Nitric oxide and indoleamine 2,3-dioxygenase mediate CTLA4Ig-induced survival in heart allografts in rats

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.     

Anti-CD40 therapy extends renal allograft survival in rhesus macaques

BACKGROUND: Organ transplant recipients currently require lifetime immunosuppressive therapy, with its accompanying side effects. Biological agents that block T-cell costimulatory pathways are important components of strategies being developed to induce transplantation tolerance. The aim of this study was to test the effect of a novel chimeric anti-human CD40 monoclonal antibody (Chi 220), either alone or in combination with CTLA4-Ig, on the survival of renal allografts in a nonhuman primate model. METHODS: Captive-bred adolescent male rhesus monkeys (Macaca mulatta) (4-10 kg) were used as recipients and donors. Four treatment protocols were tested: Chi220 monotherapy, CTLA4-Ig monotherapy, Chi220 combined with CTLA4-Ig, and H106 (anti-CD40L) combined with CTLA4-Ig. Control animals received human albumin. Recipients were followed for survival, renal allograft function as determined by measurement of serum blood urea nitrogen (BUN) and creatinine, chemistries (sodium, potassium, chloride, and bicarbonate), complete blood cell count (CBC) with differential, and the development of donor-specific alloantibody. RESULTS: Treatment with Chi220 for 14 days prolonged renal allograft survival (MST 38.5 vs. 7 days in untreated controls). Notably, simultaneous blockade of the CD28/B7 pathway did not further augment graft survival but did suppress the development of donor-specific antibodies, an effect not achieved with Chi220 alone, despite peripheral B cell depletion. Finally, treatment with Chi220 suppressed the primary immune response to cytomegalovirus, resulting in severe systemic manifestations. CONCLUSIONS: Blockade of the CD40 pathway with anti-CD40 mAb is immunosuppressive in a large animal, preclinical renal transplant model. The potential effect of this therapy on viral immune responses will be important to consider for the design of safe clinical trials.     

Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb.

BACKGROUND: We previously reported the association among donor leukocyte chimerism, apoptosis of presumedly IL-2-deficient graft-infiltrating host cells, and the spontaneous donor-specific tolerance induced by liver but not heart allografts in mice. Survival of the rejection-prone heart allografts in the same strain combination is modestly prolonged by the pretransplant infusion of immature, costimulatory molecule-(CM) deficient donor dendritic cells (DC), an effect that is markedly potentiated by concomitant CM blockade with anti-CD40L (CD154) monoclonal antibody (mAb). We investigated whether the long survival of the heart allografts in the pretreated mice was associated with donor leukocyte chimerism and apoptosis of graft-infiltrating cells, if these end points were similar to those in the spontaneously tolerant liver transplant model, and whether the pretreatment effect was dependent on sustained inhibition of CM expression of the infused immature donor DC. In addition, apoptosis was assessed in the host spleen and lymph nodes, a critical determination not reported in previous studies of either spontaneous or "treatment-aided" organ tolerance models. METHODS: Seven days before transplantation of hearts from B10 (H-2b) donors, 2x10(6) donor-derived immature DC were infused i.v. into C3H (H-2k) recipient mice with or without a concomitant i.p. injection of anti-CD40L mAb. Donor cells were detected posttransplantation by immunohistochemical staining for major histocompatibility complex class II (I-Ab) in the cells of recipient lymphoid tissue. CM expression was determined by two-color labeling. Host responses to donor alloantigen were quantified by mixed leukocyte reaction, and cytotoxic T lymphocyte (CTL) assays. Apoptotic death in graft-infiltrating cells and in areas of T-dependent lymphoid tissue was visualized by terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick-end labeling and quantitative spectrofluorometry. Interleukin-2 production and localization were estimated by immunohistochemistry. RESULTS: Compared with control heart transplantation or heart transplantation after only DC administration, concomitant pretreatment with immature donor DC and anti-CD40L mAb caused sustained elevation of donor (I-Ab+) cells (microchimerism) in the spleen including T cell areas. More than 80% of the I-Ab+ cells in combined treatment animals also were CD86+, reflecting failure of the mAb to inhibit CD40/ CD80/CD86 up-regulation on immature DC in vitro after their interaction with host T cells. Donor-specific CTL activity in graft-infiltrating cells and spleen cell populations of these animals was present on day 8, but decreased strikingly to normal control levels by day 14. The decrease was associated with enhanced apoptosis of graft-infiltrating cells and of cells in the spleen where interleukin-2 production was inhibited. The highest levels of splenic microchimerism were found in mice with long surviving grafts (>100 days). In contrast, CTL activity was persistently elevated in control heart graft recipients with comparatively low levels of apoptotic activity and high levels of interleukin-2. CONCLUSION: The donor-specific acceptance of rejection-prone heart allografts by recipients pretreated with immature donor DC and anti-CD40L mAb is not dependent on sustained inhibition of donor DC CM (CD86) expression. Instead, the pretreatment facilitates a tolerogenic cascade similar to that in spontaneously tolerant liver recipients that involves: (1) chimerism-driven immune activation, succeeded by deletion of host immune responder cells by apoptosis in the spleen and allograft that is linked to interleukin-2 deficiency in both locations and (2) persistence of comparatively large numbers of donor-derived leukocytes. These tolerogenic mechanisms are thought to be generic, explaining the tolerance induced by allografts spontaneously, or with the aid of various kinds of immunosuppression.     

Prolongation of renal allograft survival in rats by replication-defective recombinant adenovirus-mediated coexpression of CD40L and CTLA4Ig

BACKGROUND: Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. METHODS: We constructed Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. RESULTS: Successful construction of Adv-shCD40L-IRES2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2%+/-0.6% and 42.1%+/-0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES2-CTLA4Ig resulted in further inhibition of MLR to 22.0%+/-0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8+/-2.5 days and 27.3+/-3.6 days, respectively, as compared to vehicle-treated controls (7.8+/-0.3 days). Cotransfection of both genes extended graft survival to 41.8+/-3.7 days. CONCLUSIONS: Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.     

Anti-LFA-1 improves pig islet xenograft function in diabetic mice when long-term acceptance is induced by CTLA4Ig/anti-CD40L

BACKGROUND: It has been previously demonstrated that addition of anti-LFA-1 to a combination of CTLA4Ig and anti-CD40L induces the permanent acceptance of dopaminergic fetal pig xenografts when transplanted into the brain of wild-type mice. The purpose of this study was to test whether this costimulation blockade also can induce acceptance of adult pig islets transplanted to C57BL/6 mice with streptozotocin-induced diabetes. METHODS: Recipients were treated with CTLA4Ig/anti-CD40L+/-anti-LFA-1 or isotype control antibodies during the first week after transplantation. Half of the costimulation blockade-treated recipients had their grafts removed after 8 weeks. The other half was observed up to 5 months. RESULTS: Recipients treated with CTLA4Ig/anti-CD40L/anti-LFA-1 had significantly lower blood glucose and gained more weight than CTLA4Ig/anti-CD40L-treated recipients. CTLA4Ig/anti-CD40L-treated recipients exhibited unstable blood glucose. IPGTT of these recipients revealed a slow recovery to normal blood glucose levels at week 4. In comparison, CTLA4Ig/anti-CD40L/anti-LFA-1 treated recipients exhibited a significantly superior glucose clearance. CTLA4Ig/anti-CD40L+/-anti-LFA-1 treated recipients did not produce anti-pig IgG, whereas control antibody-treated mice did. CD4+ T cells from costimulation blockade-treated recipients proliferated less than CD4+ T cells from control antibody-treated mice when co-cultured with syngeneic antigen presenting cells loaded with pig islet antigens. CONCLUSIONS: CTLA4Ig/anti-CD40L/anti-LFA-1-treated recipients had superior islet function compared with CTLA4Ig/anti-CD40L-treated recipients. However, both costimulation blockade regimens led to islet graft acceptance up to 5 months after a 1-week treatment.     

Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model

BACKGROUND: The blockade of costimulatory signal pathway by anti-CD40 ligand antibody or cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) prolongs allograft survival in various vascularized organ transplantations. Because of the short half life of these agents, repeated administration of proteins is required to achieve significant graft survival. Furthermore, there is limited information regarding the effect of cosimulatory blockade on the survival of composite tissue allografts. Therefore, we examined the effect of adenovirus-mediated gene transfer of CTLA4Ig or CD40Ig gene or both in composite tissue allotransplantation. METHODS: The hind limbs removed from male ACI rats (RT1 ) were transplanted into female Lewis rats (RT1 ) heterotopically. The recombinant adenovirus carrying CTLA4Ig (AxCTLA4Ig) or CD40Ig (AxCD40Ig) was intravenously administered after limb transplantation. RESULTS: Limb allograft survival was significantly prolonged by either AxCTLA4Ig or AxCD40Ig treatment at 1 x 10 plaque forming unit (mean survival time [MST] of 39.4+/-6.0 and 13.0+/-2.9, respectively) compared with the adenovirus vector containing beta-galactosidase-treated group (MST of 4.8+/-0.8). Combination of AxCTLA4Ig and AxCD40Ig led to significant prolongation of graft survival (MST of 49.2+/-6.6). Serum levels of CD40Ig were higher in rats treated with combination therapy than those treated with AxCD40Ig alone, whereas the serum levels of CTLA4Ig in rats treated with AxCTLA4Ig alone and AxCTLA4Ig and AxCD40Ig combined were very similar. CONCLUSION: This study indicates that an adenovirus-mediated gene therapy of CTLA4Ig or CD40Ig has a therapeutic potential for preventing rejection in composite tissue transplantation. Furthermore, a combination therapy of AxCTLA4Ig and AxCD40Ig was even more effective in preventing acute rejection and prolonging the survival of allografted limbs without apparent complication.     

外用环孢素A联合CTLA4Ig延长异体移植鼠耳存活的研究

目的　探讨局部外用环孢素 A(Cs A)联合细胞毒性淋巴细胞相关抗原 4融合蛋白 (CTL A4 Ig)对异体复合组织移植的免疫抑制及诱导免疫耐受的作用。方法　建立吻合血管的同种异体大鼠耳廓移植模型 ,术后在移植耳皮肤表面外涂 Cs A并联合 CTL A4 Ig腹腔注射治疗 ,观察移植物的排斥反应及存活时间 ,检测移植后受体血清白细胞介素 - 2 (IL- 2 )含量变化。结果　对照组平均存活时间为 (7.8± 1.7)天 ;单纯用 Cs A治疗组为 (15 .2± 1.9)天 ,单纯CTL A4 Ig治疗组为 (16 .6± 2 .1)天 ;Cs A +CTL A4 Ig联合治疗组为 (2 8.8± 3.5 )天 ,与其它各组相比均有统计学意义 (P<0 .0 1) ;且联合治疗组的受体血清 IL - 2含量最低 ,尤以第 5、7天为著 ,与其它各组相比有统计学意义 (P<0 .0 1)。结论　局部外用 Cs A联合 CTL A4 Ig能有效抑制异体复合组织移植排斥反应 ,显著延长移植物存活时间。     

CTLA4-Ig abrogates the anti-globulin response and prolongs cardiac allograft survival after anti-CD2 treatment

CD2 is expressed on T cells and NK cells and is important in T cell activation, making it a potential target for immune intervention. Here, we report a series of experiments aimed at defining the ability of mAbs directed against the CD2 molecule to prevent cardiac allograft rejection in low and high responder rat strain combinations. Administration of the mouse anti-rat CD2 mAbs OX34 or OX55 around the time of transplantation prolonged survival of fully allogeneic Lewis (RT1l) cardiac allografts in low responder DA (RT1a) recipients (MST 14 days for OX55 and >100 days for OX34). Treatment with OX34 prolonged graft survival in the reciprocal high responder DA to Lewis rat strain combination (MST 19 days) and when combined with CTLA4-Ig resulted in long-term graft survival (MST>100 days). Despite these in vivo effects, OX34 had little effect on in vitro assays of lymphocyte activation. Instead, the ability of OX34 to extend allograft survival correlated with T cell depletion. Administration of OX34 induced a similar degree of CD4 T cell depletion in DA and Lewis recipients, but the CD4 depletion observed was more transient in Lewis recipients. Lewis, but not DA strain rats, developed an anti-murine Ig response. Combined treatment with CTLA4-Ig abolished the anti-globulin response to OX34 in Lewis recipients, prolonged circulation of OX34 and increased the extent and duration of CD4 depletion. We conclude that anti-CD2 treatment effectively prolongs cardiac allograft survival and addition of CTLA4-Ig increases its efficacy by abrogating the production of neutralising antibodies.     

Induction of operational tolerance to discordant dopaminergic porcine xenografts

BACKGROUND: Porcine embryonic neural tissue transplanted intracerebrally could potentially relieve the symptoms of Parkinson's disease if the immune response toward the graft could be overcome. However, conventional immunosuppressive treatments have proven inefficient in preventing rejection. An alternative is blocking the costimulatory signals for lymphocyte activation. Treatment with cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and anti-CD40L has been successful in preventing rejection of xenografts in some experimental studies, but not all. Lymphocyte function antigen (LFA)-1 is an important costimulatory molecule for CD8+ T cells, and we hypothesize that blockade with anti-LFA-1 may enhance the efficacy of CTLA4Ig and anti-CD40L therapy. METHODS: C57BL/6 mice received intracerebral transplants of ventral mesencephalic tissue from embryonic porcine donors. CTLA4Ig, anti-CD40L, and anti-LFA-1 were administered every other day on days 0 to 8, and the transplants were studied after 4 to 6 weeks. Grafts were histologically analyzed for size, survival of dopaminergic nerve cells, and immune responses. Recipients were challenged with cultured glia cells of donor origin or an allogeneic skin graft to evaluate tolerance induction. RESULTS: Mice treated with all three substances had large grafts containing high amounts of dopamine cells but a low degree of immune response. Grafts in recipients challenged with glial cells showed an increased immunologic activity but were not rejected. Triple-treated mice showed a normal rejection process of the allogeneic skin grafts. CONCLUSION: After a short course of costimulation blocking therapy, discordant neural xenografts demonstrate long-term survival, withstand immunologic challenge, yet maintain host-versus-graft reactivity. Anti-LFA-1 complements CTLA4Ig and anti-CD40L in the induction of operational tolerance to these xenografts.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

CTLA4ig induces long-term graft survival of allogeneic skin grafts and totally inhibits T-cell proliferation in LFA-1-deficient mice

BACKGROUND: It was recently shown that some strains of mice are capable of rejecting transplants independently of B7 and CD40L signaling and that this rejection is mediated by CD8(+) T cells. LFA-1 is known to be important for CD8(+) T cell activation and cytotoxicity. Therefore, blockade of LFA-1 could be important in overcoming costimulation blockade, CD8(+) T-cell-mediated, resistant rejection. The purpose of this study was to define the effect of combined blockade of the LFA-1 and B7 costimulation pathways on the alloimmune response in mice. METHODS: Allogeneic skin transplantation was performed using BALB/c mice as donors and C57BL/6J wild-type or LFA-1-deficient (CD11a(-/-)) mice as recipients. CTLA4Ig or anti-LFA-1 was administered either as an induction or a prolonged therapy. Mixed lymphocyte reactions were conducted to study the effect of CTLA4Ig on T-cell proliferation in CD11a(-/-) mice. RESULTS AND CONCLUSIONS: Administration of CTLA4Ig completely inhibits CD11a(-/-) T-cell proliferation in response to alloantigens and significantly improved skin allograft survival in CD11a(-/-) mice. Prolonged treatment of wild-type recipient mice with CTLA4Ig and anti-LFA-1 increased median survival time to 45.5 days compared with 16 days after induction therapy, but it was not sufficient to induce indefinite allograft survival in this model.     

Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation

An inducible co-stimulator (ICOS), a recently identified co-stimulatory receptor with a close structural homology of CD28 and CTLA4, is expressed on activated T cells. Anti-ICOS antibody was demonstrated to be effective on prolongation of graft survival after liver transplantation in rats. In this study, we investigated the potency of tolerance induction using the antibody combined with a recombinant adenovirus vector containing CTLA-4Ig cDNA (AdCTLA-4Ig) in rat heart transplantation model. Using a DA-to-Lewis rat heart transplantation model, an anti-rat ICOS antibody and AdCTLA-4Ig were simultaneously administered i.v. into recipients. The tissue specimens from the grafts were removed on various days after transplantation for histological evaluation. Donor-strain skin and heart grafts, and third-party heart allografts were challenged in the recipients with a long-term surviving graft. Splenocytes from the tolerance-induced recipients were used for adoptive transfer study. Anti-ICOS antibody alone did not prolong the survival of heart allograft. AdCTLA-4Ig monotherapy significantly prolonged the survival of heart allograft (Group 4). With a combination of Anti-ICOS antibody and AdCTLA-4Ig, all recipients were resulted in a long-term allograft acceptance for more than 200 days (Group 8). When challenged donor-strain skin grafts in the tolerant rats of Group 4, the skin was rejected, which also lead to a rejection of primary heart allografts. The recipients in Group 8 also rejected donor-strain skin grafts with no rejection of the primary heart grafts. These recipients accepted secondary heart grafts from donor-strain but not third-party. In Group 8 long-term survival recipients showed a high population of CD4+CD25+ regulatory T cell in peripheral blood, and in adoptive transfer study subtraction of these CD4+CD25+ T cells accelerate the rejection of heart graft in secondary irradiated recipients. The present results demonstrated that anti-ICOS antibody combined with AdCTLA-4Ig potently induces a stable immune tolerance after heart allografting in rat, which is mediated by the induction of CD4+CD25+ regulatory T cells. This strategy may be attractive for clinical employment to induce transplantation tolerance.     

T-cell response to cardiac myosin persists in the absence of an alloimmune response in recipients with chronic cardiac allograft rejection

BACKGROUND: Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. METHODS: The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. RESULTS AND CONCLUSIONS: MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to na?ve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.     

4-1BB promotes long-term survival in skin allografts treated with anti-CD45RB and anti-CD40L monoclonal antibodies

4-1BB (CD137) is a T-cell co-stimulatory molecule that promotes T cell activation. Using a skin transplantation model, we observed that simultaneous administration of monoclonal antibodies (mAb) targeting CD45RB and CD40L prolonged skin allograft in co-stimulation blockade (CTLA4-Ig and anti-CD40L mAb)-resistant mice, because of reducing CD8(+) T cells and CD4(+) CD45RB(high) T cells. Anti-CD45RB mAb (45RB) blocks the activation of T helper 1 (Th1) cells and generates regulatory T cells (T(reg)). The experimental design included five groups: group 1, control; group 2, 45RB-MR1; group 3, 45B-MR1 + 4-IBBL; group 4, anti-CD4 mAb plus group 3 treatment; group 5, anti-CD8 mAb plus group 3 treatment. In this study we highlight the involvement of 4-1BB/4-1BBL in the development of T-cell responses. C57BL/6 recipients of BALB/c skin grafts were treated with 45RB, anti-CD40L mAb (MR1), and antagonistic anti-4-1BBL mAb (4-1BBL) on days 0, 2, 4, 6, and 8 posttransplantation. Additional 4-1BBL further prolonged skin graft survival, although the percentage of splenocyte-derived CD8(+) T cells was reduced similarly in both groups. Use of 4-1BBL seems to have additive effects on T(reg) cells, which play a major role in the maintenance of tolerance. Even after immunosuppressive therapy in combination with CD4(+) T-cell depletion, we did not achieve prolonged graft survival, possibly because of the absense of T(reg) cells, which require CD4-independent CD8(+) T cells, based on the observation of increasing proportion of CD8(+) T cells in similar degree as the control group.     

Mechanisms Involved in the Establishment of Tolerance Through Costimulatory Blockade and BMT: Lack of Requirement for CD40L-Mediated Signaling for Tolerance or Deletion of Donor-reactive CD4+ Cells

We have previously shown that high levels of multiline-age mixed hematopoietic chimerism and systemic T-cell tolerance can be achieved in mice without myeloablation through the use of anti-CD40L and costimulatory blockade alone (plus CTLA4Ig) or with recipient CD8 depletion and allogeneic bone marrow transplantation. Chimeric mice permanently accept donor skin grafts (> 100 days), and rapidly reject third-party grafts. The mechanisms by which costimulatory blockade facilitates the engraftment of allogeneic hematopoietic cells have not been defined. To further understand the in vivo mechanisms by which the administration of anti-CD40L mAb facilitates the engraftment of donor bone marrow and rapidly tolerizes CD4+ T cells, we analyzed the establishment of chimerism and tolerance in CD40L -/- mice. We demonstrate here that anti-CD40L mAb treatment is required only to prevent CD40L/CD40 interactions, and that no signal to the T cell through CD40L is necessary for the induction of CD4+ tolerance. Peripheral deletion of donor-reactive CD4+ T cells occurs rapidly in CD40L -/- mice receiving bone marrow transplantation (BMT), indicating that this deletion in the presence of anti-CD40L is not due to targeting of activated CD4+ cells by the antibody. Complete CD4+ cell tolerance is observed by both skin graft acceptance and in vitro assays before deletion is complete, indicating that additional mechanisms play a role in inducing CD4+ T-cell tolerance as the result of BMT in the presence of CD40/CD40L blockade.     

Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival

BACKGROUND: The expression of costimulatory molecules on antigen-presenting cells is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODNs) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendritic cells (DCs). MATERIALS AND METHODS: DCs were propagated from C57BL/10 (B10; H2b) bone marrow cells in granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, and transduced with anti-CD80 or anti-CD86 antisense ODNs (base-mismatched ODNs as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DCs was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. RESULTS: ODNs were effectively incorporated by DCs, which were enhanced by the presence of lipofectamine. Antisense ODNs targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DCs and inhibited their capacity to elicit the proliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2k) spleen T cells. This was associated with decreased IL-2, but elevated IL-4 production, and an increase in T-cell apoptosis. In contrast with the administration of control DCs into C3H recipients that exacerbated rejection of B10 cardiac allografts, injection of DCs transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. CONCLUSION: Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective approach to modify DCs that renders them tolerogenic by inducing T-cell hyporesponsiveness and apoptosis. This may lead to the development of new therapeutic strategies in transplantation.     

New insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs

OBJECTIVE: To determine the precise in vivo interaction between T-cell costimulatory blockade and conventional immunosuppression in transplantation. SUMMARY BACKGROUND DATA: Blocking B7 or CD154 T-cell costimulatory activation pathways prevents allograft rejection in small and large animal transplant models and is considered a promising strategy for clinical organ transplantation. METHODS: A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD154 or CTLA4Ig monotherapy and conventional immunosuppressive drugs in promoting long-term graft acceptance. The frequency of alloreactive T cell was measured by ELISPOT. Chronic rejection was examined by histology. RESULTS: Cyclosporine, tacrolimus, and anti-IL-2R monoclonal antibody therapy abrogated the effect of a single-dose protocol of anti-CD154 therapy. In contrast, rapamycin acted synergistically with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose schedule of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the effect was not affected by the concomitant use of any immunosuppressive drugs. CONCLUSIONS: The widespread view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection.     

Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatment in CD40 ligand-deficient mice

Previous work has demonstrated that short-term systemic administration of cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig blocks human pancreatic islet xenograft rejection in mice and induces long-term, donor-specific tolerance, whereas studies on pig pancreatic islet rejection in mice have failed to demonstrate a role for CTLA4Ig in preventing rejection. Treatment with anti-CD40 ligand (L) monoclonal antibodies alone is somewhat effective in prolonging the survival of islet xenografts, but ineffective when applied to skin xenografts. However, simultaneous blockade of the CD28 and CD40 co-stimulatory pathways prolongs the survival of pig skin on recipient mice. To evaluate the role of CD28 and CD40 co-stimulatory pathways in pig islet-like cell cluster (ICC) xenograft rejection in mice, CD40L-deficient mice transplanted with fetal porcine ICCs were given posttransplant treatment with human (h) CTLA4Ig or a human IgG1 chimeric mAb (hL6). Xenografts were evaluated 6 or 12 days after transplantation. Fetal porcine ICC xenografts were protected from rejection in hCTLA4Ig-treated CD40L-deficient mice, whereas xenograft rejection persisted in untreated CD40L-deficient mice. Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways is mandatory to inhibit ICC xenograft rejection in the pig-to-mouse model, because the CD28 and CD40 co-stimulatory pathways seem capable of efficiently substituting for one another.     

Anti-CD45RB Monoclonal Antibody Prolongs Renal Allograft Survival in Cynomolgus Monkeys

Previously, an anti-CD45RB monoclonal antibody (mAb) has been shown to induce murine allograft tolerance. The present study was performed to assess the ability of an anti-human CD45RB mAb to prevent rejection in a monkey MHC-mismatched kidney transplant model. The recipients were allocated into the following treatment groups: (1) isotype control IgG; (2) mouse anti-human CD45RB IgG1 (6G3); (3) human-mouse chimeric anti-CD45RB-IgG1 (C6G3-IgG1); (4) human-mouse chimeric anti-CD45RB-IgG2 (C6G3-IgG2); (5) tacrolimus at a subtherapeutic dose and (6) tacrolimus and C6G3-IgG1 in combination. Monotherapy with anti-CD45RB mAb significantly prolonged renal allograft survival to a median survival of 21 days. Adding a subtherapeutic dose of tacrolimus improved the efficacy of the anti-CD45RB mAb, achieving a median survival of 85 days, whereas a subtherapeutic dose of tacrolimus alone only moderately prolonged survival to 27 days. Treatment with anti-CD45RB mAb resulted in an alteration of the CD45RB(hi) : CD45RB(lo) cell ratio in the peripheral blood. We have, for the first time, demonstrated that an anti-human CD45RB mAb (6G3) can prolong graft survival. Induction with an anti-CD45RB mAb improves the efficacy of tacrolimus in the prevention of rejection. These encouraging results indicate that an anti-CD45RB mAb may be valuable in future clinical transplantation.