Cytotoxic Antibody Titers Research Articles

Replacement of Mycophenolic Acid by Everolimus in Patients Who Become Neutropenic after Renal Transplantation

The presence of neutropenia after kidney transplantation represents a frequent adverse event (AE) that is usually treated with lowering of mycophenolic acid (MPA) dose. This leads to an increased incidence of acute rejection episodes and potential loss of renal allograft. This study evaluated the efficacy and safety of everolimus when used instead of MPA in patients who developed neutropenia (absolute neutrophil count < 2000/μL). We studied 17 patients, 12 men and 5 women, aged 40 ± 12 years old, who developed neutropenia after kidney transplantation. Five of them were patients with high immunological risk (high titer of cytotoxic antibodies, second or third transplant). All patients treated with MPA (1912 ± 196mg/day), calcineurin inhibitor (cyclosporine n=1, tacrolimus n=16) and methylprednisolone. Neutropenia occurred in 15 patients during the first 3 months after transplantation and in 2 patients between the 3rd and 6th month post-transplantation (59 ± 38 days). The 15 patients who developed neutropenia during the first 3 months after transplantation were still on valgancyclovir as prophylaxis against cytomegalovirus (CMV) infection. A total of 118 episodes of neutropenia were recorded, originally treated by reduction of MPA dose and administration of granulocyte colony-stimulating factor (G-CSF). Three patients experienced acute rejection (Banff 1A) 5 to 10 days after the reduction of MPA dose (0-1000mg/day) which was successfully treated with IV pulse of methylprednisolone. Five patients developed CMV infection, 108 ± 65 days after the onset of neutropenia and 5.5±1.5 months after transplantation. The replacement of MPA (765 ± 390mg/day) by everolimus was performed 1 to 23 months after transplantation and 5 days up to 48 months from the onset of neutropenia. After the induction of everolimus, 50 episodes of neutropenia were observed in 6 patients. In one of them discontinuation of everolimus was necessary 1.5 month later and in the rest 5 (who developed CMV infection and treated by valgancyclovir), neutropenia of less severity was observed up to the end of treatment for CMV infection. In the remaining 11 patients, no episode of neutropenia was observed. No episode of acute rejection occurred and the renal function remained stable 6 months after initiation of everolimus and during the whole follow up period of 47±30months [eGFRMDRD6: 45±14ml/min/1.73m2 → (6months):51±16ml/min/1.73m2 → (47±30months): 47±22ml/min/1.73m2]. Conclusion: The replacement of MPA by everolimus because of neutropenia in renal transplant recipients on triple immunosuppressive regimen appears to be safe and effective alternative treatment. Although neutropenia also occurred after administration of everolimus, it was less serious and possibly due to a different mechanism of action. Further research with more prospective randomized studies is necessary in order to confirm these findings.

Effect of daclizumab on TReg counts and EGFRvIII-specific immune responses in GBM

2034 Background: TRegs are increased in patients with GBM and constitutively express the high affinity interleukin-2 receptor (IL-2Rα). Treatment with an antibody that blocks IL-2Rα signaling functionally inactivates and eliminates TRegs without inducing autoimmune toxicity in murine models. We hypothesized that daclizumab, a commercially-available, IL-2Rα-specific antibody would function identically.Methods: A randomized phase II clinical trial assessed the effects of daclizumab in the context of the cancer vaccine, CDX-110, which is comprised of an EGFRvIII-specific peptide sequence linked to KLH. EGFRvIII is a constitutively activated and immunogenic mutation not expressed in normal tissues, but widely expressed in GBMs and other neoplasms. In patients with newly-diagnosed, EGFRvIII+ GBM, after resection and radiation/TMZ, patients received CDX-110 vaccinations biweekly x 3, then monthly until tumor progression in combination with TMZ (200 mg/m2 x 5/28 days). Half the patients were randomized to receive daclizumab (1mg/Kg x1) at the first vaccine. The others received saline in a double-blinded fashion.Results: There were no drug related SAEs. EGFRvIII-specific immune responses were generated in all patients, and all immune responses were sustained or enhanced during subsequent TMZ cycles. Preliminary analysis (n = 4) suggests that daclizumab reduces Treg (CD4+CD25+CD45RO+FOXP3+) numbers [change 82.4 ± 7.1% from baseline (p = 0.011; t-test)] without reducing overall CD8+ or CD4+ T-cell counts. Tregs decreased only 3.7 + 11.0% after vaccination in the saline treated group during the same interval. Preliminary analysis (n = 4) also suggest that daclizumab enhanced EGFRvIII-specific immune responses (p = 0.01; t-test) and enhanced the titer of cytotoxic EGFRvIII-specific IgG1 isotype antibodies compared to the saline treated group (p = 0.003; t-test) and compared to previously vaccinated patients who did not receive daclizumab (p = 0.0015; t-test). TTP and OS survival in both arms has not been reached. Conclusions: Daclizumab may reduce Treg counts in patients with GBM. TMZ and daclizumab may enhance EGFRvIII-targeted immune responses despite lymphodepletion. These combinations are currently under further investigation. [Table: see text]

Administration of antisense oligodeoxyribonucleotides targeting NF-kappaB prolongs allograft survival via suppression of cytotoxicity.

Presentation of alloantigens by antigen-presenting cells (APC) deficient in expression of costimulatory molecules (CM) induces alloantigen-specific hyporesponsiveness or tolerance. NF-kappaB has been shown to be a crucial transcription factor that regulates CM expression on the surface of APC. We designed an antisense phosphorothioate oligodeoxynucleotide (ODN) to specifically inhibit mRNA of mouse NF-kappaB, resulting in a deficiency in CM expression on APC that may prolong organ allograft survival. Anti-NF-kappaB ODN was delivered systemically by an osmotic pump implanted in the abdominal cavity of recipients following transplantation of vascularized heart allografts. The animals in control groups were given scrambled control ODN or were left untreated. Normal C3H (H2(k)) recipients rejected B10 (H2(b)) heart allografts at a median survival time (MST) of 15 days. A 14-day administration of 12.5 mg/kg/day anti-NF-kappaB ODN prolonged the survival of cardiac allografts to an MST of 25 days (P <0.05). In contrast, a scrambled control ODN was not effective (MST 17 days, P >0.05 compared to untreated controls). To investigate the underlying mechanisms of the immunosuppressive effect of anti-NF-kappaB ODN administration, graft infiltrating cells, spleen cells, and serum were collected from animals on day 7 posttransplant. Freshly isolated graft-infiltrating cells from anti-NF-kappaB ODN-treated recipients exhibited significantly decreased donor-specific CTL activity. Generation of CTL activity of spleen T cells from anti-NF-kappaB ODN-treated recipients was also impaired compared with untreated animals. Administration of anti-NF-kappaB ODN did not influence the titers of complement-dependent cytotoxic antibodies. These data suggest that treatment with anti-NF-kappaB ODN markedly inhibits the cellular response of allograft recipients, resulting in significant prolongation of allograft survival. Antisense ODN therapy targeting NF-kappacB may be a novel strategy for future immunosuppressive therapy.

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits.

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with M(r), of approximately 74 and approximately 23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with M(r) 39-kDa antigen. In lysates of thymocytes a weak band corresponding to M(r) of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freund's adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.

Induction of hyperacute rejection of skin allografts by CD8+ lymphocytes.

Second-set rejection is generally regarded as a phenomenon mainly mediated by humoral cytotoxic antibodies, although a few discordant data have been presented. In the reported experiments, we have taken advantage of the absence of production of specific cytotoxic alloantibodies contrasting with the normal development of transplantation cellular immunity, in two murine models: chimeric mice and RAG mice. Chimeras (BALB/c-->CBA) were obtained by transplantation of 2x10(7) fetal liver cells from BALB/c (H-2d) mice to lethally irradiated CBA (H-2k) mice. After hyperimmunization with third-party C57/ BL6 (B6) (H-2b) skin transplants and with injections of 2x10(7) B6 spleen cells, antibody production, and skin graft survival were analyzed. To identify further the factors or cells responsible for accelerated rejection of B6 skin transplants in hyperimmunized chimeras, transfer experiments were carried out involving the injection of serum, whole spleen cells, spleen T cells, spleen CD8+ T cells or spleen CD4+ T cells from chimeras into BALB/c mice that had received 6 Gy irradiation. The recipient mice were then grafted with B6 skin. Similarly, the immunodeficient RAG mice were used to construct a model of recipient animals with anti-H-2d hyperimmunized B6 T cells in the total absence of antibody. In chimeras, anti-B6 cytotoxic antibodies were not detectable in any of hyperimmunized chimeric mice, yet accelerated rejection of B6 skin transplant occurred: a graft survival of 8.6+/-0.5 days (d), comparable to 8.9+/-0.8 d survival in CBA control mice subjected to the same hyperimmunization procedure, and significantly shorter than that in nonhyperimmunized (BALB/c-->CBA) chimeras (11.6+/-0.5 d) or in non-hyperimmunized CBA control mice (12.1+/-0.6 d). High titers of anti-B6 cytotoxic antibodies were present in the serum of hyperimmunized CBA control mice. In transfer experiments, the graft survival was over 14 d in mice treated with irradiation alone, with irradiation + serum or with irradiation + CD4+ T cells. It was significantly shorter in mice treated with irradiation + whole spleen cells, with irradiation + T cells or with irradiation + CD8+ T cells (8.9+/-0.8 d). Similarly, in immunodeficient RAG mice, reconstitution of the T cell compartment with T cells from hyperimmunized B6 mice led to accelerated rejection of BALB/c skin allografts (11.4+/-1.1 d vs. 18.8+/-0.8 d when T cells were provided by nonimmunized mice). In a second transfer of cells from these reconstituted RAG mice into naive RAG mice, CD8+ T cells were shown to induce accelerated rejection of skin allografts (12.0+/-0.6 d) whereas CD4+ T cells were much less efficient (16.5+/-0.1 d). These data indicate that T cells, and especially the CD8+ subset, can be responsible for second-set rejection in the absence of anti-donor antibodies in chimeric and RAG mouse models. These sensitized CD8+ T cells are also likely to play an important role in normal mice, in addition to that of cytotoxic antibodies.

Differential effects of exogenous interleukin-10 on cardiac allograft survival: inhibition of rejection by recipient pretreatment reflects impaired host accessory cell function.

There have been conflicting reports of the influence of exogenous mammalian interleukin (IL)-10 on immune reactivity. These findings may reflect the pleiotropic effects of IL-10 on the functions of antigen-presenting cells and immune effector cells. The purpose of this study was to extend observations of the influence of the cytokine on organ allograft survival and to investigate its effects on the function of accessory and immune effector cells in a mouse cardiac transplant model. C3H (H2k) recipients of heterotopic vascularized B10 (H-2b) heart allografts were treated with recombinant (r) mouse IL-10 over a wide range of doses (0.2-200 microg/day), either before the transplant (days -3, -2, -1), peri-operatively (days -1, 0, 1), or after the transplant (days 0-6). Anti-donor cytotoxic T lymphocyte activity of host spleen and graft-infiltrating cells, and circulating complement-dependent cytotoxic antibody titers were determined by isotope release assays. Mixed leukocyte reactions were used to determine the influence of IL-10 on the function of antigen-presenting cells and allogeneic responder T cells. Recipient pre-transplant administration of IL-10 (days -3, -2, -1) prolonged graft survival at all doses tested. Donor pretreatment with IL-10 (25 microg/day; days -3, -2, -1) was also effective, but less. A pre-transplant or perioperative course of IL-10, however, did not significantly affect the immunosuppressive action of tacrolimus given on days 0-6. If given only after the transplant, IL-10 either had no effect on graft survival or (at high dosage) accelerated rejection and prevented the immunosuppressive effect of cyclosporine. Pretransplant treatment of graft recipients with IL-10 reduced splenic anti-donor cytotoxic T lymphocyte activity and the incidence of graft-infiltrating CD8+ cells. There was no significant effect on circulating alloantibody titers. MLR assays revealed that preincubation of responder cells, but not stimulator spleen cells with IL-10, inhibited T cell proliferation, whereas addition of IL-10 after the start of culture modestly enhanced proliferation. Preincubation of purified T responders with IL-10 showed no inhibitory effect. The modest and opposing effects of exogenous IL-10 on organ allograft survival are dependent on timing and dosage. Recipient pretreatment prolongs graft survival. This finding, together with the MLR results, suggest that IL-10 inhibits the function of host immune accessory cells and that the direct pathway of alloantigen presentation may be less susceptible to inhibition by IL-10.

THE TEMPORAL RELATIONSHIP BETWEEN THE CELLULAR AND HUMORAL IMMUNE RESPONSES IN THE PATHOGENESIS OF TRANSPLANT VASCULAR SCLEROSIS

249 Humoral responses against the allografts have long been contented to play a cardinal role in the initiation and perpetuation of morphological changes pathognomonic of transplant vascular sclerosis (TVS). Studies in human recipients of cadaveric renal allografts have suggested that the presence of anti-donor antibodies in the sera of transplant patients must be considered as a premonition for the eventual development of TVS. However, despite these observations, the temporal relationship between cell and antibody-mediated immune responses in the precise evolvement of this lesion is as yet ambiguous. Methods: To address this issue further, we transplanted aortic allografts across MHC disparate C57B1/10 (H2b) → C3H/HeJ (H2k) allogeneic barrier (n=24). Aorta transplanted across syngeneic animals (C3H → C3H) served as a control (n=24). Animals were not immunosuppressed. At d1, 3, 6, 10, 15, 20 and 30 post-transplantation (PTx), the grafts were harvested and examined for microscopic and immunohistochemical aberrations. To delineate the role of Th1 and Th2-specific cytokines in the genesis of this lesion, RT-PCR was performed using mRNA isolated from grafts harvested at variable time PTx. Additionally, using recipient's splenocytes and sera, α-donor cellular and humoral immunity was determined by MLR and microcytotoxicity assays respectively. Results: No morphological alterations were witnessed in syngeneic grafts harvested at various times PTx. On the contrary, mononuclear cell infiltration in the intima was first encountered in aortic allografts harvested at d6 PTx. It resolved completely by d10 post-implantation with evidence of residual patchy endothelial cell denudation and necrosis. By d20 PTx, patchy intimal thickening (which was largely due to proliferation of α-smA+ cells) became evident which progressively worsened such that, by d30 PTx, it acquired a diffuse concentric appearance characteristic of TVS. Contrary to humoral responses, α-donor proliferative responses were also heightened at d6 PTx, only to abate thereafter. Detectable titers of α-donor cytotoxic antibodies were first encountered at d20 PTx and were markedly increased (1/800) in sera obtained at d30 post-implantation. Interestingly, with the exception of IL-10, the mRNA levels of all other cytokines examined (IL-2, IL-4 and γ-IFN) were elevated in the allografts harvested at d10-15 (and beyond) PTx. Conclusions: Taken together these data suggest that antibody-mediated cytotoxicity is involved in the perpetuation but not the initiation of changes characteristic of TVS. The latter is possibly accomplished by cell-mediated immune reactivity. Additionally, the presence of markedly elevated levels of IL-10 mRNA alone at d3 and d6 PTx in allogeneic but not syngeneic grafts, suggest that this cytokine may play an important role in the precipitous self-resolution of acute cellular rejection.

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway.

Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.

THE USE OF MHC CLASS II-DEFICIENT MICE TO DETERMINE THE ROLE OF DIRECT VS INDIRECT ANTIGEN PRESENTATION IN THE PATHOGENESIS OF CHRONIC REJECTION

14 The relative contributions of direct vs indirect donor alloantigen presentation to recipients' T cells on the development of chronic rejection (CR) is as yet ambiguous. It has been argued that while direct allorecognition may play an important role in initiating acute cellular rejection (ACR); CR on the contrary, is predominantly an outcome of donor allopeptides being presented in the context of recipient MHC. Given that CR is one of the two most common causes for late organ allograft failure, it is somewhat imperative that the prevailing conundrum concerning its pathogenesis be resolved. To address unequivocally this issue, we transplanted aortas (AO) from C57B1/6 Aβb (H2b, MHC class II-1-)→naive C3H (H2k) recipients (Group V; n=6). It was reasoned that due to lack of MHC class II expression in the donor tissue, the antigen (Ag) presentation would be primarily indirect allowing us to study its role in the development of CR. Additionally, the role of direct Ag presentation in the pathogenesis of this lesion was also studied by transplanting AO from naive C3H→C57Bl/6 Aβb recipients (Group VI; n=6). Controls (n=6/group) included Tx of AO from C3H → C3H (Group 1), C57Bl/6 Aβb→C57Bl/6 Aβb (Group II), C3H→C57Bl/6 WT (Group III) and C57Bl/6 WT→C3H (Group IV). As published previously, these aortic allografts undergo self-resolving ACR with spontaneous acceptance, eventually developing changes pathognomonic of CR by d30 post-Tx. At the time of sacrifice (d30-40 post-Tx), the grafts were harvested for immunohistochemical analysis and the sera and splenocytes were also recovered for the determination of α-donor antibody and proliferative responses by complement-dependent microcytotoxicity and MLR assays respectively. Unlike that from Groups I and II, AO harvested from animals in Groups III and IV had marked circumferential intimal thickening due largely to proliferation of α-smooth muscle actin✦ (α-smA✦) cells. Additionally, both the α-donor and α-third party proliferative responses and the titers of cytotoxic antibodies remained comparable in animals in Groups III and IV. On the contrary, AO obtained from the majority of Group V animals exhibited marked reduction in intimal thickening which was largely eccentric and patchy with preservation of elastic membranes; this finding was associated with unmitigated α-donor proliferative and humoral responses. Interestingly, despite the lack of α-donor proliferative and cytotoxic antibody responses, circumferential intimal thickening of moderate severity was witnessed in the majority of aortic allografts harvested from Group VI recipients. Taken together, these data suggest that despite preservation of α-donor immune responses, presentation of Ag by indirect pathway alone does not precipitate the fulminant development of CR indicating that perhaps both pathways of Ag presentation are required for optimal immune activation. More importantly, the presence of moderate CR in recipients which lack α-donor cellular and humoral responses underscore the importance of numerous immune and non-immune pathways which operate in concert towards the development of this lesion.

VALIDATION OF THE MOUSE-TO-RABBIT COMBINATION AS A MODEL FOR PIG-TO-PRIMATE XENOTRANSPLANTATION

110 Hyperacute rejection (HAR) of porcine organs transplanted into primates is initiated by binding of preformed natural anti-porcine antibodies to the vascular endothelium of the graft and activation of the classical pathway of complement (C). Yet, in the small animal models currently employed, HAR is initiated mainly by activation of the alternative C pathway. Therefore it would be useful to develop a small animal model of more clinical relevance that reproduced the rejection process that takes place in pig-to-primate xenotransplantation. With this aim, we performed mouse-to-rabbit heterotopic cardiac transplantation to study graft survival time, histology and immunopathology of the rejected hearts. We then investigated, in vitro, the activation mechanisms that are triggered when mouse red cells are incubated with rabbit serum as a source of antibody and C. We found a mean graft survival of 37 ± 9.6 min (n=10) for mouse-to-weanling rabbit transplants and 40 ± 11.1 min (n=5) for mouse-to-adult rabbit transplants. Histology of rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage and myocyte necrosis consistent with HAR. Immunofluorescence showed moderate diffuse deposition of IgG and dense deposition of IgM and C3 on the vascular endothelium of the graft. To determine whether HAR in this combination is C-mediated, we performed 2 mouse-to-C6-deficient rabbit transplants. The first graft was rejected in 21 hr and the second recipient died with a functioning graft after 6.5 hr. Histology of the first graft showed severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate of neutrophils and some macrophages, while that of the second graft showed normal myocytes and vasculature, with minimal spotty interstitial hemorrhage. This markedly delayed rejection in C6-deficient rabbits implies that the membrane attack complex of C plays a major role in the pathogenesis of HAR in this model. To establish the role of natural antibodies and the pathway of C activation, we showed that rabbit serum 1) contains high titers of cytotoxic antibodies against mouse red cells (62 ± 11.7 units), 2) has strong classical pathway activity when tested with antibody-sensitized mouse red cells (49.6 ± 19.3 units, similar to that of human serum), and 3) has no significant alternative pathway activity against mouse red cells (in contrast to human serum which had 23.7 ± 8.5 units). Thus the cytotoxicity of rabbit serum for mouse red cells is mediated by natural antibodies and activation of the classical C pathway. In conclusion, these studies suggest that HAR in the mouse-to-rabbit combination is mediated by natural antibodies that trigger activation of the classical C pathway. Thus, the pathogenesis of HAR in this model is similar to that seen in pig-to-primate combinations. This model has potential for performing clinically relevant studies at low cost with animals that are readily accessible and easy to maintain.

T CELL ANERGY BUT NOT DELETION IS THE PREDOMINANT MECHANISM FOR INDEFINITE ISLET ALLOGRAFT SURVIVAL IN RECIPIENTS TREATED WITH rhCTLA4-Ig AND ??-gp39 ANTIBODY

48 We had previously reported that in mice, combined simultaneous blockade of costimulatory pathways by short perioperative treatment with rhCTLA4-Ig fusion protein and α-gp39 mAb results in indefinite islet allograft survival with reversal of streptozotocin (STZ)-induced diabetes. Furthermore, islet allograft survival was shown to be comparable in controls and in animals treated with either rhCTLA4-Ig or α-gp39 mAb alone, suggesting that combined simultaneous but not distinct blockade of CD28/B7 and CD40/gp39 costimulatory pathways is imperative for prolonging allograft survival. To ascertain the mechanism underlying these observations, we proceeded to transplant islets isolated from the pancreata of DBA/2 (H2d, Mlsd, Vβ6-CD4+) animals under the kidney capsule of STZ-treated diabetic MHC and MIs-disparate C3H (H2k, Mlsc, Vβ6+CD4+) recipients. The rationale for selecting this strain combination was triggered by the observation that in the donor but not the recipient, Vβ6+CD4+ were deleted during ontogeny thus prompting us to suggest that perhaps this could be used as a surrogate marker to determine the mechanism of T cell tolerance in these animals. In addition to MLR and complement-dependent microcytotoxicity assays, the proliferative status and the presence or absence of Vβ6+CD4+ T cells in the peripheral blood and splenocytes of the recipients was also determined by antibody-mediated TcR crosslinking and flow cytometric analysis respectively. The recipient treatment was as follows: Group I (n=4), isotype control mAb; Group II (n=6), anti-gp39 mAb (250µg/d on d0,2 and 4 post-Tx, i.m); Group III (n=5), rhCTLA4-Ig (200µg on d0,2,4 and 6 post-Tx, i.p) and Group IV (n=6), rhCTLA4-Ig + anti-gp39 mAb. All in vitro analysis were performed using samples obtained from the recipients either after islet allograft rejection (Groups I, II and III) or else at d81 post-Tx (Group IV). While donor-specific proliferative responses and cytotoxic antibody titers remained unchanged in Groups I, II and III, they were markedly reduced in animals in Group IV. These findings were corroborated by TcR crosslinking assays, in which the use of α-Vβ6 mAb resulted in marked proliferation of cells obtained from animals in Groups I, II and III; markedly reduced proliferation was witnessed in cells obtained from Group IV animals. Interestingly, flow cytometric analysis of T cell repertoire both in the PBMC and splenocytes of islet allograft recipients in all four groups revealed the presence of an equivalent number (∼8-9%) of donor-reactive Vβ6+CD4+ T cells arguing against deletion being the mechanism of the observed T cell tolerance. Take together, these preliminary data suggest that T cell anergy is perhaps the predominant mechanism for the indefinite islet allograft survival witnessed in animals subjected to combined simultaneous blockade of CD28/B7 and CD40/gp39 costimulatory pathways.

Xenotransplantation of adult porcine islets in diabetic mice. A study of UVB irradiation, cryopreservation and immunosuppression on graft survival time.

The major obstacle for successful xenotransplantation of islets to large animals and human diabetics is the host rejection. To address the rejection problem, we studied the efficacy of UV-B irradiation, cryopreservation and immunosuppression on the in vivo functional time and immunogenicity of adult porcine islets (PI) in outbred CD1 mice. Exposure of PI to UV-B irradiation between 300-1800J/M2 did not affect the cellular viability as assessed by fluorescein diacetate or their daily insulin secretion in vitro. Fresh PI normalized the blood glucose (BG) of diabetic CD1 mice for 3.1+/-0.6 (n = 8, mean+/-SEM) days. Islets treated with 600J/M2 UV-B irradiation or cryopreservation had similar graft functional times to fresh islets upon transplantation in diabetic CD1 mice. Immunosuppression with cyclosporin A (CsA), antilymphocyte serum (ALS) and FK506 prolonged the functional time of fresh pig islets to 7.9+/-0.9 (n = 9), 6.2+/-1.3 (n = 5) and 24.2+/-10.4 (n = 12) days, respectively. However, additional pretransplant treatment with either UV-B irradiation or cryopreservation did not further increase the functional time of pig islets in mice immunosuppressed with CsA. Furthermore, there was no apparent difference in the frequency of appearance of cytotoxic antibodies and antibody titers in the recipients of UV-B irradiated or cryopreserved pig islet compared with non-treated islets. The UV-B irradiation and cryopreservation of PI before transplantation with the present protocols did not appear to have significant effect on the islet immunogenicity when assessed by in vivo survival duration and anti-donor antibody titer production.

PROLONGATION OF ORGAN ALLOGRAFT SURVIVAL BY SYSTEMIC ANTI-IL-10 ANTIBODY ADMINISTRATION IS MEDIATED BY B CELL SUPPRESSION

394 IL-10 has been regarded as an immunosuppressive cytokine because of its capacity to inhibit the function of antigen presenting cells (APC) and downregulate the synthesis of several proinflammatory cytokines It has been demonstrated previously however, that systemic post-transplant administration of recombinant mouse IL-10 can accelerate B10 (H-2b) cardiac allograft rejection in C3H (H-2k) recipients. This is associated with enhancement of donor-specific cytotoxic T lymphocyte (CTL) and alloantibody responses. There is also evidence that systemic post-transplant administration of a neutralizing anti-IL-10 monoclonal antibody (mAb) can significantly prolong B10 mouse organ allograft survival in normal and presensitized C3H recipients(the latter animals are exposed to donor alloantigens by skin grafting 2 weeks before organ transplantation). The inhibition by anti-IL-10 of secondary allograft rejection is of particular significance and potential clinical importance, as donor presensitization remains a difficult problem due to its resistance to conventional immunosuppressive therapy. In presensitized recipients, anti-IL-10 mAb (rat IgM; Schering-Plough Research Institute, Kenilworth, NJ; purity 85-95%) at 0.5 mg/d for 6 consecutive days after organ transplantation significantly prolonged survival of both vascularized cardiac and orthotopic liver grafts. Graft median survival times were extended from 5(n = 5) to 10 days (n = 6) p<0.05, and from 4 (n = 5) to 11 days (n = 7) p<0.05, respectively. To understand the underlying mechanisms, we examined the influence of administration of anti-IL-10 mAb on anti-donor CTL activity, circulating complement-dependent cytotoxic (CDC) antibody titers and the phenotype of graft-infiltrating and splenic immunocytes in the presensitized liver allograft recipients. Prolongation of liver allograft survival in presensitized recipients was not associated with a suppressive effect on CTL activity against donor alloantigens mediated by either graft-infiltrating cells or host spleen cells. Inhibition of rejection was however, associated with suppression of circulating CDC antibody titers, and with significant reductions in the incidence of B220+ B cells in both grafts and recipient spleens. In conclusion, the data provide evidence for a potential therapeutic role of anti-IL-10 mAb in allograft transplantation in both normal and presensitized recipients. The suppressive effect of anti-IL-10 mAb may be mediated by inhibition of B cell development and cytotoxic alloantibody production.

Interleukin-2 and interleukin-12 mediate distinct effector mechanisms of liver allograft rejection.

Interleukin-2 (IL-2), interleukin-12 (IL-12) or interleukin-4 (IL-4) were administered postoperatively to otherwise spontaneously accepting mouse liver allograft recipients (C57BL/10-->C3H) to test whether TH1 cytokines are critical mediators of rejection in this model. The induction of rejection at days 5 to 7 by exogenously administered IL-2 and IL-12, but not IL-4, suggests that mouse liver allograft rejection can be induced by TH1 cytokines; however, there appeared to be differences in the mechanism by which these cytokines induce liver rejection. IL-2 administration was accompanied by an increased intragraft infiltration of CD4+ and CD8+ cells and an up-regulation of natural killer (NK), lymphokine-activated killer (LAK), allospecific cytotoxic killer (CTL) activity and perforin mRNA when compared with media-treated controls. In contrast, exogenous IL-12 treatment was associated with a suppression of CTL, NK, and LAK activity compared with controls but an enhanced infiltration of F4/80+ macrophages as determined by immunohistochemistry. Determination of cytokine mRNA profiles by semi-quantitative reverse transcription polymerase chain reaction showed the up-regulation of interferon (IFN)-gamma, IL-4, IL-6, and IL-10 mRNA with IL-2 treatment when compared with media-treated controls. Interestingly, IL-2 mRNA was down-regulated in these animals, suggesting a negative feedback mechanism in IL-2 regulation. IL-12 treatment resulted in the up-regulation of IFN-gamma, IL-6, and IL-10 mRNA, but not IL-2 or IL-4 mRNA. Higher complement-directed cytotoxic antibody titers were seen in IL-12-treated recipients compared with controls, whereas IL-2 treatment showed no apparent differences in antibody titers compared with media treatment. These in vivo observations were mimicked in a mixed leukocyte reaction by supplementing the reaction with IL-2, IL-12, or media. These results suggest that rejection of mouse liver allografts may involve more than one distinct cellular immunological effector mechanism. One is mediated by IL-2 and appears to favor alloreactive CTL, whereas the other pathway is mediated by IL-12/IFN-gamma and involves macrophages and cytotoxic antibodies largely resembling a delayed-type hypersensitivity reaction.

Long-term survival of rat to mouse cardiac xenografts with prolonged blockade of CD28-B7 interaction combined with peritransplant T-cell depletion

The hCTLA4Ig/mCTLA4Ig fusion protein of the extracellular domain of human/mouse CTLA4 and the Fc portion of the human/mouse immunoglobulin G1 block the CD28/B7 costimulatory T-cell activation pathway. We evaluated the effect of prolonged B7-CD28 blockade, T-cell depletion, or both on rat to mouse cardiac xenografts. C57BL/6 (H-2b) mice receiving infant Wistar Furth (RT1u) rat cardiac xenografts were treated with anti-CD4 (GK1.5) and anti-CD8 (2.43) monoclonal antibodies (mAb; 0.2 mg intravenous each) on days -2 and 0, hCTLA4Ig or mCTLA4Ig every other day from day 0 until day 14 and then twice a week until day 50 or day 100, or both. Changes in cellular reactivity were assayed by mixed lymphocyte culture and cell-mediated cytotoxicity and the development of cytotoxic antibodies was serially measured after transplantation. Either human CTLA4Ig or murine CTLA4Ig alone led to significant prolongation of rat to mouse cardiac xenografts (median survival time [MST], 22 or 26 days, respectively [p = 0.008], versus control). hCTLA4Ig given for 50 days in combination with two doses of anti-CD4/CD8 monoclonal antibodies further prolonged graft survival (MST, 61 days; p versus control < 0.0001). In this combination, when hCTLA4Ig was continued until day 100, the graft survival was further prolonged (MST, 119 days). mCTLA4Ig for 100 days plus anti-CD4/CD8 similarly prolonged rat xenograft survival (MST, 94 days). However, all cardiac xenografts eventually failed, primarily from humoral rejection. Cytotoxic antibody titers rose rapidly only in animals rejecting a graft, and suppressed cell-mediated immunity had completely recovered in rejecting recipients. Blockage of the CD28-B7 costimulatory interaction can inhibit both humoral and cell-mediated immune responses and result in the prolonged acceptance of rat to mouse cardiac xenografts. Longer administration of CTLA4Ig and anti-CD4/CD8 monoclonal antibodies further prolongs but does not achieve indefinite survival of rat cardiac xenografts.

Antibody-dependent cell-mediated cytotoxicity against orbital target cells in thyroid-associated ophthalmopathy and related disorders; close relationship between serum cytotoxic antibodies and parameters of eye muscle dysfunction.

We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular muscle (EOM), Müller's muscle, orbital fibroblasts and skeletal muscle in patients with thyroid-associated ophthalmopathy (TAO) and related eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves' disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves' hyperthyroidism without ophthalmopathy, 10% with Hashimoto's thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller's muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves' disease, 44% with lid lag, 19% with Graves'hyperthyroidism, 50% with Hashimoto's thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves' hyperthyroidism and in 29% of patients with Hashimoto's thyroiditis, but in no patient with euthyroid Graves' disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the eye muscle dysfunction expressed as an eye muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller's muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller's muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related eye disorders is unknown.

Mechanisms of hyperacute rejection in porcine liver transplantation. Antibody-mediated endothelial injury.

Hyperacute rejection after OLT is an unusual event. We have demonstrated previously that hepatic hyperacute rejection can occur in the presence of high titers of donor-specific cytotoxic antibody. This study addresses the issues of antibody localization within the allograft and the consequences of antibody deposition and complement activation within the transplanted organ. A porcine model of liver transplantation was used and 3 experimental groups were studied. Group I recipients (n = 6) were specifically sensitized to their liver donors with skin grafts from their liver donors before hepatic grafting. Group II recipients (n = 6) underwent third-party skin graft sensitization before liver transplantation. Group III recipients (n = 6) underwent liver grafting without prior sensitization. After liver transplantation, serum complement (CH50) levels declined promptly in all groups; a statistically significant drop as compared with control animals was seen only in group I (P < 0.05). Liver biopsies from donor-specific sensitized recipients showed massive injury by light microscopy within 30 min of revascularization, demonstrating fibrinoid necrosis of vessels and neutrophil influx. On immunofluorescent examination, liver specimens from donor-specific sensitized animals showed intense IgG, IgM, and C3 deposition in the vessels of the portal triads. Antibody deposition was not seen in third-party sensitized animals or control animals. On electron microscopy, control animals and third-party sensitized animals showed minimal ultrastructural alterations. In comparison, livers from donor-specific sensitized animals showed severe microvascular injury with destruction of endothelial cells, edema, hemorrhage, and hepatocyte necrosis. In a passive serum transfer experiment carried out by infusing serum from a skin graft-sensitized pig directly into the portal vein of the skin graft donor, severe liver injury was evident, with identification of antibody deposition by light and electron microscopy and by immunofluorescence. These studies demonstrate that tissue injury in hyperacute hepatic rejection is mediated by antibody deposition within the grafted liver and is associated with systemic activation of the complement cascade.

Effect of immunomodulators on specific tumor immunity induced by liposome-encapsulated tumor-associated antigens

Reconstituted membranes consist of liposomal structures formed by removal of detergent from solubilized membrane constituents. The membrane-like configuration of reconstituted membranes makes them attractive as vehicles for presentation of tumor-associated antigens and induction of immune responses. In this study the potential of immunomodulators was assessed to enhance the specific immune response induced by immunization with reconstituted membranes prepared from SL2 lymphosarcoma cells. Reconstituted membranes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) provided better protection against a challenge with SL2 cells than did reconstituted membranes containing alternative immunomodulators. Local administration of IL-2 at the immunization sites further augmented the protection induced by reconstituted membranes with MTP-PE, but was ineffective when administered with plain reconstituted membranes. Immunity elicited by the triple modality of reconstituted SL2 membranes with MTP-PE and IL-2 was specific for SL2 cells. Systemic immunity was obtained against a challenge with a 100-fold higher number of SL2 cells than was reached after immunization with reconstituted membranes alone (10(5) vs. 10(3) SL2 cells). Macrophages isolated from the peritoneal cavity of immunized mice 5 to 7 days after tumor challenge expressed high in vitro cytotoxicity. However, in contrast to the observed specificity of the systemic immunity, macrophages killed both SL2 cells and non-related P815 cells. Neither major cytotoxic lymphocyte activity nor substantial cytotoxic antibody titers were detectable. These results clearly indicate that the approach using reconstituted membranes combined with particular immunomodulators warrants further exploration for the development of safe, well-characterized cancer vaccines.

Evaluation of cyclosporine, mycophenolate mofetil, and Brequinar sodium combination therapy on hamster-to-rat cardiac xenotransplantation.

We examined the effect of CsA, mycophenolate mofetil (MM), and Brequinar sodium (BQR) combination therapy on hamster-to-rat cardiac xenotransplantation survival. Since the mechanism of rejection in concordant cardiac xenotransplantation may be antibody mediated as well as cellularly (CD4) mediated, we also examined the effect of these agents on antidonor antibody levels. In untreated controls, rejection occurred within 4 days, with elevation of cytotoxic antibody titers and severe humoral destruction of the xenografted hearts. It involved both IgM and IgG antibody-mediated humoral immunity. CsA alone (20 mg/kg/day) could not modify this pattern of rejection. High-dose MM (40/20 mg/kg/day)+BQR (12/6 mg/kg 3 times a week) combination therapy achieved slight prolongation of survival and suppressed the elevation of cytotoxic antibody titers relative to controls. While these grafts were rejected within 2 weeks, humoral destruction in the rejected xenografts and antibody deposition were reduced. Combination of CsA (20 mg/kg/day) with BQR (3 or 12 ng 3 times/week) dramatically increased graft survival. When CsA (20 mg/kg/day) was combined with MM (20 mg/kg/day) and BQR (3 mg/kg/day), xenograft survival was significantly prolonged (P < 0.002); however, significant toxicity was observed. Cytotoxic antibody formation was delayed for 1 month in most cases. Histological examination revealed cellular rejection with little evidence of humoral rejection. Thus, combination therapy consisting of CsA and BQR or CsA, MM, and BQR was effective in delaying the rejection of hamster-to-LEW rat concordant cardiac xenografts. The mechanism of prolonged graft survival may involve delayed humoral response and prevention of the cellular response.

Renal transplantation following immunoadsorption in highly sensitized recipients.

Five highly sensitized patients, with panel reactivity greater than 80% for 1.75-5 years, were treated by extracorporeal staphylococcal protein-A immunoadsorption, prednisolone, and cyclophosphamide. The five patients underwent treatment of 18-40 (mean 31) liters of plasma, respectively in 4-7 (mean 5.6) sessions. This reduced the titer of cytotoxic antibodies to sensitizing antigens to < 1/8 in all cases and abolished reactivity to crossreacting antigens. Two patients required retreatment following resynthesis of cytotoxic antibodies. All five patients have been transplanted, and four of these now have stable serum creatinines of 168 mumol/L at 34 months, 208 mumol/L at 29 months, 96 mumol/L at 5 months, and 125 mumol/L at 3 months posttransplantation. One patient had primary graft dysfunction due to acute tubular necrosis; the kidney was removed after eight weeks and showed cortical necrosis without evidence of acute rejection.