of B6 hearts (MST, >60 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in recipients given fumigation of TJ-23. In conclusion naïve but not olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 displayed prolonged survival of fully allogeneic cardiac allografts and generation of regulatory cells.     

Induction of indefinite survival of fully allogeneic cardiac grafts and generation of regulatory cells by intratracheal delivery of alloantigens under blockade of the CD40 pathway

BACKGROUND: The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance. METHODS: CBA mice were given donor splenocytes (1x107) or Kb peptide (100 microg) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 microg) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5 x 107) from primary recipient CBA mice given ITD of donor splenocytes or Kb peptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day. RESULTS: ITD of donor splenocytes and Kb peptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment. CONCLUSIONS: ITD of donor splenocytes or Kb peptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.     

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model.MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS).ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α⁺ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α⁻ DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS.ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.     

Seven Japanese Herbals Prolonged Cardiac Allograft Survival

Japanese herbal medicines have long been used as alternative therapy because of their immunomodulatory effects. In recent years, use herbal medicines is rapidly increasing worldwide. In this study, we investigated the effect of 17 components of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA mice by using microsurgical techniques. Artemisiae capillaris herba (Inchinko) was given to CBA recipients at a dosage of 1 g/kg/day from the day of transplantation until 7 days afterward. The other 16 components were given at a dosage of 2 g/kg/day for the same time period. Naïve CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST] of 7 days). CBA transplant recipients given 2 g/kg/day of Glycyrrhizae radix (Kanzou), Poria sclerotium (Bukuryo), Pinellia tuber (Hange), Cnidii rhizome (Senkyu), Paeoniae radix (Shakuyaku), and Scutellariae radix (Ogon) had prolonged C57BL/6 allograft survival significantly (MSTs were 18, 18, 17, 14, 12, and 12 days, respectively). Moreover, CBA transplant recipients given 1g/kg/day of Artemisiae capillaris herba had prolonged C57BL/6 allograft survival (MST >100 days); however, none of other 10 components prolonged allograft survival. In conclusion, administration of 7 components of traditional Japanese herbal medicines might induce prolongation of fully major histocompatibility complex–mismatched cardiac allografts.     

Effects of Glycyrrhizic Acid in Licorice on Prolongation of Murine Cardiac Allograft Survival

Recent evidence has pointed to the promising benefits of using specific immunosuppressive herbal compounds to prolong transplant allograft survival. In this study, we investigated the effects of glycyrrhizic acid (GA), a major component of licorice, in a model of murine heart transplantation. CBA (H2^k) mice were transplanted with a fully-MHC mismatched C57BL/6 (H2^b) heart allograft and subsequently received daily intraperitoneal administration of normal saline or 0.02, 0.2, or 2.0 mg/d of GA for 7 consecutive days. Untreated CBA recipients, with a median survival time (MST) of 7 days, and groups receiving 0.02mg/d (MST, 8 days) or 0.2mg/d (MST, 9 days) of GA acutely rejected C57BL/6 cardiac allografts. But mice treated with 2.0 mg/d of GA demonstrated significant prolongation of allografts (MST, 23 days). Histologic studies showed that cardiac allografts from GA-treated CBA recipients had preserved graft and vessel structure. Moreover, flow cytometric study showed that the percentage of CD4⁺CD25⁺Foxp3⁺ cell (regulatory T cell [Treg]) populations were increased in GA-treated CBA recipients. In a mixed leukocyte culture, splenocytes from GA-treated mice demonstrated suppressed allo-proliferation, in which interleukin (IL)-2 and interferon gamma production were downregulated and IL-10 secretion was upregulated. In conclusion, GA may be a novel promising therapeutic agent to prolong cardiac allograft survival through direct anti-inflammatory effects and induction of Treg populations.     

Graft Protective Effect of HMG-CoA Reductase Inhibitor Pravastatin in Murine Cardiac Allograft Transplantation

The HMG-CoA reductase inhibitor (statin), which reduces serum cholesterol, has been demonstrated in the control of immune responses and may potentially play an important role in the regulation of acute and chronic rejection in organ transplantations. We investigated the graft-protective effect of a kind of statin, pravastatin, in the survival of fully major histocompatibility complex--mismatched murine cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients through microsurgical techniques. CBA recipients transplanted with a C57BL/6 heart received oral administration of 40, 120, or 400 μg/kg/day of pravastatin from the day of transplantation to 7 days afterward. Immunohistochemical staining studies were performed to determine whether intimal formation of coronary arteries in the transplanted cardiac allografts was preserved and also to conduct morphometric analysis. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] 7 days). CBA recipients exposed with 40 and 120 μg/kg/day of pravastatin had a small prolonged allograft survival (MSTs of 10 and 9 days, respectively). However, the MST of CBA recipients exposed to 400 μg/kg/day of pravastatin was significantly effective for allograft survival (MST 50 days). Immunohistochemical staining assessments on 4 weeks after grafting showed suppression of intimal hyperplasia in allograft coronary arteries. Pravastatin could induce the prolongation of fully major histocompatibility complex--mismatched cardiac allograft through the protection of the coronary artery.     

Effects of immunosuppressants on induction of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery (ITD) of alloantigen generated regulatory cells in mice. Here, we examined the effect of various doses of conventional immunosuppressants (FK506, cyclosporine A, azathioprine, mycophenolate mofetil, and rapamycin) on inducing regulatory cells in our model. METHODS: CBA mice (primary recipients) were given C57BL/6 splenocytes by ITD and either no additional treatment or various doses of an immunosuppressant. Seven days later, splenocytes from these mice were adoptively transferred into naive secondary CBA recipients that underwent C57BL/6 cardiac grafting the same day. RESULTS: Adoptive transfer from primary recipients given ITD of splenocytes alone induced prolonged allograft survival in secondary recipients (median survival time [MST], 50 days), suggesting that regulatory cells were generated. When ITD of alloantigen was combined with daily administration of 0.1 mg/kg FK506 or 0.2 mg/kg rapamycin, graft survival was similarly prolonged (MST 55 and 50 days, respectively). When combined with 20 or 40 mg/kg MMF or 0.4 mg/kg rapamycin, the majority of recipients demonstrated indefinite survival (MST, >100 days in all groups). When ITD of alloantigen was combined with 0.3, 0.5, or 1.0 mg/kg FK506; 5, 10, or 25 mg/kg cyclosporine A; or 1.0 or 2.0 mg/kg azathioprine, allografts were rejected acutely (MST 7-13 days). CONCLUSION: Generation of regulatory cells by ITD of alloantigen was facilitated by mycophenolate mofetil and high doses of rapamycin but abrogated by cyclosporine A, azathioprine, and high doses of FK506. Low doses of rapamycin and of FK506 did not interfere with generation of regulatory cells.     

CD27/CD70, CD134/CD134 ligand, and CD30/CD153 pathways are independently essential for generation of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We investigated whether blockade of tumor necrosis factor receptor-ligand pathways could generate regulatory cells induced by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1x10(7)) from C57BL/10 (H-2b) mice and intraperitoneal administration of monoclonal antibody (mAb) specific for CD70, CD134 ligand (CD134L), CD153, or CD137L. Seven days later, C57BL/10 hearts were transplanted into pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes (5x10(7)) from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST] 12 days). Pretreatment with intratracheal delivery of C57BL/10 donor splenocytes prolonged graft survival significantly (MST 84 days). Mice given intratracheal delivery of alloantigen plus anti-CD70, anti-CD134L, or anti-CD153 mAb, but not those given intratracheal delivery of alloantigen plus anti-CD137L mAb, rejected their graft acutely (MST 16, 14, 10, and 65 days, respectively). Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of alloantigen plus anti-CD70, CD134L, or CD153 mAb did not prolong survival of C57BL/10 cardiac grafts in naive secondary CBA recipients (MST 14, 11, and 11 days, respectively), whereas adoptive transfer of splenocytes from mice given intratracheal delivery of alloantigen plus anti-CD137L mAb did (MST 75 days). CONCLUSION: The CD27/CD70, CD134/CD134L, and CD30/CD153 pathways are independently required for generation of regulatory cells in our model.     

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen

BACKGROUND: Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti-PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti-PD-1 or anti-PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti-PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti-PD-1 or anti-PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). CONCLUSIONS: PD-1-PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.     

Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. METHODS: CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). CONCLUSION: IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.     

Artemisiae capillaris herba induces prolonged survival of fully cardiac allografts and generates regulatory cells in mice

Inchingorei-san (TJ-117), a 6-component herbal medicine, is used in Japan for the treatment of vomiting, urticaria, and liver and kidney disorders with few side effects. In this study we investigated the effect of TJ-117 on alloimmune responses in murine cardiac allograft transplantation. CBA (H2^k) mice underwent transplantation of a C57BL/6 (B6, H2^b) heart with oral administration of TJ-117 (or 1 component of TJ-117) from the day of transplantation for 7 days. CBA recipients given 1 g/kg/d of TJ-117 showed prolonged B6 allograft survival (median survival time [MST], 23 days). Naive CBA mice rejected B6 cardiac grafts acutely (MST, 7 days). Moreover, Artemisiae capillaris herba (ACH; 1g/kg/d) 1 component of TJ-117, significantly prolonged B6 allograft survival (MST, > 100 days). However, the other 5 components of TJ-117 were individually less effective than TJ-117 or ACH. ACH also suppressed splenocyte proliferation and interferon-γ production. Secondary CBA recipient showed prolonged survival of B6 hearts after treatments with whole splenocytes from primary ACH-treated CBA recipients carrying B6 cardiac allografts for 30 days (MST, >50 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in transplant recipients given ACH. In conclusion, ACH, 1 component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts with generation of CD4⁺CD25⁺Foxp3⁺ regulatory cells.     

Induction of hyporesponsiveness to fully allogeneic cardiac grafts by intratracheal delivery of alloantigen

BACKGROUND: Soluble protein delivered through the mucosal surface can induce immunological unresponsiveness. The purpose of this study was to determine if prior exposure to alloantigen via the trachea could modulate the immune response to subsequent cardiac allografts. METHODS: Hearts from C57BL/10(H2b) mice were transplanted into CBA(H2k) recipients. Recipient mice were given donor 1x10(7) splenocytes into the trachea with or without antibody specific for mouse CD80 (1G10) and/or CD86 (GL1) (100 microg each) 7 days before transplantation. RESULTS: All grafts survived in recipients treated with intratracheal delivery of alloantigen for over 35 days (mean survival time [MST], 56 days), whereas naive control mice and mice treated with syngeneic antigen rejected grafts acutely (MST, 8 and 7 days, respectively). Interestingly, when 1G10, GL1, or both of them were combined with the protocol, the majority of grafts were rejected within 21 days after grafting (MST, 7, 15, and 17 days, respectively). CONCLUSION: Intratracheal delivery of alloantigen induced significantly prolonged survival of fully mismatched cardiac allografts and the effect was abrogated by the blockade CD80 and/or CD86 pathway.     

胸腺内注射可溶性异基因主要组织相容性复合物抗原诱导皮肤移植特异性耐受

用３ｍｏｌ／ＬＫＣｌ从Ｃ５７ＢＬ／６小鼠脾细胞提取可溶性主要组织相容性复合物（ＭＨＣ）抗原，注射到ＢＡＬＢ／Ｃ受体鼠胸腺内，诱导了成年小鼠对该异基因小鼠皮肤移植物的耐受。除在胸腺注射当天及第３天给予抗Ｔ细胞单克隆抗体外，不使用免疫抑制剂。实验组移植皮肤平均存活时间（ＭＳＴ）为８３天，对照组ＭＳＴ为１１天。诱导耐受的小鼠对第３供体的移植皮肤仍正常排斥（ＭＳＴ为１２天）。单向混合淋巴细胞反应，耐受小鼠脾脏淋巴细胞对特异供体的脾细胞无反应，对第３供体的脾细胞反应正常，对丝裂原刺激的增殖反应正常。显示诱导的耐受是供体特异性的，无非特异性免疫抑制。     

Administration of Thrombomodulin (CD141) Could Improve Cardiac Allograft Survival in Mice

Thrombomodulin (TM) is a promising natural anti-coagulant therapeutic protein that is effective in the treatment of disseminated intravascular coagulation. However, the mechanisms by which TM on micro-vessels enable the regulation of intimal hyperplasia remain elusive. We investigated the graft-protective effects of TM in a fully major histocompatibility complex-mismatched murine cardiac allograft transplantation model. CBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of 0.2, 2.0, and 20.0 μg/day of TM for 8 days. Histological staining was conducted to assess the degree of inflammation and infiltration in the transplanted cardiac grafts. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] was 7 days). CBA recipients exposed to the above dosages had significantly prolonged allograft survival (MSTs were 16, 21, and 37.5 days, respectively). Histologic assessments from TM-exposed recipients 2 weeks after grafting showed that the myocardium and vessel structure in their allografts were clearly preserved, and that the infiltration of inflammatory cells around coronary arteries was suppressed. TM can induce the prolongation of fully major histocompatibility complex-mismatched cardiac allograft by exerting graft protective effects within the myocardium and coronary arteries.     

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.