A specific natural killer cells phenotypic signature associated to long term elite control of HIV infection.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16dimCD56dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV.

Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites.

Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.

Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.

Identification of miRNAs that target Fcγ receptor-mediated phagocytosis during macrophage activation syndrome.

Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile arthritis, accompanied by cytokine storm and hemophagocytosis. In addition, COVID-19-related hyperinflammation shares clinical features of MAS. Mechanisms that activate macrophages in MAS remain unclear. Here, we identify the role of miRNA in increased phagocytosis and interleukin-12 (IL-12) production by macrophages in a murine model of MAS. MAS significantly increased F4/80+ macrophages and phagocytosis in the mouse liver. Gene expression profile revealed the induction of Fcγ receptor-mediated phagocytosis (FGRP) and IL-12 production in the liver. Phagocytosis pathways such as High-affinity IgE receptor is known as Fc epsilon RI -signaling and pattern recognition receptors involved in the recognition of bacteria and viruses and phagosome formation were also significantly upregulated. In MAS, miR-136-5p and miR-501-3p targeted and caused increased expression of Fcgr3, Fcgr4, and Fcgr1 genes in FGRP pathway and consequent increase in phagocytosis by macrophages, whereas miR-129-1-3p and miR-150-3p targeted and induced Il-12. Transcriptome analysis of patients with MAS revealed the upregulation of FGRP and FCGR gene expression. A target analysis of gene expression data from a patient with MAS discovered that miR-136-5p targets FCGR2A and FCGR3A/3B, the human orthologs of mouse Fcgr3 and Fcgr4, and miR-501-3p targets FCGR1A, the human ortholog of mouse Fcgr1. Together, we demonstrate the novel role of miRNAs during MAS pathogenesis, thereby suggesting miRNA mimic-based therapy to control the hyperactivation of macrophages in patients with MAS as well as use overexpression of FCGR genes as a marker for MAS classification.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Elk1 enhances inflammatory cell infiltration and exacerbates acute lung injury/acute respiratory distress syndrome by suppressing Fcgr2b transcription.

OBJECTIVE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant mortality rates. The role of Fcgr2b in the pathogenesis of ALI/ARDS is not fully elucidated. This study aimed to investigate the functions of Fcgr2b in ALI/ARDS and explore its underlying mechanisms. METHODS: Methods: In this study, rat models of ARDS and pulmonary microvascular endothelial cell (PMVEC) injury models were established through the administration of lipopolysaccharide (LPS). The expression levels of Fcgr2b and Elk1 were quantified in both LPS-induced ARDS rats and PMVECs. Subsequent gain- and loss-of-function experiments were conducted, followed by comprehensive assessments of lung tissue for pathomorphological changes, edema, glycogen storage, fibrosis, and infiltration of inflammatory cells. Additionally, bronchoalveolar lavage fluid was analyzed for T-helper 17 (Th17) cell infiltration, inflammatory response, and microvascular permeability to evaluate lung injury severity in ARDS models. Furthermore, the activity, cytotoxicity, apoptosis, and angiogenic potential of PMVECs were assessed to gauge cell injury. The interaction between Elk1 and Fcgr2b was also examined to confirm their regulatory relationship. RESULTS: In the context of LPS-induced ARDS and PMVEC injury, Fcgr2b expression was markedly reduced, whereas Elk1 expression was elevated. Overexpression of Fcgr2b led to a decrease in Th17 cell infiltration and mitigated lung tissue damage in ARDS models, in addition to reducing LPS-induced injury in PMVECs. Elk1 was found to suppress Fcgr2b transcription through the recruitment of histone 3 lysine 9 trimethylation (H3K9me3). Knockdown of Elk1 diminished Th17 cell infiltration and lung tissue damage in ARDS models, and alleviated LPS-induced injury in PMVECs, effects that were reversed upon Fcgr2b upregulation. CONCLUSION: Elk1 negatively regulates Fcgr2b transcription, thereby augmenting the inflammatory response and exacerbating lung injury in LPS-induced ALI/ARDS.

Immunoglobulin genes and severity of COVID-19.

There is tremendous interindividual and interracial variability in the outcome of SARS-CoV-2 infection, suggesting the involvement of host genetic factors. Here, we investigated whether IgG allotypes GM (γ marker) 3 and GM 17, genetic markers of IgG1, contributed to the severity of COVID-19. IgG1 plays a pivotal role in response against SARS-CoV-2 infection. We also investigated whether these GM alleles synergistically/epistatically with IGHG3 and FCGR2A alleles-which have been previously implicated in COVID-19-modulated the extent of COVID-19 severity. The study population consisted of 316 COVID-19 patients who needed treatment in the intensive care unit of Hospital Universitario Central de Asturias. All individuals were genotyped for GM 3/17, IGHG3 hinge length, and FCGR2A rs1801274 A/G polymorphisms. Among the 316 critical patients, there were 86 deaths. The risk of death among critical patients was significantly higher in subjects with GM 17 (IgG1) and short hinge length (IgG3). GM 17-carriers were at almost three-fold higher risk of death than non-carriers (p < 0.001; OR = 2.86, CI 1.58-5.16). Subjects with short hinge length of IgG3 had a two-fold higher risk of death than those with medium hinge length (p = 0.01; OR = 2.16, CI 1.19-3.90). GM 3/3 and IGHG3 (MM) genotypes were less frequent among death vs. survivors (9% vs 36%, p < 0.001) and associated with protective effect (OR = 0.18, 95% CI = 0.08-0.39). This is the first report implicating IgG1 allotypes in COVID-19-spurred death. It needs to be replicated in an independent study population.

FCGR3A-V158F gene polymorphism: A potential predictor for rituximab dosing optimization in Chinese patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Rituximab (RTX), an anti-CD20 monoclonal antibody, has shown promise in managing neuromyelitis optica spectrum disorders (NMOSD) by depleting B cells and reducing relapses. However, there is no consensus on the optimal RTX dosing regimen, and genetic factors, such as FCGR3A-V158F polymorphism, may influence treatment outcomes. This study investigates how FCGR3A-V158F genotypes influence RTX efficacy in Chinese NMOSD patients under varying dosing regimens and aims to optimize treatment protocols. METHODS: We conducted a retrospective analysis of 25 Chinese NMOSD patients treated with RTX, grouped into standardized and low-dosage regimens. FCGR3A-V158F genotypes were determined, and treatment responses were evaluated, including relapse rates, time to first relapse (TFR), B-cell depletion, dose adjustments, and treatment retention. RESULTS: Among all patients, 15 received standardized dosages, while 10 received varied induction doses (500 mg to 1200 mg) in low-dose regimens. For FCGR3A-V158F genotypes, 15 had the FF genotype, and 10 were V carriers (3 VV genotype, 7 VF genotype). Regardless of dosing, FF genotype patients had a higher relapse rate post-RTX treatment compared to V carriers (P < 0.05). None of the 3 VV genotype patients in either dose group experienced relapses post-RTX. In both dose groups, FF genotype patients had significantly shorter TFR and required more RTX dose adjustments post-RTX treatment compared to V carriers in the standardized dosage group (P < 0.05). FF genotype patients in the low dosage group were more likely to experience insufficient B-cell depletion, had lower treatment retention rates, and more discontinuations than V carriers in the standardized dosage group (P < 0.05). Insufficient B-cell depletion significantly predicted clinical relapses after RTX treatment (P < 0.05). In survival analysis, FF genotype patients, regardless of dosing, experienced earlier relapses post-RTX treatment (P < 0.05). CONCLUSIONS: This study highlights the importance of RTX dosage selection in NMOSD treatment, particularly for FCGR3A-FF genotype patients. Standard-dose RTX therapy with vigilant monitoring of peripheral blood B-cell levels is recommended for these individuals to optimize treatment efficacy.

Novel peptide-based oncolytic vaccine for enhancement of adaptive antitumor immune response via co-engagement of innate Fcγ and Fcα receptors.

BACKGROUND: Cancer immunotherapy relies on using the immune system to recognize and eradicate cancer cells. Adaptive immunity, which consists of mainly antigen-specific cytotoxic T cells, plays a pivotal role in controlling cancer progression. However, innate immunity is a necessary component of the cancer immune response to support an immunomodulatory state, enabling T-cell immunosurveillance. METHODS: Here, we elucidated and exploited innate immune cells to sustain the generation of antigen-specific T cells on the use of our cancer vaccine platform. We explored a previously developed oncolytic adenovirus (AdCab) encoding for a PD-L1 (Programmed-Death Ligand 1) checkpoint inhibitor, which consists of a PD-1 (Programmed Cell Death Protein 1) ectodomain fused to an IgG/A cross-hybrid Fc. We coated AdCab with major histocompatibility complex (MHC-I)-restricted tumor peptides, generating a vaccine platform (named PeptiCab); the latter takes advantage of viral immunogenicity, peptide cancer specificity to prime T-cell responses, and antibody-mediated effector functions. RESULTS: As proof of concept, PeptiCab was used in murine models of melanoma and colon cancer, resulting in tumor growth control and generation of systemic T-cell-mediated antitumor responses. In specific, PeptiCab was able to generate antitumor T effector memory cells able to secrete various inflammatory cytokines. Moreover, PeptiCab was able to polarize neutrophils to attain an antigen-presenting phenotype by upregulating MHC-II, CD80 and CD86 resulting in an enhanced T-cell expansion. CONCLUSION: Our data suggest that exploiting innate immunity activates T-cell antitumor responses, enhancing the efficiency of a vaccine platform based on oncolytic adenovirus coated with MHC-I-restricted tumor peptides.

Essential role of local antibody distribution in mediating bone-resorbing effects.

The link between antibodies and bone mass is debated. Activated IgG, which interacts directly with Fc gamma receptors, stimulates osteoclastogenesis in vitro, and local injection in immune-activated mice leads to bone loss. Multiple myeloma patients with high serum IgG levels have induced osteoclast activation and display bone loss. In addition, bone loss has been linked to serum autoantibodies in autoimmune diseases, including anti-citrullinated protein antibodies (ACPA) in individuals with rheumatoid arthritis (RA). Whether serum IgG or autoantibodies regulate bone mass under healthy conditions is poorly studied. In elderly men, neither serum levels of polyclonal IgG nor autoantibody were associated with areal bone mineral density in the MrOS Sweden study. Repetitive systemic injections of high-dose polyclonal IgG complexes in mice did not exert any discernible impact on bone mineral density. However, repetitive local intra-articular injection of the same IgG complexes led to a localized reduction of trabecular bone density. These results indicate antibodies may only impact bone density when close to the bone, such as within the synovial joint.

Therapeutic strategies in FcγIIA receptor-dependent thrombosis and thromboinflammation as seen in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT).

INTRODUCTION: Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. AREAS COVERED: Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. EXPERT OPINION: Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.

Persistence of a Skewed Repertoire of NK Cells in People with HIV-1 on Long-Term Antiretroviral Therapy.

HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH.

Genetic variation of FcγRIIa induces higher uptake of Leishmania infantum and modulates cytokine production by adherent mononuclear cells in vitro.

Introduction: Single nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production. Methods: We genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants. Results and discussion: In participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.

Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding.

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.

Regulatory mechanism of FCGR2A in macrophage polarization and its effects on intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co-expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein-protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF-κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. KEY POINTS: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD). FCGR2A knockdown mitigates IDD in cellular and animal models. Single-cell RNA-sequencing uncovers diverse macrophage subpopulations in degenerated IVDs. This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization. This study confirms the role of the NF-κB/STAT3 pathway in regulating macrophage polarization in IDD.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Fcgr2b and Fcgr3 are the major genetic factors for cartilage antibody-induced arthritis, overriding the effect of Hc encoding complement C5.

Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b-- mice but not in Fcgr3-- mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b++.Hc** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b--.Hc** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency.

A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.