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.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

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.     

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.     

Oral delivery of xeno-antigen combined with non-depleting anti-CD4 monoclonal antibody induces significantly prolonged survival of concordant skin xenograft

Abstract: Oral administration can induce unresponsiveness to protein antigens. Therefore, we examined whether oral administration of xeno-antigen could induce the prolonged survival of xenogeneic skin grafts. CBA mice were given 1 × 10⁷ SD rat splenocytes orally, 7 days before transplantation of a SD rat skin in the presence or absence of a non-depleting anti-CD4 monoclonal antibody (mAb) (YTS177, 200 µg/dose, −8 and −7 days relative to transplantation). All skin grafts survived with a median survival time (MST) of 62 days when xeno-antigens were administered orally in combination with anti-CD4 mAb. Mice treated with anti-CD4 mAb alone or oral administration of xeno-antigen alone induced modest prolonged survival of rat skin grafts (MST = 18 and 19 days, respectively) while naive mice rejected rat skin acutely (MST = 12 days). Oral administration alone or combined with anti-CD4 mAb reduced the level of xeno-antibody production compared with that in untreated mice after transplantation. Xenogeneic mixed leukocyte response was reduced when splenocytes from mice pre-treated with oral administration of xenogeneic cells were used as the responder compared with that in untreated mice. Oral delivery of xeno-antigen plus non-depleting anti-CD4 mAb can induce prolongation of concordant xenogeneic skin grafts.     

Blockade of the PD-1/PD-1L pathway reverses the protective effect of anti-CD40L therapy in a rat to mouse concordant islet xenotransplantation model

BACKGROUND: We have previously demonstrated that costimulatory blockade with anti-CD40L monoclonal antibody (mAb) prolongs the survival of non-vascularized concordant rat to mouse islet xenografts. Here, we examine whether signaling through the PD-1/PD-1L pathway is required for the anti-CD40L therapy to prolong concordant islet graft survival using a novel anti-murine PD-1 mAb (clone 4F10). METHODS: C57BL/6 mice received a cellular concordant islet xenograft under the left kidney capsule and four experimental groups were prepared. Group I: untreated control; group II: recipient mice were treated with three doses of 0.5 mg of anti-CD40L mAb (clone MR1) on days 0, 2 and 4; group III: mice were treated with 0.5 mg of anti-PD-1 (CD279) mAb (clone 4F10) every other day for 8 days; and finally group IV: mice received the combined treatment that consisted of anti-CD40L plus anti-PD-1 mAb. RESULTS: Concordant islet xenografts transplanted in control untreated mice showed a median survival time (MST) of 17 +/- 7.43 days, whereas anti-CD40L treatment led to a significant prolongation of graft survival (MST: 154 +/- 65.56, P < 0.0001). The administration of anti-PD-1 alone significantly accelerated graft rejection compared to non-treated controls (MST: 10 +/- 2.24 vs. MST: 17 +/- 7.43, P < 0.0004). Remarkably, the combined administration of anti-CD40L and anti-PD-1 reversed the protective effect obtained with anti-CD40L alone (anti-CD40L, MST: 154 +/- 65.56 vs. anti-CD40L plus anti-PD-1, MST: 10 +/- 7.72, P < 0.0002). CONCLUSION: Overall, our data indicate that the PD-1/PD-1L pathway is required for the achievement of prolonged graft survival in anti-CD40L-treated mice in a setting of rat to mouse concordant islet xenotransplantation.     

Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into na?ve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.     

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.     

Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection.

INTRODUCTION: This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice. METHODS: "Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses. RESULTS: The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and >100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts. CONCLUSIONS: These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.     

Application of recipient-derived dendritic cells to induce donor-specific T-cell hyporesponsiveness

Administration of donor-derived immature dendritic cells (DC) treated with NF-kappaB oligodeoxyribonucleotides (ODN) prevents allograft rejection. We attempted to explore the use of recipient-derived DC pulsed with donor antigens, in which the donor antigens were presented to host T cells via an indirect pathway (cross-priming). Expression of CD40, CD80, and CD86 on DC was significantly inhibited by treatment with NF-kappaB ODN, whereas MHC class I and II were minimally affected. Normal C3H DC pulsed with B10 antigens stimulated proliferative responses and donor-specific CTL activity in C3H T cells, both of which were, however, markedly inhibited when DC were treated with NF-kappaB ODN. This manipulation was associated with reduced IFN-gamma and increased IL-10 production in the supernate, suggesting a Th2 bias. More frequent apoptotic T cells were observed in cultures with NF-kappaB ODN DC. In contrast to administration of normal DC pulsed with donor antigens that accelerated rejection of B10 cardiac allografts (median survival time [MST] 7 days versus 10 days in no-DC treatment control, P < .05), a single injection of 2 x 10(6) NF-kappaB ODN DC significantly prolonged allograft survival (MST 50 days, P < .05 compared with no-DC treatment control). The anti-donor CTL activity in infiltrating T cells isolated from cardiac grafts in recipients that received NF-kappaB ODN DC was significantly suppressed. These data indicate that vaccination with immature DC, propagated from recipient BM is an attractive approach to induce T-cell hyporesponsiveness.     

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.     

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies

ABSTRACT: Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS 177.9. Our data show that mAb YTS 177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three penoperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8±37.1 days compared with controls (8.3±1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7±5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS 177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS 177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.     

Normal Donor Bone Marrow is Superior to Flt3 Ligand-Mobilized Bone Marrow in Prolonging Heart Allograft Survival when Combined with Anti-CD40L (CD154)

Flt3 ligand (FL) administration markedly increases bone marrow (BM) stem cells and immature dendritic cells. We investigated the influence of CD40-CD40Ligand (CD154) pathway blockade on antidonor immunity, cytokine production, microchimerism and heart graft survival in BALB/c (H2d) recipients of fully allogeneic C57BL/10 (H2b) FL-mobilized BM (FL-BM) or normal BM. Anti-CD40L mAb strongly suppressed anti-donor T-cell proliferative responses in recipients of either normal or FL-BM, but was less efficient in inhibiting antidonor cytolytic T-cell (CTL) activity, especially in recipients of FL-BM. Interestingly, CD40L blockade was more effective in recipients of multiple compared with single donor BM infusions. Anti-donor cytokine responses revealed complete impairment of IFN-gamma, IL-4 and IL-10 production in recipients of normal BM and CD40L mAb. By contrast, and in agreement with the CTL data, mice given FL-BM retained ability to produce IFN-gamma CD40-CD40L blockade did not promote microchimerism, as evidenced by immunohistology and real time polymerase chain reaction. Nevertheless, anti-CD40L mAb enhanced heart allograft survival in recipients of FL-BM, but the effect was inferior to that achieved with normal BM. These data provide insight into the influence of growth factor-expanded donor BM and costimulation blockade on antidonor immune reactivity and transplant outcome. The comparatively poor outcome obtained using FL-BM plus anti-CD40L mAb in this model may be ascribed to the failure of effectively interdicting antidonor CTL activity.     

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.     

Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates

BACKGROUND: Costimulation blockade has been proposed to induce allograft tolerance. We combined an antagonist anti-CD40 monoclonal antibody (mAb) with an antagonist anti-CD86 mAb in a rhesus monkey kidney allograft model. We chose this combination because it leaves CD80-CD152 signaling unimpaired, allowing for the down-regulatory effect of CD152 signaling to take place through this pathway. METHODS: Rhesus monkeys underwent transplantation with a major histocompatibility complex-mismatched kidney. One group of animals received anti-CD40 alone, and a second group received the combination of anti-CD40 and anti-CD86, twice weekly for 56 days. RESULTS: Three animals with low levels of anti-CD40 rejected the transplanted kidney while still receiving treatment. Three animals with high levels of anti-CD40 rejected at days 91, 134, and 217 with signs of chronic rejection. Animals treated with the combination of anti-CD40 and anti-CD86 mAbs rejected their kidneys at days 61, 75, and 78, shortly after cessation of treatment. Two animals were killed on days 71 and 116 with a blocked ureter. These animals developed virtually no signs of tubulitis or infiltration during treatment and no donor-specific alloantibodies. CONCLUSIONS: Both treatment protocols prevented rejection for the duration of the treatment in most animals. Blocking costimulation by anti-CD40 or by anti-CD40 plus anti-CD86 may be an effective method to prevent graft rejection and may obviate the need for other immunosuppressive drugs, especially in the immediate posttransplantation period.     

表达IκBα突变体的树突状细胞对大鼠移植肾存活时间的影响

目的 观察IκBα突变体基因修饰的供体树突状细胞(IκBαM-Dc)对大鼠移植肾存活时间的影响.方法 利用重组腺病毒载体将IκBαM基因转染WF大鼠骨髓DC,流式细胞仪检测DC共刺激分子CD80、CD86表达.以wF大鼠为供体,Lewis大鼠为受体,行同种肾移植.术前7 d,受体输注供体IκBαM-DC作为IκBαM组,未输注IκBαM-DC的受体作为对照组,观察移植肾存活时间和术后肾功能变化.术后第14天检测受体T细胞对供体成熟DC及第三方大鼠成熟DC的反应性.结果 IκBαM明显抑制DC共刺激分子CD80、CD86表达.IκBαM组受体鼠移植肾存活时间为(32.5±7.8)d,较对照组移植肾存活时间(8.5±1.7)d明显延长(P<0.01);而其术后7 d血清肌酐为(57.3±8.2)μmol/L,较对照组血清肌酐(498.0±46.3)μmol/L明显减低.肾移植术后,IκBαM组受体T细胞对供体成熟DC反应明显低于对照组,而对第三方大鼠成熟DC的反应性与对照组比较,差异无统计学意义(P>0.05).结论 表达IκBα突变体的供体树突状细胞能诱导受体大鼠T细胞对供体抗原的免疫低反应,显著延长大鼠移植肾存活时间.     

A negative regulatory role in mouse cardiac transplantation for a splice variant of CD80

BACKGROUND: Members of the B7 costimulatory protein family (CD80 and CD86) play a determining role in allograft rejection. Both CD80 and CD86 have naturally occurring splice variants whose roles in transplantation are unknown. Full length CD80 has two immunoglobulin (Ig)-like domains in the extracellular portion, IgC and IgV. In mouse, the isoform IgV-CD80 lacks the IgC-like domain. Here we analyzed the role of mouse IgV-CD80 in heart allograft rejection and search for equivalent splice variants in human. METHODS: Mice made deficient for full-length CD80 but which retain expression of the shorter IgV-CD80 (CD80 mice) were used as donor or recipient of a heart allograft. Recipient animals were untreated or pretreated with alloantigen expressing cells and/or treated with CD80 and CTLA4 monoclonal antibodies (mAbs). RESULTS: Recipients expressing IgV-CD80 but not full length CD80 exhibited a slight prolongation in survival of either wild-type (Wt) or CD80 grafts. More dramatically, CD80 animals pretreated with donor alloantigen exhibited permanent graft survival, whereas their Wt counterparts rejected their grafts with a median survival of 24 days. This prolonged survival was due to the expression of IgV-CD80 in recipients since treatment with CD80 mAb abrogated the beneficial effect observed. We identified and report here a similar isoform of CD80 from human cDNA encoding a putative soluble, IgV-containing protein. CONCLUSIONS: IgV-CD80 bearing recipients show enhanced allograft survival especially after donor alloantigen pretreatment. This together with data from other species suggests that regulation delivered by splice variants of CD80 significantly modulates immunity and may be common across the species.     

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.