FCGR2A Research Articles

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles.

Genetic variation in the FCGR3B gene is responsible for different variants of human neutrophil antigen 1 (HNA-1). Laboratory techniques currently utilized for routine HNA-1 genotyping, predominantly PCR-sequence-specific primer (PCR-SSP) and PCR-sequence-based typing (PCR-SBT), lack specificity for FCGR3B. This study compares the capabilities and limitations of existing technologies including an in-house TaqMan PCR, a commercial PCR-SSP test, PCR-SBT and multiplex ligation-dependent probe amplification (MLPA) with those of a long-read nanopore sequencing assay. Testing was performed with both related and unrelated Danish samples with different copy numbers and/or rare alleles. Long-read nanopore sequencing was validated by blind testing of ten English samples. The results showed that FCGR3B copy numbers correlate with a dose-dependent distribution of alleles that complicates genotyping by TaqMan PCR, PCR-SSP and PCR-SBT, due to co-amplification of the homologous FCGR3A gene. MLPA can correctly quantify the dose-dependent distribution but not detect novel variants. Long-read nanopore sequencing showed high specificity for FCGR3B and was able to detect dosage-dependent distribution, and rare and novel variants that were previously not described. Current HNA-1 genotyping methods cannot produce unambiguous allele-level results, whereas long-read nanopore sequencing has shown the potential to resolve observed ambiguities, identify new HNA-1 variants and allow definitive allele assignment.

Botensilimab, an Fc-enhanced anti-CTLA-4 antibody, is effective against tumors poorly responsive to conventional immunotherapy.

Conventional immune checkpoint inhibitors (ICI) targeting CTLA-4 elicit durable survival, but primarily in patients with immune-inflamed tumors. Although the mechanisms underlying response to anti-CTLA-4 remain poorly understood, Fc-gamma receptor (FcγR) IIIA co-engagement appears critical for activity, potentially explaining the modest clinical benefits of approved anti-CTLA-4 antibodies. We demonstrate that anti-CTLA-4 engineered for enhanced FcγR affinity leverages FcγR-dependent mechanisms to potentiate T cell responsiveness, reduce intratumoral Tregs, and enhance antigen presenting cell activation. Fc-enhanced anti-CTLA-4 promoted superior efficacy in mouse models and remodeled innate and adaptive immunity versus conventional anti-CTLA-4. These findings extend to patients treated with botensilimab, an Fc-enhanced anti-CTLA-4 antibody, with clinical activity across multiple poorly immunogenic and ICI treatment-refractory cancers. Efficacy was independent of tumor neoantigen burden or FcγRIIIA genotype. However, FcγRIIA and FcγRIIIA expression emerged as potential response biomarkers. These data highlight the therapeutic potential of Fc-enhanced anti-CTLA-4 antibodies in cancers unresponsive to conventional ICI therapy.

SingleNucleotide Polymorphisms as Biomarkers of Mepolizumab and Benralizumab Treatment Response in Severe Eosinophilic Asthma.

The most promising treatment options for severe uncontrolled asthma (SUA) have emerged in recent years with the development of monoclonal antibodies for blocking selective targets responsible for the underlying inflammation, such as mepolizumab and benralizumab. However, there is variability in treatment response that is not fully controlled. The variability of the response to mepolizumab and benralizumab could be influenced by single-nucleotide polymorphisms (SNPs), and it would be useful to detect these and use them as predictive biomarkers of response. We conducted a retrospective observational cohort study of 72 Caucasian patients recruited from a tertiary hospital with severe uncontrolled eosinophilic asthma treated with mepolizumab and benralizumab. Polymorphisms in the IL5 (rs4143832, rs17690122), RAD50 (rs11739623, rs4705959), IL1RL1 (rs1420101, rs17026974, rs1921622), GATA2 (rs4857855), IKZF2 (rs12619285), FCGR2A (rs1801274), FCGR2B (rs3219018, rs1050501), FCGR3A (rs10127939, rs396991), FCER1A (rs2251746, rs2427837), FCER1B (rs1441586, rs573790, rs569108), and ZNF415 (rs1054485) genes were analyzed by real-time polymerase chain reaction (PCR) using Taqman probes. The response was analyzed after 12 months of treatment. In patients under mepolizumab treatment, a treatment response defined as a reduction in exacerbations was associated with ZNF415 rs1054485-T (p = 0.042; OR = 5.33; 95% CI = 1.06-30.02), treatment response defined as a reduction in oral corticosteroids use was associated with the number of exacerbations in the previous year (p = 0.029; OR = 3.89; 95% CI = 1.24-14.92), and treatment response defined as improvement in lung function was associated with the age at the beginning of biological therapy (p = 0.002; OR = 1.10; 95% CI = 1.04-1.18), FCER1B rs569108-AA (p < 0.001; OR = 171.06; 95% CI = 12.94-6264.11), and FCER1A rs2427837-A (p = 0.021; OR = 8.61; 95% CI = 1.71-76.62). On the other hand, in patients under benralizumab treatment, treatment response, defined as a reduction in exacerbations, was associated with ZNF415 rs1054485-T (p = 0.073; OR = 1.3 × 108; 95% CI = 1.8 × 10-19-NA), FCER1B rs569108-AA (p = 0.050; OR = 11.51; 95% CI = 1.19-269.78), allergies (p = 0.045; OR = 4.02; 95% CI = 1.05-16.74), and sex (p = 0.028; OR = 4.78; 95% CI = 1.22-20.63); and treatment response defined as improvement in lung function was associated with polyposis (p = 0.027; OR = 9.16; 95% CI = 1.58-91.4), IKZF2 rs12619285-AA (p = 0.019; OR = 9.1; 95% CI = 1.7-75.78), IL5 rs4143832-T (p = 0.017; OR = 11.1; 95% CI = 1.9-112.17), and FCER1B rs1441586-C (p = 0.045; OR = 7.81; 95% CI = 1.16-73.45). The results of this study show the potential influence of the studied polymorphisms on the response to mepolizumab and benralizumab and the clinical benefit that could be obtained by defining predictive biomarkers of treatment response.

NK Cytotoxicity Mediated by NK-92 Cell Lines Expressing Combinations of Two Allelic Variants for FCGR3.

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors, respectively. Allelic variants of the FCGR3A gene include the high-affinity single-nucleotide polymorphism (SNP) rs396991 (V176F), which is associated with the efficacy of monoclonal antibody (mAb) therapies, and the SNP rs10127939 (L66H/R). The contribution of FCGR3A SNPs to NK cell effector functions remains controversial; therefore, we generated a panel of eight NK-92 cell lines expressing specific combinations of these SNPs and tested their cytotoxicities. NK-92 cells were stably transfected with plasmids containing different combinations of FCGR3A SNPs. Messenger RNA and FcγRIIIa/CD16 cell surface expressions were detected using new generation sequencing (NGS) and flow cytometry, respectively. All FcγRIIIa/CD16-transfected NK-92 cell lines exhibited robust ADCC against three different target cell lines with minor differences. In addition, enhanced direct NK cytotoxicity against K562 target cells was observed, suggesting a mechanistic role of FcγRIIIa/CD16 in direct NK cytotoxicity. In conclusion, we generated eight FcγRIIIa/CD16-transfected NK-92 cell lines carrying different combinations of two of the most studied FCGR3A SNPs, representing the major genotypes described in the European population. The functional characterization of these cell lines revealed differences in ADCC and direct NK cytotoxicity that may have implications for the design of adoptive cancer immunotherapies using NK cells and tumor antigen-directed mAbs.

Natural Killer Cell Presence in Antibody-Mediated Rejection.

Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm2 glomerular area (NKglom) and PTC per mm2 cortical area (NKPTC) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NKglom Spearman's correlation coefficient [SCC] = 0.55, p < 0.001, NKPTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMRprob: NKglom 0.59, NKPTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NKPTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR.

Agalactosyl IgG induces liver fibrogenesis via Fc gamma receptor 3a on human hepatic stellate cells.

The relevance of aberrant serum IgG N-glycosylation in liver fibrosis has been identified; however, its causal effect remains unclear. Because hepatic stellate cells (HSCs) contribute substantially to liver fibrosis, we investigated whether and through which mechanisms IgG N-glycosylation affects the fibrogenic properties of HSCs. Analysis of serum IgG1 N-glycome from 151 patients with chronic hepatitis B or liver cirrhosis revealed a positive correlation between Ishak fibrosis grading and IgG1 with agalactosyl N-glycoforms on the crystallizable fragment (Fc). Fc gamma receptor (FcγR) IIIa was observed in cultured human HSCs and HSCs in human liver tissues, and levels of FcγRIIIa in HSCs correlated with the severity of liver fibrosis. Additionally, agalactosyl IgG treatment caused HSCs to have a fibroblast-like morphology, enhanced migration and invasion capabilities, and enhanced expression of the FcγRIIIa downstream tyrosine-protein kinase SYK. Furthermore, agalactosyl IgG treatment increased fibrogenic factors in HSCs, including transforming growth factor (TGF)-β1, total collagen, platelet-derived growth factor subunit B and its receptors, pro-collagen I-α1, α-smooth muscle actin, and matrix metalloproteinase 9. These effects were more pronounced in HSCs that stably expressed FCGR3A and were reduced in FCGR3A knockout cells. Agalactosyl IgG and TGF-β1 each increased FCGR3A in HSCs. Furthermore, serum TGF-β1 concentrations in patients were positively correlated with agalactosyl IgG1 levels and liver fibrosis severity, indicating a positive feedback loop involving agalactosyl IgG, HSC-FcγRIIIa, and TGF-β1. In conclusion, agalactosyl IgG promotes fibrogenic characteristics in HSCs through FcγRIIIa. © 2024 The Pathological Society of Great Britain and Ireland.

Correlations of FCGR2A 131R/H and FCGR3A 158V/F Polymorphisms with the Susceptibility of Peri-implantitis in Chinese Han Population.

The purpose of the study is to investigate the relationship of peri-implantitis (PI) with FCGR2A and FCGR3A gene polymorphisms. One hundred and forty-four patients with PI and 136 patients without PI infection were selected. Gingival crevicular fluid samples were collected from the two groups. The FCGR2A and FCGR3A polymorphism in the two groups were measured. All volunteers were evaluated for periodontal status. The effect of polymorphisms on PI susceptibility was investigated by chi-square analysis and logistic regression. The frequency of FCGR2A rs1801274 GG genotype of PI group was higher than that of the control group, while the GA and AA genotype carriers were less in PI group. After adjusting for other clinical indicators, rs1801274 GA genotype, AA genotype, and the A allele were still negatively correlated with the onset of PI. FCGR3A rs396991 polymorphism was not associated with PI. FCGR2A rs1801274 polymorphism was significantly associated with PI in the Chinese Han population, and GG genotype might be a genetic risk factor for PI.

Genetic predisposition to vaccine-induced immune thrombotic thrombocytopenia: is there a family link?

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare life-threatening thrombotic reaction to COVID-19 vaccines. Case description: Two young male first cousins, with a family history of idiopathic thrombocytopenic purpura, developed VITT after the Ad26.COV2.S vaccine. Both had a favourable clinical and analytical outcome. We investigated the genetic factors that could be associated with a genetic predisposition to VITT. Conclusions: There are no published cases where the VITT patients were relatives. The genetic study did not reveal any likely pathogenic variants, although the prevalent polymorphism c.497A&gt;G (p.(His166Arg)) in the FCGR2A gene was found in a homozygous state. More studies are required to better understand VITT’s pathophysiology and any underlying genetic predispositions.

Genetic biomarkers for diagnosis and treatment of spondyloarthropathies in Moroccan population

BackgroundThe spondyloarthropathies (SpA) are inflammatory rheumatologic diseases whose pathogenesis comes from interaction between genetic and environmental factors. MethodsThis is a case-control analysis that aims to explore in Moroccan population the relationship of some allelic polymorphisms of the CTLA-4 CT60, FCGR3A-158G/T SNPs and HLA-B locus with SpA disease. Eighty-four Spa patients grouped according to pain site and 95 healthy individuals from Morocco were typed by sequence-based-typing methods. ResultsThe CTLA-4 CT60 investigation showed a significantly increased frequency of rs11571319 A allele in male patients with sacroiliitis (pcorr.=0.048, OR=2.756) and a protective role of A/G genotype in SPA patients with thoracic spine (pcorr.=0.016, OR=0.055) and peripheral joints pain (pcorr.=0.036 OR=0.068), compared to controls. Furthermore, a significant increase of A/A in rs3087243 was shown in male patients with peripheral joints pain (p = 0.035 OR = 4.45). Data from FCGR3A-158 analysis evidenced a reduction of T/T (F/F) genotype in patients with thoracic spine pain (p = 0.030, OR = 0.09) and a higher frequency of G/T (F/V) (p = 0.038 OR = 5.045) towards controls. With regard to HLA-B locus, we noticed a protective role of HLA-B*45:01 (pcorr. = 0.033,OR = 0.041) and an increased frequency of HLA-B*51 alleles (51:01, 51:02, 51:08, 51:09, 51:29) and B*52:01:01 (p = 0.041,OR = 2.714), in addition to HLA-B*27 alleles (p = 0.028, OR = 2.593) commonly associated with AS pathologies. ConclusionsOur data suggest the role of some additional CTLA-4, FCGR3A and HLA-B gene polymorphisms in SPA pathogenesis of Moroccan population, useful as new diagnostic biomarkers and targets for immunotherapy.

Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP

AbstractFc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody’s Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum–sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

Association of NK cell subsets and cytotoxicity with FCGR3A gene polymorphism in functional NK cell deficiency.

The purpose of this study was to assess the association between clinical, laboratory, and functional analyses and polymorphism in the FCGR3A gene in individuals with functional NK cell deficiency. A total of 15 functional NK cell deficiency patients and 10 age-matched healthy controls underwent NK cell subgroup, cytotoxicity, and FCGR3A whole-exome analysis with next-generation sequencing. Three different NK cell subsets (CD56brightCD16neg, CD56brightCD16int, and CD56dimCD16hi) were identified. No statistically significant difference was found in the ratio of CD56brightCD16neg cells between patients and controls. CD56brightCD16int and CD56dimCD16hi ratios were found to be significantly lower in patients. As a result of NK cell cytotoxicity analysis, a proportional decrease of K562 amount between patients and controls was found to be statistically significant (p<0.001). In the FCGR3A whole-exome analysis, all patients were found to be homozygous mutant for the c.526G > T (p.V176F) in exon 4, while three patients were homozygous wild type and 12 patients were heterozygous for the c.197T>A (p.L66H) in exon 3. In this study, a group of pediatric patients with suspected functional NK cell deficiency were evaluated and the findings indicated that NK subsets, cytotoxicity results, and FCGR3A gene polymorphism were found to be correlated with the clinical features. We conclude that this kind of study might contribute to follow-up the patients in time.

A Non-Coding Fc Gamma Receptor Cis-Regulatory Variant within the 1q23 Gene Cluster Is Associated with Plasmodium falciparum Infection in Children Residing in Burkina Faso

Antibodies play a crucial role in activating protective immunity against malaria by interacting with Fc-gamma receptors (FcγRs). Genetic variations in genes encoding FcγRs can affect immune cell responses to the parasite. In this study, our aim was to investigate whether non-coding variants that regulate FcγR expression could influence the prevalence of Plasmodium falciparum infection. Through bioinformatics approaches, we selected expression quantitative trait loci (eQTL) for FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes encoding FcγRs (FCGR), in whole blood. We prioritized two regulatory variants, rs2099684 and rs1771575, located in open genomic regions. These variants were identified using RegVar, ImmuNexUT, and transcription factor annotations specific to immune cells. In addition to these, we genotyped the coding variants FCGR2A/rs1801274 and FCGR2B/rs1050501 in 234 individuals from a malaria-endemic area in Burkina Faso. We conducted age and family-based analyses to evaluate associations with the prevalence of malarial infection in both children and adults. The analysis revealed that the regulatory rs1771575-CC genotype was predicted to influence FCGR2B/FCGR2C/FCGR3A transcripts in immune cells and was the sole variant associated with a higher prevalence of malarial infection in children. In conclusion, this study identifies the rs1771575 cis-regulatory variant affecting several FcγRs in myeloid and neutrophil cells and associates it with the inter-individual capacity of children living in Burkina Faso to control malarial infection.

Inebilizumab reduces neuromyelitis optica spectrum disorder risk independent of FCGR3A polymorphism.

Inebilizumab, a humanized, glycoengineered, IgG1 monoclonal antibody that depletes CD19+ B-cells, is approved to treat aquaporin 4 (AQP4) IgG-seropositive neuromyelitis optica spectrum disorder (NMOSD). Inebilizumab is afucosylated and engineered for enhanced affinity to Fc receptor III-A (FCGR3A) receptors on natural killer cells to maximize antibody-dependent cellular cytotoxicity. Previously, the F allele polymorphism at amino acid 158 of the FCGR3A gene (F158) was shown to decrease IgG-binding affinity and reduce rituximab (anti-CD20) efficacy for NMOSD attack prevention. In contrast, our current findings from inebilizumab-treated NMOSD patients indicate similar clinical outcomes between those with F158 and V158 allele genotypes.

Apoptosis and inflammatory genes variants in primary non-response to anti-TNF therapy in Crohn's disease patients.

Anti-TNF therapy has indeed revolutionized the treatment of Crohn's disease, leading to higher rates of response and remission in patients. However, a significant proportion of 20-40% of patients do not respond to the initial therapy, others experience a secondary loss of response with ongoing treatment. Adverse drug reactions also occur in some patients. The effectiveness of anti-TNF treatment may be influenced by genetic variability, including FCGR3A, ADAM17, TNFRSF1A, TNFRSF1B, FAS, FASL, IL1B, CASP9 , and MIF genes. In this article, we provide an overview of the current knowledge and findings in the pharmacogenetics of anti-TNF drugs in CD focusing on the aspect of apoptosis and inflammatory genes variants in primary non-response. Pharmacogenetic investigations have been conducted to identify genetic markers that can predict response to anti-TNF therapy. However, large multi-center validation studies and multi-loci algorithms development are required to effectively prognose the treatment effect. The identification of predictive markers of response to anti-TNF therapy can help clinicians make informed decisions about treatment options and minimize adverse drug reactions in patients.

CLEC18A Impairs Phagocytosis by Reducing FcγRIIA Expression and Arresting Autophagosome-Lysosome Fusion.

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome.

Analysis of FCGR3A-p.176Val variants in women with recurrent pregnancy loss and the association with CD16a expression and anti-HLA antibody status

Natural Killer (NK) cells have been implicated in recurrent pregnancy loss (RPL). The p.Val176Phe (or Val158Phe) Single Nucleotide Polymorphism (SNP) in the FCGR3A gene encoding the FcγRIIIA or CD16a receptor has been associated with an enhanced affinity for IgG and stronger NK-mediated antibody-dependent cellular cytotoxicity. We hypothesized that the presence of at least one p.176Val variant associates with RPL and increased CD16a expression and alloantibodies e.g., against paternal human leukocyte antigen (HLA). In 50 women with RPL, we studied frequencies of the p.Val176Phe FCGR3A polymorphisms. Additionally, CD16a expression and anti-HLA antibody status were analyzed by flowcytometry and Luminex Single Antigens. In woman with RPL, frequencies were: 20% (VV), 42% (VF) and 38% (FF). This was comparable to frequencies from the European population in the NCBI SNP database and in an independent Dutch cohort of healthy women. NK cells from RPL women with a VV (22,575 [18731-24607]) and VF (24,294 [20157-26637]) polymorphism showed a higher expression of the CD16a receptor than NK cells from RPL women with FF (17,367 [13257-19730]). No difference in frequencies of the FCGR3A-p.176 SNP were detected when comparing women with or without class I and class II anti-HLA antibodies. Our study does not provide strong evidence for an association between the p.Val176Phe FCGR3A SNP and RPL.

Association of the ILR1 and FAS gene variants with primary nonresponse to anti-tumor necrosis factor therapy in patients with Crohn disease.

Crohn disease (CD) is a chronic inflammatory disease characterized by an uncontrolled immune response of the intestinal mucosal cells to antigens derived from the gut lumen. Specifically, the introduction of anti-tumor necrosis factor (TNF) drugs has changed the approach to the treatment of inflammatory bowel disease, and set new therapeutic goals, such as that of controlling clinical symptoms while simultaneously achieving complete endoscopic and mucosal remission. The mechanisms of action of anti-TNF drugs-and consequently the mechanisms of resistance to anti‑TNF therapy-are unknown. Our study was an attempt to discover whether the potential mechanism of nonresponse may be conditioned by polymorphisms in the genes involved in independent inflammatory or apoptotic pathways. The study included 196 diagnosed and clinically characterized Polish patients with CD treated with anti‑TNF therapy. Variants rs7539036, rs2041747, rs5746053, rs5746054, rs1061624, rs1143634, rs7896789, and rs55790676 of the FCGR3A, IL1R, TNFRSF1B, IL1B, FAS, and ADAM17 genes were genotyped using Sanger sequencing, and analyzed in the context of response to biologic treatment. We observed that 33 patients (16.8%) did not respond to the therapy, which was associated with carrying the rs2041747 G allele variant of the ILR1 gene (odds ratio [OR], 3.72; P = 0.009). Moreover, the presence of the FAS rs7896789 homozygous CC genotype correlated with increased susceptibility to the lack of response to the anti‑TNF therapy (OR, 15.22; P = 0.003), whereas TT was identified as a potentially protective genotype. In patients with CD treated with anti‑TNF drugs, complex pathways with multigene conditioning participate in the mechanism underlying treatment resistance. The genes involved in apoptosis, FAS and ILR1, seem to play an essential role in the lack of response to the treatment, and would be interesting objects of further population and functional research.

Abstract P5-02-40: Anthracycline improves the outcome of neoadjuvant trastuzumab-treated breast cancer patients with FcγRIIIA F/F genotype by decreasing the ratios of CD8+PD1+ and CD68+PDL1+ cells

Abstract Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the most important mechanisms of trastuzumab. Fragment C Gamma receptor (FcγR) IIA and IIIA polymorphisms influence the affinity of immunoglobin G (IgG). Recently, FcγRIIA and FcγRIIIA polymorphisms have identified with the efficacy of trastuzumab. However, whether FcγR polymorphisms are associated with the efficacy of trastuzumab in the neoadjuvant setting was unclear. Patients and methods: We retrospectively enrolled 101 patients with HER2-positive breast cancer receiving chemotherapy plus trastuzumab at least four cycles as neoadjuvant therapy and mastectomy in Sun Yat-sen university cancer center from May 2015 to March 2021. Among them, twenty patients were excluded because lacking of blood samples. Polymorphisms of FcγRIIA(rs1801274) and FcγRIIIA(rs396991) were examined by nested polymerase chain reaction (PCR) and sanger sequencing. Lastly, we performed multiple immunohistochemistry (mIHC) to examine the expressions of CD8, CD68, CD57, PD1 and PDL1. Results: Blood samples (n=81) were successfully detected. No significant differences between FcγRIIA/FcγRIIIA genotypes and clinical characteristics, including age, clinical stages, menstrual status, molecular subtyping, treatment and pathological complete response (pCR) rate. In paclitaxel-based treatment subgroup (n=34), FcγRIIIA polymorphism was significantly correlated with pCR rate (P&amp;lt; 0.05,Table1), moreover, the disease-free survivals (DFS) in FcγRIIIA-158V carriers subgroup (high affinity) were significantly longer than that in FcγRIIIA-158 F/F genotype group (low affinity) (P=0.036), while that was not significant difference in patients with anthracycline-based treatment (n=47, P=0.248), indicating that anthracycline enhanced the efficacy of neoadjuvant therapy in patients with FcγRIIIA F/F genotype(low affinity). So far, there is no recurrence events in paclitaxel-based treatment group with FcγRIIIA-158V carriers genotype. The mIHC showed that the ratios of stroma CD8+PD1+ and CD68+PDL1+ cells were significantly higher in anthracycline-based treatment group (Table 2), respectively, indicating that anthracycline improved the efficacy of neoadjuvant targeted therapy by decreasing the ratios of exhausted immune cells. Conclusions: FcγRIIIA-158V genotype is associated with better outcome in HER2-positive breast cancer patients who received paclitaxel combined with trastuzumab as neoadjuvant therapy. Anthracycline improves the outcome of neoadjuvant trastuzumab-treated breast cancer patients with FcγRIIIA F/F genotype by decreasing the ratios of CD8+PD1+ and CD68+PDL1+ cells. Correlation between FcγRIIIA and pCR Correlation between FcγRIIIA and pCR in total population and paclitaxel-based treatment population. Ratios of immune cells Ratios of immune cells in anthracycline-based and paclitaxel-based treatment group. Citation Format: Kaping Lee, Rongzhen Luo, Qianyi Lu, Shusen Wang, Fei Xu. Anthracycline improves the outcome of neoadjuvant trastuzumab-treated breast cancer patients with FcγRIIIA F/F genotype by decreasing the ratios of CD8+PD1+ and CD68+PDL1+ cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-40.

FcγRIIIA activation-mediated up-regulation of glycolysis alters MDSCs modulation in CD4+ T cell subsets of Sjögren syndrome

Our and other researchers’ previous studies found that myeloid-derived suppressor cells (MDSCs) were increased, and these MDSCs, supposed to play immunosuppressive roles, showed significant pro-inflammatory effects in Sjögren’s syndrome (SS). However, the key factors and potential mechanisms leading MDSCs to be inflammatory remain unclear. In this study, we found that MDSCs from SS patients were positively correlated with the percentages of Th17 cells, disease activity and serum autoantibodies, and showed higher levels of Fc gamma receptor (FcγR) IIIA and glycolysis. Most importantly, SS MDSCs or heat-aggregated IgG (HAIG)-treated MDSCs down-regulated Th1/Th2 ratio and up-regulated Th17/Treg ratio, which could be obviously rescued by IgG monomer or glycolysis inhibitor 2-DG. As well, the levels of FcγRIV and glycolysis in MDSCs and the ratio of Th17/Treg were increased, and the ratio of Th1/Th2 was decreased in SS-like NOD mice. Our study indicated that MDSCs showed pro-inflammatory phenotypes by disturbing CD4+ T-cell balances in SS. The pro-inflammatory effects of MDSCs might be directly linked to the enhanced glycolysis mediated by FcγRIIIA activation.