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.     

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.     

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.     

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 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.     

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.     

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.     

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.     

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.     

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.     

B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice

BACKGROUND: The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice. METHODS: In CBA (H2 ) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2 ) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7-1, B7-2, or CTLA4 were adoptively transferred to na?ve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC). RESULTS: Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days; P<0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7-1, B7-2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased. CONCLUSIONS: Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7-1, B7-2, and CTLA4 signals.     

A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells

BACKGROUND: Peritransplant treatment with antithymocyte serum (ATS) and posttransplantation administration of donor bone marrow or donor splenocytes results in extended skin allograft survival. In this study, we examined the molecular basis of the tolerance promoting effect of donor bone marrow (BMC) cells and splenocytes with emphasis on the role of CD8 expression on the donor cells. METHODS: (C57BL/6J x A/J)F1 mice were treated on days -1 and +2 with ATS relative to transplantation with C3H/HeJ skin. On day +7, they were infused with CD8+ BMC, CD8- BMC, CD8+ splenocytes, or CD8- splenocyte donor subpopulations isolated by magnetic or fluorescence-based sorting. In additional experiments, B10.D2(R107) mice were treated in the same manner with C57BL/6 skin and BMC or splenocytes from C57BL/6 mice in which the CD8alpha gene had been inactivated. RESULTS: CD8+ donor bone marrow cells induced operational tolerance (defined as graft acceptance in the absence of chronic immunosuppression) in skin graft recipients at a dose that was reduced by 250-fold relative to unfractionated bone marrow cells (1.0x10(5) cells per recipient, median survival time (MST)=41 days vs. 2.5x10(7) cells per recipient, MST=49 days, P=0.40). Similarly, donor bone marrow cells from CD8 knockout mice did not promote graft acceptance (MST=98 days vs. animals not treated with bone marrow cells, MST=70 days, P=0.16). In contrast, the extension of graft survival by donor splenocytes did not require the presence of CD8+ donor cells because splenocytes depleted of CD8+ cells extended graft survival (MST=55 days) as well as unsorted splenocytes (44 days, P=0.2), and splenocytes from CD8 knockout animals (MST=145 days) extended graft survival at least as well as unsorted splenocytes (MST=74 days, P=0.4) CONCLUSIONS: These results suggest that the prolongation of graft survival by donor bone marrow is dependent on the presence of the CD8 molecule, whereas prolongation by donor splenocytes is not. Therefore, we suggest that the prolongation of graft survival by these cell types occurs via distinct molecular mechanisms probably mediated by different cell types.     

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).     

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.     

Various costimulatory pathways are essential for induction of regulatory cells by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen induced regulatory cells in a mouse heart transplantation model. We investigated the roles of costimulatory pathways in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1 x 10(7)) from C57BL/10 (H-2b) mice and administration of monoclonal antibodies (mAb) specific for programmed death (PD)-1 and its ligands, programmed death-ligand (PD-L)1 and PD-L2, CD70, CD134 ligand (CD134L), CD153, CD137L, or receptor activator of nuclear factor-kappaB (NF-kappaB) (RANK). Seven days later, naive CBA mice underwent adoptive transfer of splenocytes (5 x 10(7)) from the pretreated CBA mice and transplantation of C57BL/10 heart. 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). Concomitant administration of control immunoglobulin (Ig)G, anti-PD-L2 mAb, or anti-CD137L along with intratracheal delivery did not significantly affect the prolongation (MST, 72, 68, and 65 days, respectively). In contrast, anti-PD-1, anti-PD-L1, anti-CD70, anti-CD134L, anti-CD153, or anti-RANK mAb abrogated the prolongation induced by adoptive transfer from the pretreated mice with intratracheal delivery (MST, 18, 17, 16, 14, 10, and 18 days, respectively). CONCLUSION: The PD-1/PD-L1, CD27/CD70, CD134/CD134L, CD30/CD153, and tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE)/RANK interactions are independently required for generation of regulatory cells by intratracheal delivery of alloantigen.     

Operational tolerance induced by pretreatment with donor dendritic cells under blockade of CD40 pathway.

BACKGROUND: Dendritic cells can mount immune response as competent antigen presenting cells. Recently, it has been reported that immature dendritic cells induce prolongation of allograft survival. However, the ability of mature dendritic cells to induce operational tolerance is unclear. Therefore, in this study, we examined the ability of splenic mature dendritic cells to induce operational tolerance to fully allogeneic antigens using mouse heterotopic heart transplantation model. METHODS: CBA (H2k) mice received i.v. injections with donor splenic dendritic cells or B cells in the absence or presence of monoclonal antibody (mAb) specific for CD40 ligand or CD80/CD86 2 weeks before transplantation of a C57BL/10 (H2b) heart. RESULTS: When donor dendritic cells were injected i.v. 2 weeks before transplantation, rejection response was accelerated compared with that of naive mice [median survival time (MST) = 7 and 8 days, respectively]. However, when CD40 pathway was blocked by anti-CD40 ligand mAb, i.v. injection of donor dendritic cells but not B cells induced indefinite graft survival (MST >100 and 20 days, respectively). Mice treated with anti-CD40 ligand mAb alone rejected their grafts with a MST of 18 days. Intravenous injection of donor dendritic cells and B cells in combination with anti-CD80/CD86 mAbs was less effective to induce graft prolongation (MST = 9.5 and 13 days, respectively). CONCLUSIONS: Therefore, under blockade of CD40 pathway, mature dendritic cells were tolerogens in vivo independent of CD80/86 pathways.     

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。     

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.     

阻断ICOS/B7h信号的供体特异性输血对移植受体CD4+CD25+调节性T细胞的影响

目的 观察阻断ICOS/B7h信号的供体特异性输血(DST)对异基因小鼠心脏移植术后体内CD4+CD25+调节性T细胞(Treg)的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组:供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组:移植当天给予受体鼠(C57BL/6)尾静脉注射5×106 ICOS-Fc靶定的供体(BALB/C)脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4+CD25+Treg的亚群比例,利用逆转录-聚合酶链反应(RT-PCR)检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4+CD25+Treg对CD4+CD25-效应T细胞(Teff)的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[(84.38±29.14)d比(7.00±0.76)d,P＜0.01].各组中,治疗组受体外周血中CD4+CD25+Treg亚群比例显著上调[(15.60±5.69)%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4+CD25+Treg能够更高效地抑制CD4+CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4+CD25+Treg在耐受的形成与维持中均起着重要作用.