Complement-independent Mechanisms Research Articles

Development of novel multi-protein chimeric immunogens that protect against infection with the Lyme disease agent, Borreliella burgdorferi.

Lyme disease is the most common tick-borne disease in North America. A vaccine for use in humans is not available. Here, we detail the development of two chimeric vaccine antigens, BAF and Chv2M. BAF elicits Abs that target proteins and protein variants produced by Borreliella species in ticks (OspB and OspA) and mammals (FtlA/B). OspB serves as the backbone structure for the BAF chimeric. Two OspA221-240 epitope-containing domain (ECD) variants (#A1 and #A15) were inserted into a loop in OspB. The N-terminal region of the FtlA protein was joined to the C-terminus of the chimeric. The second chimeric, Chv2M, consists of L5 (loop 5) and H5 (helix 5) ECDs derived from diverse OspC proteins. Borreliella species produce OspC upon exposure to the bloodmeal and during early infection in mammals. Here, we demonstrate that BAF and Chv2M are potent immunogens that elicit Abs that bind to each component protein (FtlA, FtlB, OspB, and multiple OspA and OspC variants). Anti-BAF and anti-Chv2M Abs kill Borreliella burgdorferi strains through Ab-mediated complement-dependent and complement-independent mechanisms. Eighty percent (32/40) of mice that received a three-dose vaccine regimen were protected from infection with B. burgdorferi B31. Efficacy increased to 90% (18/20) when the amount of Chv2M was increased in the third vaccine dose. Readouts for infection were flaB PCR and seroconversion to VlsE. This study establishes proof of principle for a chimeric immunogen vaccine formulation that elicits Abs to multiple targets on the B. burgdorferi cell surface produced during tick and mammalian stages of the enzootic cycle.IMPORTANCELyme disease is a growing public health threat across parts of the Northern Hemisphere. Regions that can support sustained tick populations are expanding, and the incidence of tick-borne diseases is increasing. In light of the increasing risk of Lyme disease, effective preventive strategies are needed. Most vaccine development efforts have focused on outer surface protein A, a Borreliella burgdorferi protein produced only in ticks. Herein, we describe the development of a novel vaccine formulation consisting of two multivalent chimeric proteins that are immunogenic and elicit antibodies with bactericidal activity that target several cell surface proteins produced by the Lyme disease spirochetes in feeding ticks and mammals. In a broader sense, this study advances efforts to develop custom-designed vaccinogens comprised of epitope-containing domains from multiple proteins.

Targeting antibody-mediated complement-independent mechanism in bullous pemphigoid with diacerein

BackgroundBullous pemphigoid (BP) is an antibody-mediated blistering disease predominantly affecting the elderly. The pathogenesis involves both complement-dependent and complement-independent mechanisms. The therapeutic potential of targeting complement-independent mechanism has not yet been determined. The mainstay of treatment, corticosteroid, has many side effects, indicating the needs of better treatments. ObjectiveWe tempted to establish an in vitro model of BP which resembles complement-independent mechanism and to examine the therapeutic potential of a novel anti-inflammatory agent, diacerein. MethodsCultured HaCaT cells were treated with purified antibodies from BP patients, with or without diacerein to measure the cell interface presence of BP180, protein kinase C, and the production of proinflammatory cytokines. An open-label, randomized, phase 2 trial was conducted to compare topical diacerein and clobetasol ointments in patients with mild-to-moderate BP (NCT03286582). ResultsThe reduced presentation of BP180 at cell interface after treating with BP autoantibodies was noticed in immunofluorescence and western blotting studies. The phenomenon was restored by diacerein. Diacerein also reduced the autoantibody-induced increase of pro-inflammatory cytokines. Reciprocal changes of BP180 and protein kinase C at the cell interface were found after treating with BP autoantibodies. This phenomenon was also reversed by diacerein in a dose-dependent manner. The phase 2 trial showed that topical diacerein reduced the clinical symptoms which were comparable to those of topical clobetasol. ConclusionDiacerein inhibited BP autoantibody-induced reduction of BP180 and production of proinflammatory cytokines in vitro and showed therapeutic potential in patients with BP. It is a novel drug worthy of further investigations.

The Role of Fc Gamma Receptors in Antibody-Mediated Rejection of Kidney Transplants.

For the past decades, complement activation and complement-mediated destruction of allograft cells were considered to play a central role in anti-HLA antibody-mediated rejection (AMR) of kidney transplants. However, also complement-independent mechanisms are relevant in the downstream immune activation induced by donor-specific antibodies, such as Fc-gamma receptor (FcγR)-mediated direct cellular activation. This article reviews the literature regarding FcγR involvement in AMR, and the potential contribution of FcγR gene polymorphisms to the risk for antibody mediated rejection of kidney transplants. There is large heterogeneity between the studies, both in the definition of the clinical phenotypes and in the technical aspects. The study populations were generally quite small, except for two larger study cohorts, which obviates drawing firm conclusions regarding the associations between AMR and specific FcγR polymorphisms. Although FcγR are central in the pathophysiology of AMR, it remains difficult to identify genetic risk factors for AMR in the recipient’s genome, independent of clinical risk factors, independent of the donor-recipient genetic mismatch, and in the presence of powerful immunosuppressive agents. There is a need for larger, multi-center studies with standardised methods and endpoints to identify potentially relevant FcγR gene polymorphisms that represent an increased risk for AMR after kidney transplantation.

Pathomechanisms in demyelination and astrocytopathy: autoantibodies to AQP4, MOG, GFAP, GRP78 and beyond

The purpose of this review is to highlight the recently emerging pathomechanisms of diseases associated with autoantibodies to AQP4, MOG, GFAP, GRP78 and further novel targets. We discuss novel biomarkers and therapeutic approaches. Although complement-mediated cytotoxicity (CDC) is regarded as the major effector mechanism for AQP4-IgG in neuromyelitis optica spectrum disorders (NMOSD), recent studies helped to understand the relevance of complement-independent effector mechanisms. For MOG-IgG mediated diseases the role of CDC is less clear. MOG-IgG may trigger a tightly controlled FcR and BTK-driven microglia proliferative response in MOG-antibody-associated diseases. Differences of antibody-mediated tissue damage may reflect differential response to therapy. In addition, antibodies to GFAP, GRP78 and further novel targets have been implicated in demyelination and astrocytopathy. Elucidating the whole spectrum of effector functions in diseases mediated by AQP4-IgG and MOG-IgG and understanding the role of additional novel autoantibodies involved in demyelination and astrocytopathy may guide further novel treatment decisions.

Treatment of Neuromyelitis Optica Spectrum Disorders.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune central nervous system (CNS) inflammatory disorder that can lead to serious disability and mortality. Females are predominantly affected, including those within the reproductive age. Most patients develop relapsing attacks of optic neuritis; longitudinally extensive transverse myelitis; and encephalitis, especially brainstem encephalitis. The majority of NMOSD patients are seropositive for IgG autoantibodies against the water channel protein aquaporin-4 (AQP4-IgG), reflecting underlying aquaporin-4 autoimmunity. Histological findings of the affected CNS tissues of patients from in-vitro and in-vivo studies support that AQP4-IgG is directly pathogenic in NMOSD. It is believed that the binding of AQP4-IgG to CNS aquaporin-4 (abundantly expressed at the endfoot processes of astrocytes) triggers astrocytopathy and neuroinflammation, resulting in acute attacks. These attacks of neuroinflammation can lead to pathologies, including aquaporin-4 loss, astrocytic activation, injury and loss, glutamate excitotoxicity, microglial activation, neuroinflammation, demyelination, and neuronal injury, via both complement-dependent and complement-independent pathophysiological mechanisms. With the increased understanding of these mechanisms underlying this serious autoimmune astrocytopathy, effective treatments for both active attacks and long-term immunosuppression to prevent relapses in NMOSD are increasingly available based on the evidence from retrospective observational data and prospective clinical trials. Knowledge on the indications and potential side effects of these medications are essential for a clear evaluation of the potential benefits and risks to NMOSD patients in a personalized manner. Special issues such as pregnancy and the coexistence of other autoimmune diseases require additional concern and meticulous care. Future directions include the identification of clinically useful biomarkers for the prediction of relapse and monitoring of the therapeutic response, as well as the development of effective medications with minimal side effects, especially opportunistic infections complicated by long-term immunosuppression.

P-MAPA, a Fungi-Derived Immunomodulatory Compound, Induces a Proinflammatory Response in a Human Whole Blood Model.

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF-α, IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models.

Sox7 is involved in antibody-dependent endothelial cell activation and renal allograft injury via the Jagged1-Notch1 pathway

BackgroundAntibody-mediated rejection (AMR) can cause graft loss and reduces long-term graft survival after kidney transplantation. Human leukocyte antigen (HLA) and/or non-HLA antibodies play a key role in the pathogenesis of AMR by targeting the allograft epithelium via complement activation and complement-independent mechanisms. However, the precise mechanisms of AMR remain unclear and treatment is still limited. MethodsIn this study, we investigated the role of the endothelial-associated transcription factor Sox7 in AMR, using the anti-HLA antibody W6/32, shRNA-mediated Sox7 knockdown, and by manipulating the Notch pathway. We used an in vitro human kidney glomerular endothelial cells (HKGECs) model and an in vivo MHC-mismatched kidney transplantation model. ResultsSox7 expression was upregulated and the Jagged1-Notch1 pathway was activated in HKGECs after W6/32 activation. W6/32 antibodies increased the expression of adhesion molecules (VCAM-1, ICAM-1), inflammatory cytokines (IL-6, TNF-α), and chemokines (CXCL8, CXCL10), and Sox7 knockdown and inhibition of the Notch pathway by DAPT significantly reduced these effects. Jagged1 overexpression rescued the inhibitory effects of Sox7 knockdown. In addition, Sox7 knockdown attenuated acute allograft kidney injury in mice and reduced the expression of adhesion molecules and Jagged1-Notch1 signaling after transplantation. ConclusionsOur findings suggest that Sox7 plays an important role in mediating HLA I antibody-dependent endothelial cell activation and acute kidney allograft rejection via the Jagged1-Notch1 pathway. Manipulating Sox7 in donor organs may represent a useful treatment for AMR in solid organ transplantation.

Aquaporin-4 Autoantibodies From Neuromyelitis Optica Spectrum Disorder Patients Induce Complement-Independent Immunopathologies in Mice

Neuromyelitis optica spectrum disorders (NMOSD) are central nervous system inflammatory disorders causing significant morbidities and mortality. The majority of NMOSD patients have autoimmunity against aquaporin-4 (AQP4), evidenced by seropositivity for autoantibodies against aquaporin-4 (AQP4–IgG). AQP4–IgG is pathogenic with neuroinflammation initiated upon binding of AQP4–IgG to astrocytic AQP4. Complement activation contributes to astrocytic cytotoxicity, neuroinflammation, and tissue necrosis in NMOSD, but the role of complement-independent mechanisms is uncertain. We studied the complement-independent pathogenic effects of AQP4–IgG by passive transfer of IgG from NMOSD patients to mice with breached blood–brain barrier (BBB). Mice, pretreated with bacterial proteins, received daily intraperitoneal injections of IgG purified from AQP4–IgG-seropositive NMOSD patients [IgG(AQP4+)], or IgG from AQP4–IgG-seronegative patients [IgG(AQP4−)] or healthy subjects [IgG(Healthy)] for 8 days. Motor function was tested by walking across narrow beams, and spinal cords were collected for immunofluorescent analysis. We found that human IgG infiltrated into cord parenchyma of mice with breached BBB without deposition of complement activation products. Spinal cord of mice that received IgG(AQP4+) demonstrated loss of AQP4 and glial fibrillary acidic protein (suggestive of astrocyte loss), decrease in excitatory amino acid transporter 2, microglial/macrophage activation, neutrophil infiltration, patchy demyelination, and loss in axonal integrity. Mice that received IgG(AQP4+) required longer time with more paw slips to walk across narrow beams indicative of motor slowing and incoordination. Our findings suggest that AQP4–IgG induces complement-independent cord pathologies, including astrocytopathy, neuroinflammation, demyelination, and axonal injuries/loss, which are associated with subtle motor impairments. These complement-independent pathophysiologies likely contribute to early NMOSD lesion development.

048 Possible complement-independent mechanisms in tissue destruction in bullous pemphigoid

048 Possible complement-independent mechanisms in tissue destruction in bullous pemphigoid

Tissue Destruction in Bullous Pemphigoid Can Be Complement Independent and May Be Mitigated by C5aR2.

Bullous pemphigoid (BP), the most frequent autoimmune bullous disorder, is a paradigmatic autoantibody-mediated disease associated with autoantibodies against BP180 (type XVII collagen, Col17). Several animal models have been developed that reflect important clinical and immunological features of human BP. Complement activation has been described as a prerequisite for blister formation, however, the recent finding that skin lesions can be induced by anti-Col17 F(ab′)2 fragments indicates complement-independent mechanisms to contribute to blister formation in BP. Here, C5−/− mice injected with anti-Col17 IgG showed a reduction of skin lesions by about 50% associated with significantly less skin-infiltrating neutrophils compared to wild-type mice. Reduction of skin lesions and neutrophil infiltration was seen independently of the employed anti-Col17 IgG dose. Further, C5ar1−/− mice were protected from disease development, whereas the extent of skin lesions was increased in C5ar2−/− animals. Pharmacological inhibition of C5a receptor 1 (C5aR1) by PMX53 led to reduced disease activity when applied in a prophylactic setting. In contrast, PMX-53 treatment had no effect when first skin lesions had already developed. While C5aR1 was critically involved in neutrophil migration in vitro, its role for Col17-anti-Col17 IgG immune complex-mediated release of reactive oxygen species from neutrophils was less pronounced. Our data demonstrate that complement-dependent and -independent mechanisms coexist in anti-Col17-autoantibody-mediated tissue destruction. C5aR1 and C5aR2 seem to play opposing roles in this process with C5aR1 exerting its primary effect in recruiting inflammatory cells to the skin during the early phase of the disease. Further studies are required to fully understand the role of C5aR2 in autoantibody-mediated skin inflammation.

Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies.

Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of immunosuppressive treatments, robust ADCC responsiveness can be maintained in some KTRs. Because it evaluates both the Fab recognition of alloantigens and Fc-driven NK cell activation, the NK-CHAT represents a potentially valuable tool for the non-invasive and individualized evaluation of humoral risk during transplantation.

Complement inhibition as potential new therapy for antibody-mediated rejection.

Antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. While the exact mechanisms contributing to donor-specific antibody (DSA)-triggered tissue injury are still incompletely understood, complement activation via the classical pathway is believed to be one of the key players. There is now growing interest in complement blockade as an antirejection treatment. One attractive strategy may be inhibition of terminal complex formation using anti-C5 antibody eculizumab. Anecdotal reports, case series, and a unique cohort of flow crossmatch-positive live donor kidney transplant recipients subjected to eculizumab-based desensitization have demonstrated successful prevention and reversal of acute clinical ABMR. Nevertheless, maybe due to complement activation steps proximal of C5 or even complement-independent mechanisms, subclinical rejection processes that might culminate in chronic injury were found to escape inhibition. Larger studies designed to clarify the actual clinical value of terminal complement inhibition as an antirejection treatment are currently underway. In addition, alternative concepts, such as therapies that target key component C1, are currently under development, and we will see in the near future whether new strategies in the pipeline will have the potential to beneficially impact clinical practice.

Role of complement and complement regulatory proteins in the complications of diabetes.

It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

New insights regarding chronic antibody-mediated rejection and its progression to transplant glomerulopathy.

To discuss new insights regarding chronic antibody-mediated rejection (CAMR) and its progression to transplant glomerulopathy. We will describe the progression to transplant glomerulopathy from a histologic perspective and provide updates on what is known about its pathophysiology, prognosis, and potential therapy. Transplant glomerulopathy is a major contributor to long-term renal allograft loss and is most often associated with CAMR. On the basis of protocol biopsies, we have found that 3.5% of conventional transplants and 27.5% of positive crossmatch kidney transplants have transplant glomerulopathy at 1 year. The pathophysiology of the process is largely unknown, but complement activation was previously thought to be essential. However, CAMR appears to develop despite terminal complement blockade and many C4d negative cases of CAMR have been identified. Thus, complement independent mechanisms, such as direct endothelial cell activation and the infiltration of natural killer cells and monocytes, are likely key to the development of transplant glomerulopathy. Transplant glomerulopathy is often the result of CAMR and leads to allograft loss. It is characterized by distinctive histologic changes, and its pathophysiology is a multifaceted process involving both innate and adaptive immunity. Despite advances in the understanding of this condition, no effective therapy exists.

Antibody-mediated graft injury

Antibody-mediated rejection (AMR) is emerging as the leading cause of chronic rejection and allograft failure. Traditionally, the mechanisms of graft injury mediated by donor-specific antibodies beyond complement activation were not well appreciated. However, an evolving paradigm of Fc-independent antibody functions, along with clinical recognition of C4d-negative AMR, has increased awareness of the action of antibodies leading to endothelial activation and dysfunction. Herein, we address current clinical trends, including the signature of microvascular inflammation in biopsies of grafts undergoing AMR, the prevalence of antibodies to human leukocyte antigen class II DQ locus (HLA-DQ) and non-HLA targets, and the functional characterization of HLA immunoglobulin G (IgG) subclasses and complement-fixing capacity. We also discuss recent experimental evidence revealing new mechanisms of endothelial and smooth muscle cell activation by HLA antibodies, which may contribute to vascular inflammation and chronic rejection. Finally, we touch upon novel discoveries of the interplay between antibodies, the complement system, and CD4 T-cell-mediated alloimmunity. The current literature suggests that, although complement-fixing antibodies may have some prognostic value for graft outcome, complement-independent mechanisms of graft injury are increasingly relevant. Therapeutic strategies, which target endothelial activation induced by antibodies may ameliorate vascular inflammation and mononuclear cell infiltration characteristic of AMR.

Cellular Protection Against Antibodies Occurs Through a Complement-Independent Pathway

Abstract 3290 Background:Although mechanisms exist whereby T cells differentiate self from non-self, occasionally tolerance breaks down and T cells target self-antigens. Under these circumstances, healthy cells possess unique receptors that serve to inhibit potential cell-mediated destruction. However, unlike cell-mediated tissue injury, antibody-secreting cells elicit their effects at remote sites, precluding direct feedback from target tissue to secreting cells. As a result, target cells appear to have evolved mechanisms of inhibiting antibody-mediated destruction. Indeed, patients with reduced levels of complement inhibitory factors experience severe hemolysis following antibody engagement. However, regulatory components responsible for protection against complement-independent antibody effecter functions remain poorly understood. RBCs provide a unique tool in the examination of cellular fate following antibody engagement as RBCs fail to synthesize new antigen and also fail to proliferate. As a result, we utilized a model of antibody-RBC binding to determine whether RBCs possess complement-independent mechanisms of cellular resistance to antibody-induced hemolysis. Methods:B6, C3 KO or FcγR KO mice were passively immunized with an anti-Fy3 IgG2a monoclonal antibody. Fluorescent dye labeled RBCs that transgenically express a chimeric antigen including the Fy3 epitope from human Duffy (HOD mice) were then transfused into immunized or nonimmunized control mice. Over a timecourse, peripheral blood was obtained and transfused RBCs were visualized by flow cytometry, followed by examination of clearance, surface C3, bound IgG and Fy3 antigen. To discriminate old from new RBCs following transfusion, RBCs were harvested from mice 35 days post-biotinylation, transfused into immunized or nonimmunized mice and examined for differential clearance by strepavidin staining and flow cytometry. To further evaluate RBC resistance to antibody-induced clearance, HOD RBCs were harvested 2 days following transfusion and re-transfused into immunized or nonimmunized mice followed by examination for clearance, surface C3, bound IgG and Fy3 antigen. Results:Immunized mice rapidly cleared over half of HOD RBCs within 2 hours; however, a significant number of resistant cells remained in circulation despite the persistence of antibody levels capable of clearing additional RBCs following subsequent transfusion. No clearance was observed in FcγR KO mice but normal clearance and resistance was observed in C3 KO mice. Moreover, no complement degradation products were detected following transfusion into wt mice. RBCs 35 days and older displayed comparable antibody-induced clearance as younger RBCs, suggesting that resistance did not reflect preferential loss of older RBCs. Furthermore, HOD RBCs remained resistant to clearance following re-transfusion into recently immunized recipients. Examination of HOD antigen levels demonstrated a rapid decrease in HOD antigen that correlated with the development of resistance. Similar to clearance, HOD antigen levels decreased in C3 KO but not FcγR KO. Conclusion:These results suggest that in addition to known complement-dependent mechanisms of cellular resistance to antibody-induced hemolysis, a novel pathway exists whereby Fcγ receptors not only participate in clearance, but also appear to facilitate specific loss of antibody reactive antigen, which may be responsible for a population of RBCs resistant to further clearance. Taken together, these results indicate that RBCs possess multiple mechanisms for cellular protection against antibodies that may be relevant in autoimmune hemolytic anemia and other antibody-mediated processes. Disclosures:No relevant conflicts of interest to declare.

HLA-DR Expressing Endothelial Cell Expansion of Allogeneic CD4+ T Lymphocytes Towards Either Pro-Inflammatory Th17 or Regulatory Populations

Introduction: The major obstacle to organ transplantation remained chronic rejection due to the generation and the persistence of an alloimmune response principally mediated by Major Histocompatibility Complex molecules (MHC) present on donor cells. Graft microvascular endothelial cells (ECs) express MHC class I and II molecules and have therefore a dual role as both a stimulator of, and a target for allo-immune responses. Recently, we have shown that ECs generate either regulatory CD4+ T cells (Tregs) or pro-inflammatory CD4+IL-17+ cells (Th17) but the factors which orient this CD4+ T cell allo-immune response remain to be determinate. Methods: We have established a model of either inducible or constitutive HLA-DR expression in microvascular ECs using lentiviral vector infection. This model was used to study (1) the mechanisms of EC induced activation of allogeneic CD4+ T cells and (2) to determine the effect of EC pre-activation with an HLA-DR antibody on the orientation of the allogeneic CD4+ T cell response. Results: Under inflammatory conditions, HLA-DR expression by human microvascular ECs allows them to orient the CD4+ T cell response towards either a Th17 pro-inflammatory response or a regulatory T cell response. Expansion of Th17 depended upon IL-6 secretion by ECs which led to STAT-3 activation in allogeneic CD4+ T cells associated with their proliferation, while Treg expansion was dependent upon their interaction with ECs mediated by the adhesion molecule ICAM-1 (CD54). HLA-DR antibody induced signaling in ECs leading to an increase in their secretion of IL-6 and their ability to expand alloreactive Th17 cells while lessening the regulatory T cell response. Conclusion: These data reveal that firstly human microvascular EC regulates polarization of the memory CD4+ T cell response towards either cytokine-dependent inflammation or contact-dependent regulation and secondly that HLA-DR antibody pre-activation of EC increases their ability to induce a pro-inflammatory Th17 response. Together these results provide evidence for a link between the roles of EC as a target of HLA-DR antibody binding and as an allo-stimulator of the pro-inflammatory Th17 response implicated in chronic rejection. Moreover, these data may contribute to our understanding of HLA class II allo-antibodies mediated complement independent mechanisms of EC damage.

Non-HLA-antibodies targeting Angiotensin type 1 receptor and antibody mediated rejection

Antibody-mediated mechanisms directed against non-HLA related targets may exert negative impact on allograft function and survival. Angiotensin type 1 receptor (AT(1)R) emerges as a functional target for non-HLA allo- and autoantibodies (AT(1)R-Abs) comprising of IgG1 and IgG3 subclasses. Proof of concept for pathophysiologic relevance of AT(1)R-Abs in antibody mediated rejection (AMR) in renal transplants was provided by passive transfer studies in animal model and therapeutic rescue of patients. Although AT(1)R-Abs may belong to complement fixing IgG subclasses, C4d positivity in renal transplant biopsies was not frequently detected implicating complement independent mechanisms of injury. AT(1)R-Abs exert direct effects on endothelial and vascular smooth muscle cells by induction of Erk1/2 signaling and increased DNA binding of transcription factors associated with pro-inflammatory and pro-coagulatory responses. Establishment of enzyme-linked immunosorbent assay employing extracts of cells overexpressing AT(1)R in its native conformation was instrumental for recent studies in independent cohorts. Assessing the AT(1)R-Ab-status along with the HLA-antibodies may help to identify patients at particular risk for irreversible acute or chronic allograft injuries and improve overall outcomes. This review summarizes the current state of research in AT(1)R biology, development in diagnostic strategies, discusses recent clinical studies, and provides perspectives on further refinements in understanding AT(1)R-Ab-actions.

Targeting C/EBPβ sensitises tumours to killing via complement-dependent and complement-independent mechanisms

Targeting C/EBPβ sensitises tumours to killing via complement-dependent and complement-independent mechanisms

Invasion of Erythrocytes by Francisella tularensis

Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.