Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.     

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.     

Anti-CD40L monoclonal antibody treatment induces long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts

The CD40/CD40L costimulatory pathway plays a crucial role in allograft rejection. The purpose of this study was to determine the effectiveness of anti-CD40L monoclonal antibody (mAb) treatment as a method to induce long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts. Sciatic nerve allografts were performed from BALB/c donor mice into C57BL/6 recipients. Anti-CD40L mAb (1 mg) was administered intraperitoneally to recipient mice on postoperative days 0, 1, and 2. After a 14-, 28-, or 60-day recovery period, the mice were rechallenged with either a BALB/c cardiac or peripheral nerve allograft. Rejection was assessed by measuring the production of interferon gamma (IFN-gamma), interleukin (IL)-2, -4, and -5, and alloantibodies immunoglobulin (Ig) M and IgG. IFN-gamma, IL-2, IL-4, IL-5, IgM, and IgG responses were much lower in the anti-CD40L mAb group compared with controls. Nerve allograft and nerve isograft rechallenge 60 days following the original nerve allotransplantation produced low cytokine responses, whereas cardiac allograft rechallenge produced high cytokine production, indicative of acute rejection. Short-term anti-CD40L treatment may cause long-term, tissue-specific, immunologic hyporesponsiveness. This may allow time for native axons to traverse the transplanted nerve allograft and replace the graft with autogenous peripheral nerve tissue.     

A novel bispecific antihuman CD40/CD86 fusion protein with t-cell tolerizing potential

Clinical trials designed to achieve tolerance in humans by selectively antagonizing one of the T-cell costimulatory pathways, CD40-CD40L or CD80/CD86-CD28, are pending. However, simultaneous blockade of both pathways synergistically prevented graft rejection and successfully induced donor-specific tolerance in animal models. Synergism is also supported in human T-cells in vitro following anti-CD86 mAb and anti-CD40 mAb blockade. Therefore, in our view the most promising clinical strategy would be to antagonize both CD40 and CD86. Fast clinical entrance of this anti-CD86 and anti-CD40 bidirectional concept is highly facilitated by a single molecule approach. In the present study, a single bispecific fusion protein was constructed that specifically binds human CD40 and CD86 and which combines the antagonistic activities of both anti-CD40 and anti-CD86 humanized mAb. The anti-CD40/86 fusion protein showed tolerance inducing potential as it prevented both allogeneic T-cell expansion and generation of cytotoxic effector T cells and induced anergic antigen specific regulatory T cells. These data provide proof of concept in successfully combining the antagonistic activity of two humanized mAb with great clinical potential in transplantation and autoimmunity, in one single molecule.     

CD2 is a Dominant Target for Allogeneic Responses

CD2 and 2B4 (CD244) are members of the immunoglobulin gene superfamily and are both ligands for another family member, CD48. CD2 is widely distributed on T, NK, and B cells and some antigen-presenting cells, while 2B4 is expressed on NK and some T cells and monocytes and is known to participate in NK cytotoxicity. Since indefinite allograft survival could be obtained by a combination of anti-CD48 plus anti-CD2 mAb administration, it was important to determine the role of 2B4 blockade in allograft rejection. MAbs directed against CD2, CD48, or 2B4 were administered singly or in pairs to cardiac allograft recipients. The experiments show that only anti-CD2 plus anti-CD48 mAbs result in indefinite allograft survival, while anti-CD2 plus anti-2B4 mAbs substantially prolong graft survival, and anti-CD48 plus anti-2B4 mAbs were no better than each mAb alone. The effect of these mAbs on anti-CD3 mAb and alloantigen-driven proliferation and IFN-gamma production were also assessed. In general, anti-CD2 inhibited both anti-CD3 mAb and alloantigen-driven responses, while anti-CD48 inhibited only anti-CD3 mAb but not alloantigen-driven proliferative and cytokine responses. Anti-2B4 mAbs were generally ineffective alone. Combinations of mAbs were more effective than single mAbs only in alloantigen-driven proliferation, commensurate with allograft survival results. Using CD2-/- and CD48-/- T cells and antigen-presenting cells, we also demonstrate that these inhibitory mAbs act primarily by blocking intercellular interactions, rather than directly delivering negative signals to T cells. These results suggest that, unlike CD2, 2B4 is not a potent regulatory molecule or ligand for CD48 in the response to alloantigen. Blocking the 2B4-CD48 receptor-ligand pair does not inhibit T-cell responses and alloreactivity to the same degree as CD2-CD48 blockade.     

Earlier Low-Dose TBI or DST Overcomes CD8+ T-Cell-Mediated Alloresistance to Allogeneic Marrow in Recipients of Anti-CD40L

Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day -1 or day -2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.     

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.     

Costimulation and Autoimmune Diabetes in BB Rats

Costimulatory signals regulate T-cell activation. To investigate the role of costimulation in autoimmunity and transplantation, we studied the BB rat model of type 1 diabetes. Diabetes-prone BB (BBDP) rats spontaneously develop disease when 55–120 days of age. We observed that two anti-CD28 monoclonal antibodies (mAb) with different functional activities completely prevented diabetes in BBDP rats. Anti-CD154 mAb delayed diabetes, whereas treatment with CTLA4-Ig or anti-CD80 mAb accelerated disease. Anti-CD86 or anti-CD134L mAbs had no effect. Diabetes resistant BB (BBDR) rats are disease-free, but >95% of them develop diabetes after treatment with polyinosinic-polycytidylic acid and an mAb that depletes Treg cells. In the induced BBDR model, anti-CD154 mAb delayed onset of diabetes, whereas CTLA4-Ig, anti-CD134L or either of the anti-CD28 mAbs had little or no effect. In contrast, blockade of the CD134-CD134L pathway was highly effective for preventing autoimmune recurrence against syngeneic islet grafts in diabetic BBDR hosts. Blockade of the CD40-CD154 pathway was also effective, but less so. These data suggest that the effectiveness of costimulation blockade in the treatment of type 1 diabetes is dependent on both the costimulatory pathway targeted and the mechanism of induction, stage, intensity and duration of the pathogenic process.     

Promises and obstacles for the blockade of CD40-CD40L interactions in allotransplantation

Although clinical trials using anti-CD40L mAb have been halted, blockade of CD40-CD40L interactions has given promising results in rodent and nonhuman primate models. Recently, new insights into the consequences of blocking CD40-CD40L interactions have led to the possibility of overcoming the thromboembolic complications previously associated with this strategy and may pave the way for successful tolerance induction. This mini review will address the issues relating to CD40-CD40L blockade in allotransplantation.     

Changes in expression of T-cell activation-related molecules and cytokines during tolerance induction in an allogeneic skin transplantation murine model

Bone marrow transplantation after treatment with busulfan and costimulatory blockade with monoclonal antibodies (mAb) cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig and anti-CD154 mAb or two-signal blockade using anti-CD45RB and anti-CD154 mAb are nonmyeloablative treatment regimens for allogeneic transplantation. There may be differences in the mechanisms of donor cell engraftment and reactive cell deletion by which these regimens induce donor-specific tolerance. Therefore, this study was performed to investigate changes in T cells and cytokines during tolerance induction toward allogeneic skin grafts. BALB/c and C57BL/6 mice were used as donors and recipients, respectively. Skin and bone marrow transplantations were performed and busulfan was administered. Three groups were treated with mAb as follows: group 1, anti-CD154 mAb; group 2, anti-CD154 plus anti-CD45RB mAb; and group 3, anti-CD154 mAb plus CTLA4-Ig. The proportions of CD4+ or CD8+ T cells and the expression of CD45RB isoforms on splenocytes were measured using flow cytometry and the production of cytokines by CD4+ T cells using enzyme-linked immunosorbent assay. Group 2 showed a significant reduction in the proportions of CD8+ T cells and CD45RB high isoforms compared with groups 1 and 3. The levels of interleukin (IL)-2 and IL-4 in group 2 were lower and higher than those of groups 1 and 3, respectively. In conclusion, the combined use of anti-CD154 and anti-CD45RB mAb decreases the CD8+ T-cell population and the expression of CD45RB, resulting in a Th2 cytokine profile, which may be a characteristic mechanism leading to donor cell engraftment and reactive cell deletion for donor-specific tolerance.     

CTLA4 engagement is required for induction of murine liver transplant spontaneous tolerance.

Liver transplantation in mice is accepted spontaneously in all strain combinations. The mechanisms remain largely undefined. We hypothesize that signaling via the B7-CTLA4 receptor pathway is required for induction of liver transplant tolerance. Liver transplantation was performed from B10 (H2(b)) to C3H (H2(k)) mice. The recipients received anti-mouse CTLA4 mAb 0.25 mg i.p. every other day post-operatively. Liver grafts in anti-CTLA4 mAb treated recipients were acutely rejected. The allo-specific proliferative responses, anti-donor CTL and NK cell activities of GIC and SC and the serum levels of IFN-gamma and IL-2 from anti-CTLA4 mAb treated recipients were elevated significantly in comparison to the control mice. The frequency of IFN-gamma and IL-2 producing cells were markedly increased also in the anti-CTLA4 treated recipients. The immunohistology of liver grafts from anti-CTLA4 mAb treated mice showed extensively increased lymphocyte infiltration in the portal and general parenchymal areas, and expanded T-cell area in the spleen, with a reduction in the frequency of apoptotic cells observed by TUNEL staining compared with control mice. Thus CTLA4 signaling is critical for murine liver transplant tolerance induction. CTLA4 blockade promotes donor specific T-cell activation, cytotoxicity and Th1 polarization; protects alloreactive T cells from apoptotic death and induces liver allograft acute rejection.     

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.     

The Classical Complement Pathway in Transplantation: Unanticipated Protective Effects of C1q and Role in Inductive Antibody Therapy

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.     

Anti-CD40L monoclonal antibodies can replace anti-CD4 monoclonal antibodies for the nonmyeloablative induction of mixed xenogeneic chimerism

BACKGROUND: We have previously demonstrated that xenogeneic bone marrow engraftment and donor-specific tolerance can be induced in mice receiving anti-CD4, -CD8, -Thy-1.2, and -NK1.1 monoclonal antibodies (mAbs) on Days -6 and -1, 3 Gy total body irradiation (TBI), and 7 Gy thymic irradiation on Day 0, followed by injection of T-cell depleted (TCD) rat bone marrow cells. We have recently demonstrated that anti-CD40L mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance in an allogeneic combination. METHODS: We investigated the ability of anti-CD40L mAb to promote mixed xenogeneic chimerism and donor-specific tolerance in B6 mice receiving anti-CD8, -Thy1.2 and -NK1.1 mAbs and 3 Gy TBI followed by TCD bone marrow transplantation (BMT) from F344 rats. RESULTS: Administration of anti-CD4 mAb in this model could be completely replaced by one injection of anti-CD40L mAb. Evidence for deletional tolerance was obtained in mixed chimeras prepared with this anti-CD40L-based regimen. However, anti-NK1.1 and anti-Thy1.2 mAb could not be replaced by anti-CD40L mAb. CONCLUSIONS: These results demonstrate that anti-CD40L in combination with xenogeneic BMT can tolerize preexisting peripheral and intrathymic CD4 cells to xenoantigens. However, anti-CD40L does not prevent NK cell and/or gammaDelta cell-mediated rejection of xenogeneic bone marrow.     

Anti-mouse CD154 antibody treatment facilitates generation of mixed xenogeneic rat hematopoietic chimerism, prevents wasting disease and prolongs xenograft survival in mice

BACKGROUND: The induction of xenogeneic hematopoietic chimerism is an attractive approach for overcoming the host response to xenografts, but establishing xenogeneic chimerism requires severe myeloablative conditioning of the recipient. The goal of this study was to determine if co-stimulation blockade would facilitate chimerism and xenograft tolerance in irradiation-conditioned concordant recipients. METHODS: Wistar Furth rat bone marrow (BM) cells were injected into irradiation-conditioned C57BL/6 mice with or without co-administration of anti-mouse CD154 monoclonal antibody (mAb). Chimerism was quantified by flow cytometry, and mice were transplanted with WF rat skin and islet xenografts. RESULTS: Blockade of CD40-CD154 interaction facilitated establishment of xenogeneic chimerism in mice conditioned with 600 cGy irradiation. Anti-CD154 mAb was not required for establishment of chimerism in mice treated with 700 cGy. However, mice irradiated with 700 cGy but not treated with anti-CD154 mAb developed a "graft-versus-host disease (GVHD)-like" wasting syndrome and died, irrespective of their development of chimerism. Xenogeneic chimeras established with irradiation and anti-CD154 mAb treatment exhibited prolonged skin and, in many cases, permanent islet xenograft survival. Chimerism was unstable and eventually lost in most recipients. Skin xenografts were rejected even in mice that remained chimeric, whereas most islet xenografts survived to the end of the observation period. CONCLUSIONS: Blockade of host CD40-CD154 interaction facilitates the establishment of xenogeneic chimerism and prevents wasting disease and death. Chimerism permits prolonged xenograft survival, but chimerism generated in this way is unstable over time. Skin xenografts are eventually rejected, whereas most islet xenografts survive long term and perhaps permanently.