Non-Complement- and Complement-Activating Antibodies Synergize to Cause Rejection of Cardiac Allografts

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50-200-microg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 microg of IgG2b or a single 100-200-microg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene alpha (KC). TNF-alpha augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.     

Expression of CR1/2 receptor on alloantigen-stimulated mouse T cells

Antibodies can mediate injury of organ transplants by several mechanisms, including complement activation and interaction with Fc receptors on cells. We tested the hypothesis that antibodies could also cause up-regulation of complement receptors on cells to increase the responses to complement activation by interaction with split products of C3. In our experimental model, B10.A (H-2(a)) cardiac transplants survive significantly longer in C57BL/6 (H-2(b)) immunoglobulin knockout recipients (IgKO) than in their wild-type counterparts. Passive transfer of specific antibodies to donor MHC class I to IgKO recipients of cardiac allografts at the time coinciding with a vigorous cellular infiltration reconstituted acute rejection. We tested the effects of alloantibodies on CR1/2 expression by alloantigen-stimulated T cells. Both CD4(+)/CR1/2(+) and CD8(+)/CR1/2(+) populations of T cells were expanded in C57BL/6 splenocytes stimulated by B10.A alloantigen in 7-day MLR after coculture with endothelial cells sensitized with IgG1 and IgG2b mAb specific to MHC. Endothelial cells sensitized with antibodies also caused an expansion of CD8(+) T cells expressing CR1/2 in lymph node lymphocytes harvested from a C57BL/6 recipient of a B10.A cardiac allograft. These data suggest that antibodies can augment the cellular rejection process through expanding the population of T cells interacting with complement split products.     

Agonistic and antagonistic interactions of anti-interleukin 2 receptor monoclonal antibodies in rat recipients of cardiac allografts

A panel of five mouse mAbs recognizing 4 distinct epitopes (R1-4) of the rat 55kD IL-2R molecule were tested for their influence on acute rejection (8 days) of (LEWxBN)F1 cardiac allografts in LEW hosts. IL-2R1 targeted therapy with ART-18 (IgG1, inhibits IL-2-dependent responses) prolonged graft survival to ca.21 days. IL-2R2 is recognized by ART-65 and ART-75, mAbs that do not inhibit T cell growth. Treatment with ART-65 (IgG1) but not ART-75 (IgG2a) abrogated acute rejection (ca. 16 days and 9 days, respectively). ART-35, an anti-IL-2R3 mAb (IgG1, does not inhibit T cell function) extended graft survival marginally to ca. 12 days. Finally, therapy with OX-39, (anti-R4 IgG1 mAb, inhibits IL-2 binding, but not IL-2-driven growth) was completely ineffectual. Simultaneous targeting of two IL-2R epitopes increased the therapeutic index synergistically (ART-18 [R1] + ART-65 [R2]--60% permanent graft acceptance), additively (ART-75 [R1] + ART-35 [R3]--graft survival ca. 18 days), or did not improve further graft survival at all (ART-18 [R1] + OX-39 [R4]--graft survival ca. 18 days). Thus, the cellular targeting patterns and isotype of mAbs are crucial: (1) targeting at functionally distinct epitopes controls rejection most effectively; (2) IgG1 and IgG2b mAbs are more influential in vivo than IgG2a, data supported by the studies employing the family of ART-18 isotype switch variants. Treatment with anti-IL-2R mAb did not depress Ts activity as tested both in vitro and in vivo. Sparing of putative Ts by mAb is also shown by thymectomy of graft recipients before or during ART-18 therapy, which shortened graft survival to 13-15 days; thymectomy after ART-18 therapy did not influence graft survival. However, infusion of IFN-gamma recreated classic acute rejection within 10 days in otherwise longstanding cardiac allografts in ART-18 treated hosts. Upregulation of MHC class II antigen and IL-2R expression seems to be primarily responsible for this striking biological effect of IFN-gamma in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection.

BACKGROUND: Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. METHODS: The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. RESULTS: Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. CONCLUSIONS: Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.     

Synergistic Deposition of C4d by Complement-Activating and Non-activating Antibodies in Cardiac Transplants

The role of non-complement-activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non-complement-activating alloantibodies synergistically activate complement in combination with complement-activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig-KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig-KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL-A/C knock out (MBL-KO) serum with C1q-knock out (C1q-KO) serum reestablished the synergistic activity. This suggests a novel role for non-complement-activating alloantibodies and MBL in humoral rejection.     

The involvement of FcR mechanisms in antibody-mediated rejection

BACKGROUND: Antibody-mediated rejection is characterized by macrophage margination against vascular endothelium. The potential interactions triggered by antibodies between endothelial cells (EC) and macrophages have not been examined thoroughly in transplants. We used in vivo and in vitro models of antibody-mediated rejection. METHODS: Passive transfer of monoclonal alloantibodies (Allo-mAbs) to donor major histocompatibility complex-class I antigens was used to restore acute rejection of B10.A (H-2a) hearts to C57BL/6 (H-2b) immunoglobulin knockout (IgKO) recipients. Intragraft cytokine mRNA expression was measured by real-time polymerase chain reaction. In vitro, mouse EC were cultured in the presence of Allo-mAbs to donor major histocompatibility complex class I antigens and mononuclear cells. Levels of cytokines in culture supernatants were determined in enzyme-linked immunosorbent assay. RESULTS: Expression of MCP-1, IL-6 and IL-1alpha mRNA was higher in rejecting transplants from recipients treated with Allo-mAbs compared to non-rejecting transplants. EC sensitized with Allo-mAbs produced high levels of MCP-1 and KC. The addition of macrophages to sensitized EC stimulated high levels of IL-6 in addition to MCP-1, KC, Rantes, and TIMP-1. The levels of MCP-1 and IL-6 were significantly lower in co-cultures of EC sensitized with IgG1 Allo-mAbs in the presence of mononuclear cells from Fcgamma-Receptor III KO (FcgammaRIII-KO) graft recipients compared to co-cultures with wild-type cells. The levels of both cytokines were also lower in co-cultures of EC stimulated with F(ab')2 fragments of antibody. CONCLUSIONS: Our findings indicate that IgG1 Allo-mAbs to major histocompatibility complex class I antigens can augment graft injury by stimulating EC to produce MCP-1 and by activating mononuclear cells through their Fc receptors.     

Clinical Significance of MHC-Reactive Alloantibodies that Develop after Kidney or Kidney–pancreas Transplantation

The purpose of this study was to determine the relationships between acute rejection, anti-major histocompatibility complex (MHC) class I and/or class II-reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney-pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post-transplant (mean 2.6years). The presence of anti-MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC-reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti-MHC class II, but not anti-MHC class I, alloantibodies detected post-transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II-reactive, but not MHC class I-reactive, alloantibodies post-transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post-transplant detectable MHC class II-reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post-transplant alloantibody production, and avoidance of acute rejection episodes, may improve long-term graft survival by reducing the incidence of chronic allograft rejection.     

Donor and recipient contributions of ICAM-1 and P-selectin in parenchymal rejection and graft arteriosclerosis: insights from double knockout mice.

BACKGROUND: Mice with target gene deletions were used in an immunosuppressed, heterotopic mouse cardiac transplant model to investigate the effects of simultaneous deficiencies of ICAM-1 and P-selectin on late cardiac rejection. METHODS: To determine the contribution of donor sources of ICAM-1 and P-selectin, ICAM-1/P-selectin gene deficient (I/P -/-) (n = 7) or wild type (n = 6) donor hearts were placed into CBA recipients. To study recipient sources of ICAM-1 and P-selectin, wild type donor hearts were placed into I/P -/- (n = 7) or wild type (n = 13) recipients. Recipients received a 30-day course of anti-CD4/8 mAb. RESULTS: I/P -/- donor allografts had prolonged survival (52-57 days) compared with wild type allografts (49-51 days). I/P -/- donor allografts underwent parenchymal rejection with mononuclear cell infiltration and developed alpha-smooth muscle actin positive vascular thickening (30 +/- 7% luminal occlusion, n = 78 vessels). Wild type allografts had parenchymal rejection with vascular medial necrosis and an absence of arteriosclerotic thickening (10 +/- 8%, n = 75, p = 0.008). Using the reverse combination, allografts from I/P -/- or wild type recipients had similar graft survival (50-57 days), comparable but variable degrees of parenchymal rejection, and comparable vascular occlusion (22 +/- 15% vs 28 +/- 19%, p = 0.442). CONCLUSION: We have shown that donor and recipient sources of ICAM-1 and P-selectin may have independent roles in leukocyte trafficking to the graft. Simultaneous interruption of donor ICAM-1 and P-selectin delays onset of parenchymal rejection. However, donor I/P deficiency permits arteriosclerotic development, perhaps by attenuating the alloimmune injury. In contrast, recipient deficiency alone does not altergraft outcomes suggesting that the donor is the critical site of ICAM-1 and P-selectin.     

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.     

Cellular and humoral mechanisms of vascularized allograft rejection induced by indirect recognition of donor MHC allopeptides.

INTRODUCTION: To investigate the role and mechanisms of indirect allorecognition in allograft rejection, we studied whether priming T cells with donor-derived MHC allopeptides could accelerate rejection in a vascularized allograft model. METHODS: Lewis recipients of fully mismatched Wistar Furth cardiac allografts were immunized before transplantation with donor MHC allopeptides. RESULTS: Animals immunized with immunogenic class II MHC allopeptides rejected their grafts in a significantly accelerated fashion compared with controls. Additional studies demonstrated that a single immunodominant RT1.D (HLA-DR like) allopeptide was responsible for accelerating the rejection process. Histological analysis of rejected allografts revealed marked vascular rejection in the accelerated, although not the control, group as well as severe cellular rejection. Peak production of IgM and IgG donor-specific alloantibodies was detected by flow cytometry 1 week earlier in the sera of the accelerated group compared with the control group. Immunohistological analysis of grafts from the accelerated compared with the control group showed increased endothelial deposition of IgG2b, C3, and fibrin, and up-regulation of class II MHC molecule expression. Increased intragraft expression of interferon-y and the interferon-gamma-induced chemokines, inducible protein-10 and Mig, and infiltration by activated mononuclear cells expressing CXCR3, the receptor for inducible protein-10 and Mig, was also seen. CONCLUSION: These novel data provide evidence of a definitive link between indirect allorecognition of donor-derived MHC class II peptides and the cellular and humoral mechanisms of vascularized allograft rejection.     

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN‐γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti‐LFA‐1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG‐treated recipients on day 7 posttransplant, allografts from anti‐LFA‐1 mAb‐treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor‐reactive CD8 T cells producing IFN‐γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti‐LFA‐1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG‐3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.     

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.     

Allochimeric class I MHC molecules prevent chronic rejection and attenuate alloantibody responses.

BACKGROUND: We have shown that treatment with molecularly engineered, allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.Au) amino acid substitutions for host-type ACI (RTI.Aa) sequences in the alpha1-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. This study examined the effect of allochimeric molecules on the development of chronic rejection. METHODS: Allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigenic extracts (1 mg) were administered via the portal vein into ACI recipients of WF hearts on the day of transplantation in conjunction with subtherapeutic oral cyclosporine (CsA, 10 mg/kg/day, days 0-2). Control groups included recipients of syngeneic grafts and ACI recipients of WF heart allografts treated with high-dose CsA (10 mg/kg/day, days 0-6). RESULTS: WF hearts in ACI rats receiving 7 days of CsA exhibited myocardial fibrosis, perivascular inflammation, and intimal hyperplasia at day 80. At day 120, these grafts displayed severe chronic rejection with global architectural disorganization, ventricular fibrosis, intimal hyperplasia, and progressive luminal narrowing. In contrast, WF hearts in rats treated with [alpha1 hl/u]-RT1.Aa molecules revealed only mild perivascular fibrosis, minimal intimal thickening, and preserved myocardial architecture. Alloantibody analysis demonstrated no IgM alloantibodies in all groups. An attenuated, but detectable, anti-WF IgG response was present in recipients receiving allochimeric molecules, with IgG1 and IgG2a subclasses predominating. Immunohistochemical analysis of allografts demonstrated minimal T cell infiltration and IgG binding to vascular endothelium. CONCLUSION: Treatment with allochimeric molecules prevents the development of chronic rejection. Such effect may be in part caused by deviation of host alloantibody responses.     

Fetal bone grafts do not elicit allograft rejection because of protecting anti-Ia alloantibodies. Implications to the immune survival of fetuses in allogeneic mothers.

In a previous study we showed that allografts of BN fetal bone, unlike allografts of adult bone, are not rejected by allogeneic recipients of the Lewis strain in spite of the existence of major histocompatibility complex (MHC) incompatibility between donors and hosts. In the present study, we analyzed the relationships existing between the host and fetal tissue that determine graft survival. We found that (1) the fetal BN graft, unlike adult grafts, induces in Lewis recipients a vigorous humoral response consisting mainly in the production of IgG antibody that seems to be directed against antigens of Ia-like specificities. (2) The BN rats are genetically defective in their capacity to respond to determinants and thus are not capable of producing anti-Ia antibodies; in accordance, Lewis fetal bone grafts are rejected by the BN recipients. (3) Chondrocytes isolated from fetal mouse bones do express Ia antigenic determinants. We suggest that the survival of an allogeneic fetal graft in an immunologically intact recipient depends on an active and selective immune response directed against the Ia components associated with the MHC on the embryonic and fetal cells. On the basis of these notions, we propose that the capacity of Ia determinants expressed on cells of the embryo, to elicit anti-Ia and IgG alloantibodies in the pregnant mother, determines the capacity of the embryo to escape rejection by the histoincompatible mother.     

Complement activation by anti-endothelial cell antibodies in MHC-mismatched and MHC-matched heart allograft rejection: Anti-MHC-, but not anti non-MHC alloantibodies are effective in complement activation

Classical complement activation is a major effector mechanism in the development of vascular lesions contributing to allograft rejection. We investigated complement activation by alloantibodies reactive with graft endothelial cell (EC) alloantigens in settings of MHC-mismatched and MHC-matched (non-MHC-mismatched) rat heart transplantation (Tx). Allosera and heart allografts were collected at the day of rejection (day 7–8 and day 28–35 in MHC-mismatched and non-MHC-mismatched Tx respectively) or earlier. Allosera reactivity was studied in vitro using rat-heart-endothelial-cell (RHEC) lines expressing the appropriate donor MHC and non-MHC alloantigen profile. Immunohistochemical analysis of rejected heart allografts showed deposition of alloantibodies in both MHC-mismatched and MHC-matched heart allografts, but expression of C3 was only seen in the vasculature of MHC-mismatched grafts. FACS analysis showed that anti MHC as well as anti non-MHC allosera were reactive with donor EC cell surface antigens. Both sera had similar IgG subclass profiles of anti-endothelial cell antibodies. Complement activation by anti MHC and anti non-MHC alloantibodies on EC was measured by FACS analysis of C3 and C5b-9 (MAC) expression. Distinct expression of C3 was noticed for EC incubated with anti-MHC allosera, but hardly for EC incubated with anti non-MHC allosera. C5b-9 was low but showed no difference between the two allosera. However, complement-mediated cytotoxicity experiments showed that functional (lytic) MAC was induced with anti MHC allosera but hardly with anti non-MHC allosera. Our data show that in settings of MHC-matched heart transplantation alloantibodies against endothelial non-MHC alloantigens are generated, but, in contrast to alloantibodies to MHC alloantigens, these alloantibodies have only poor complement-activating and lytic potentials. Whether anti non-MHC allolantibodies effect other biological processes relevant for heart allograft vasculopathy, including development of graft arteriosclerosis, needs further elucidation. Received: 5 November 1999/Accepted: 17 May 2000     

Evidence That Humoral Allograft Rejection in Lung Transplant Patients Is Not Histocompatibility Antigen-Related

We have recently recognized humoral rejection (HR) in lung allograft recipients and its association with acute and chronic graft dysfunction. We have shown that C4d, a stable marker of classic complement activation, is deposited in lung allografts, correlating with clinical rejection and parenchymal injury. The antigenic target may be endothelium in the setting of recurrent acute rejection while varying components of the bronchial wall may be important in chronic graft dysfunction. We sought to establish whether there is a role for antibodies with histocompatibility antigen specificity in the lung humoral allograft phenomenon. Flow cytometric and ELISA assays to assess donor-specific antigens were conducted on sera from 25 lung transplant recipients who had experienced one or more episodes of clinical rejection; in addition, the serum samples were tested for evidence of antiendothelial cell antibody activity. Morphologically, each case had biopsies showing septal capillary injury with significant deposits of immunoreactants with microvascular localization and positive indirect immunofluorescent antiendothelial cell antibody assay. Panel-reactive antibody testing showed absence of MHC Class I/II alloantibodies; ELISA based crossmatch detecting donor-specific MHC Class I/II specific antibodies was negative. HR can occur in the absence of antibodies with HLA specificity; antigenic targets may be of endothelial cell origin.     

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)–approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1β maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.     

C4d Deposition and Clearance in Cardiac Transplants Correlates With Alloantibody Levels and Rejection in Rats

Antibody-mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement-activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.