Monocyte recruitment by HLA IgG-activated endothelium: the relationship between IgG subclass and Fc?RIIa polymorphisms.
ABSTRACT: It is currently unclear which donor specific HLA antibodies confer the highest risk of antibody-mediated rejection (AMR) and allograft loss. In this study, we hypothesized that two distinct features (HLA IgG subclass and Fc? receptor [Fc?R] polymorphisms) which vary from patient to patient, influence the process of monocyte trafficking to and macrophage accumulation in the allograft during AMR in an interrelated fashion. Here, we investigated the contribution of human IgG subclass and Fc?R polymorphisms in monocyte recruitment in vitro by primary human aortic endothelium activated with chimeric anti-HLA I human IgG1 and IgG2. Both subclasses triggered monocyte adhesion to endothelial cells, via a two-step process. First, HLA I crosslinking by antibodies stimulated upregulation of P-selectin on endothelium irrespective of IgG subclass. P-selectin-induced monocyte adhesion was enhanced by secondary interactions of IgG with Fc?Rs, which was highly dependent upon subclass. IgG1 was more potent than IgG2 through differential engagement of Fc?Rs. Monocytes homozygous for Fc?RIIa-H131 adhered more readily to HLA antibody-activated endothelium compared with Fc?RIIa-R131 homozygous. Finally, direct modification of HLA I antibodies with immunomodulatory enzymes EndoS and IdeS dampened recruitment by eliminating antibody-Fc?R binding, an approach that may have clinical utility in reducing AMR and other forms of antibody-induced inflammation.
Project description:Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human Fc?Rs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through Fc?RI, and, to a lesser extent, Fc?RIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by Fc?R interactions.
Project description:In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.
Project description:Vascular-deposited IgG immune complexes promote neutrophil recruitment, but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1, in its bent state, interacts with the IgG receptor Fc?RIIA in cis to reduce the affinity of Fc?RIIA for IgG and inhibit Fc?RIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of Fc?RIIA, as does a Mac-1 ligand and a mutation in Mac-1's ligand binding ?I-domain. Sialylated complex glycans on Fc?RIIA interact with the ?I-domain via divalent cations, and this interaction is required for Fc?RIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented Fc?RIIA-dependent recruitment to IgG-coated endothelium. In mice, CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary, cis interaction between sialylated Fc?RIIA and the ?I-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases.
Project description:BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the Fc?RIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.
Project description:Fc?RIIa is an activating Fc?R, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as Fc?RIIa1. A unique splice variant, we designated Fc?RIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this Fc?RIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. Fc?RIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. Fc?RIIa3 was identified in macaque and human PBMC. The Fc?RIIa3 is distinguished from the canonical Fc?RIIa1 by a unique 19-amino acid cytoplasmic insertion and these two Fc?RIIa forms responded distinctly to antibody ligation. Whereas Fc?RIIa1 was rapidly internalized, Fc?RIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of Fc?RIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced Fc?RIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.
Project description:Antibody-mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long-term survival of solid organ transplants. AMR is caused by donor-specific antibodies to HLA, which contribute to TAV by initiating outside-in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab-mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab-activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM-1) clustering, and monocyte firm adhesion to HLA I Ab-activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1-/- recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM-1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell-monocyte interactions during AMR.
Project description:Humoral alloimmunity, particularly that triggered by preformed antibodies against human leukocyte antigens (HLA), is associated with an increased prevalence of rejection and reduced transplant survival. The high sensitivity of solid phase assays, based on microbeads coated with single antigens (SAB), consolidated them as the gold-standard method to characterize anti-HLA antibodies, ensuring a successful allograft allocation. Mean fluorescence intensity (MFI) provided by SAB is regularly used to stratify the immunological risk, assuming it as a reliable estimation of the antibody-level, but it is often limited by artifacts. Beyond MFI, other properties, such as the complement-binding ability or the IgG1-4 subclass profile have been examined to more accurately define the clinical relevance of antibodies and clarify their functional properties. However, there are still unresolved issues. Neat serum-samples from 20 highly-sensitized patients were analyzed by SAB-panIgG, SAB-IgG1-4 subclass and SAB-C1q assays. All 1:16 diluted serum-samples were additionally analyzed by SAB-panIgG and SAB-IgG1-4 subclass assays. A total of 1,285 anti-HLA antibodies were identified as positive, 473 (36.8%) of which were C1q-binding. As expected, serum-dilution enhanced the correlation between the C1q-binding ability and the antibody-strength, measured as the MFI (rneat = 0.248 vs. rdiluted = 0.817). SAB-subclass assay revealed at least one IgG1-4 subclass in 1,012 (78.8%) positive antibody-specificities. Among them, strong complement-binding subclasses, mainly IgG1, were particularly frequent (98.9%) and no differences were found between C1q- and non-C1q-binding antibodies regarding their presence (99.4 vs. 98.5%; p = 0.193). In contrast, weak or non-C1q-binding subclasses (IgG2/IgG4) were more commonly detected in C1q-binding antibodies (78.9 vs. 38.6%; p < 0.001). Interestingly, a strong association was found between the C1q-binding ability and the IgG1 strength (rIgG1dil = 0.796). Though lower, the correlation between the IgG2 strength and the C1q-binding ability was also strong (rIgG2dil = 0.758), being both subclasses closely related (rIgG1-IgG2 = 0.817). We did not find any correlation with the C1q-binding ability considering the remaining subclasses. In conclusion, we demonstrate that a particular profile of IgG subclasses (IgG1/IgG3) itself does not determine at all the ability to bind complement of anti-HLA antibodies assessed by SAB-C1q assay. It is the IgG subclass strength, mainly of IgG1, which usually appears in combination with IgG2, that best correlates with it.
Project description:Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei-specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing Fc?RIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2-affinity Fc?RIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.
Project description:While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and Fc?RIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.
Project description:Hemolytic anemia resulting from IV Immunoglobulin (IVIG) treatment can be a serious complication, especially for those with underlying conditions with a high level of inflammation and after administration of high IVIG dosages, such as Kawasaki disease (KD), a multisystem vasculitis affecting young children. This hemolysis is caused by antibodies against blood groups A and B, but the precise mechanism for hemolysis is not known. We performed a single center, partly retrospective, partly prospective study of a cohort of 581 patients who received IVIG for treatment of KD from 2006 to 2013. Factors associated with hemolysis were identified through univariable and multivariable logistic regression. Six IVIG preparations were assayed for their hemolytic effect with serological and cellular assays to clarify the mechanism of red cell destruction. During the study period, a sudden increase in the incidence of hemolysis was observed, which coincided with the introduction of new IVIG preparations in North America that contained relatively high titers of anti-A and anti-B. These blood-group-specific antibodies were of the immunoglobulin G2 (IgG2) subclass and resulted in phagocytosis by monocyte-derived macrophages in an Fc?RIIa-dependent manner. Phagocytosis was increased in the presence of proinflammatory mediators that mimicked the inflammatory state of KD. An increased frequency of severe hemolysis following IVIG administration was caused by ABO blood-group-specific IgG2 antibodies leading to Fc?RIIa-dependent clearance of erythrocytes. This increase in adverse events necessitates a reconsideration of the criteria for maximum titer (1:64) of anti-A and anti-B in IVIG preparations.