Arthritis in K/BxN mice is provoked by pathogenic autoantibodies to glucose-6-phosphate isomerase (G6PI), which is a ubiquitously expressed enzyme that is present in cells, in the circulation and on articular cartilage. When G6PI autoantibodies (auto-Abs) deposit on the articular cartilage of K/BxN mice, arthritis ensues due to the activation of various components of the innate immune system. Recent studies have investigated the in vivo efficacy of recombinant fragment-crystallizable (Fc) protein-based therapeutics. Many recombinant Fc proteins evaluated provide protection against inflammation in mouse models of arthritis, such as the K/BxN serum-transfer model. More recently, rFc-µTP-L309C, a recombinant human IgG1-Fc with an additional point mutation at position L309C fused to the human IgM tailpiece to form a hexamer, has been shown to ameliorate the arthritis in K/BxN mice. Additional studies have shown that rFc-µTP-L309C has multiple effects that work together to ameliorate the arthritis, including inhibition of neutrophil migration into the joint, inhibition of IL-1β production, downregulation of Th1 and Th17 cells, and increases in T regulatory cells and synovial fluid IL-10. In this work, rFc-µTP-L309C was shown to effectively prevent arthritis in the K/BxN serum-transfer model, significantly downregulate inflammatory cytokines/chemokines, and ameliorate the arthritis in the endogenous K/BxN model. This amelioration of the arthritis was associated with a significant decrease in autoantibody levels, which was independent of the neonatal Fc receptor (FcRn). rFc-µTP-L309C was shown to specifically inhibit G6PI autoantibody secretion from B-cells with a concomitant increase in TGFβ and decrease in B-cell activating factor (BAFF). These new findings suggest that rFc-µTP-L309C may provide a therapeutic benefit for other antibody-mediated autoimmune diseases through its effects on B-cells.

Background: Currently, the duration of tourniquet time in total knee arthroplasty is chosen by the surgeons and varies between 0 and 120 minutes. Studies evaluating the effect of tourniquet time in this operation are lacking. The purpose of this study was, therefore, to determine whether the duration of tourniquet-induced limb ischemia during total knee arthroplasty influences reperfusion injury, resulting in pain, swelling and the release of proinflammatory markers. Methods: In 40 patients undergoing total knee arthroplasty, tourniquet was applied for up to 30 minutes (group A, short tourniquet) or 90-120 minutes (group B, long tourniquet). Postoperative pain and swelling served as primary outcome parameters. The levels of pro- and anti-inflammatory markers (D-dimers, C3a, C5a, TAT, fetuin-A, PAI-I/tPA complexes, CK-MM, IP10, M-CSF, MIG, MIP-1α, and sC5b9) before surgery and 4 hours, 24 hours and 48 hours after surgery, were used as secondary outcome parameters. Results: Patients in group B, with the long tourniquet time, required patient-controlled intravenous analgesia more frequently than group A patients (47% versus 5%, group B vs. group A, p < 0.0001). However, there were no differences in numeric rating pain scale (NRS) scores and calf circumference between groups A and B. In group B, a significantly higher increase of C3a levels between 4 h and 48 h, a significantly higher increase for MIG between 4 h and 48 h as well as 24 h and 48 h, and a significantly higher increase in M-CSF levels between 24 h and 48 h were observed when compared to group A. Conclusions: Tourniquet times between 90 and 120 minutes, despite currently being accepted in the clinical setting, were associated with an increased need for intravenous analgesia and higher increase of the pro-inflammatory markers C3a, MIG, and M-CSF, suggesting a more pronounced ischemia/reperfusion injury with tourniquet times longer than 90 minutes.

High-dose intravenous immunoglobulin (IVIG) is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the fragment crystallizable (Fc) portion. Further, recent data indicate that recombinant multimeric Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of different recombinant human IgG1 Fc molecules with increasing valency and avidity, combined with mutations to increase binding affinity to complement protein C1q. These molecules were investigated for their potential dual antagonism: to antagonize Fcγ receptor (FcγR) effector functions (Ab-dependent cellular phagocytosis) in vitro, and to inhibit the activation of the classical complement pathway. C1q-binding mutants demonstrated an exponential increase in potency to inhibit the classical pathway in correlation with increasing multimerization. Importantly, in contrast to other multimeric Fc constructs such as Fc hexamers, no generation of complement C4a was observed. Reducing the binding affinity to FcγRIIB resulted in a half-life extension of the trivalent hIgG1-Fc molecules in human neonatal Fc receptor transgenic (hFcRn Tg) mice. Our data demonstrate a potent anti-inflammatory effect of recombinant human IgG1-Fc C1q-binding mutants in vitro and in vivo, mediated by blockade of FcγRs and inhibition of complement activation.

Background: Currently, the duration of tourniquet time in total knee arthroplasty is chosen by the surgeons and varies between 0 and 120 min. Studies evaluating the effect of tourniquet time in this surgery are heterogeneous, and there is limited information on molecular/complement profiling. The purpose of this study was, therefore, to determine whether the duration of tourniquet-induced limb ischemia during total knee arthroplasty influences reperfusion injury, resulting in pain, swelling, and the release of pro-inflammatory markers. Methods: In 40 patients undergoing total knee arthroplasty, a tourniquet was applied for up to 30 min (group A, short tourniquet) or 90-120 min (group B, long tourniquet). Postoperative pain and swelling served as primary outcome parameters. The levels of pro- and anti-inflammatory markers before surgery and 4 h, 24 h, and 48 h after surgery were used as secondary outcome parameters for exploratory testing. Results: There were no differences in numeric rating pain scale (NRS) scores and calf circumference between groups A and B. Patients in group B required patient-controlled intravenous analgesia more frequently than group A patients (47% versus 5%, group B vs. group A, p < 0.0001). In group B, a significantly higher increase in C3a and MIG levels between 4 h and 48 h, and a significantly higher increase for MIG and M-CSF between 24 h and 48 h, were observed. Conclusions: Tourniquet times between 90 and 120 min were not associated with higher pain levels or more swelling, but an increased need for intravenous analgesia and a higher increase in pro-inflammatory markers. This might be a consequence of a more pronounced ischemia/reperfusion injury with tourniquet times longer than 90 min.

Background/Objectives: Recombinant Fc proteins have been produced that have a protective effect in mouse models of arthritis, such as the K/BxN rheumatoid arthritis model. We have previously shown that a recombinant human IgG1 Fc with a point mutation at position 309, replacing a leucine with a cysteine, fused to the human IgM tailpiece to form a human IgG1 Fc hexamer, rFc-µTP-L309C, effectively prevents neutrophil infiltration into the joints and ameliorates arthritis in the K/BxN serum transfer model and in the endogenous chronic arthritis K/BxN model. We have now investigated the effect of rFc-µTP-L309C on T-cells in the K/BxN chronic arthritis mouse model. Methods: PBMCs were isolated from the spleen, lymph nodes and joint synovial fluid from K/BxN mice having severe chronic arthritis that had been treated with 200 mg/kg rFc-µTP-L309C or human serum albumin (HSA). Flow cytometry was used to isolate the activated CD4+CD44+ T-cells and T-regulatory cells (Tregs). Intracellular staining was used to identify Th1 and Th17 T-cell subsets, and CD4+CD25+FoxP3+ Tregs. ELISA was used to measure levels of IL-10 and TGF-β in synovial fluid. Results: We find that amelioration of the arthritis occurs after treatment with rFc-µTP-L309C and results in a decrease in Th1 cells’ production of IFNγ and Th17 cells’ production of IL-17. Amelioration also results in decreased production of GM-CSF. Moreover, amelioration results in increased Tregs and IL-10 production in the synovial fluid. Conclusions: rFc-µTP-L309C reduces the inflammatory T-cells and increases the regulatory anti-inflammatory T-cells in the chronic arthritis K/BxN mouse model. This effect explains, in part, the ability of rFc-µTP-L309C to ameliorate the arthritis and reduce damage on the articular cartilage of K/BxN mice.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury -inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement -mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer "decoy" therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody -mediated diseases.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

BackgroundIschemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI.MethodsWe conducted our study on amputated pig forelimbs exposed to 1 h or 9 h of ischemia and then reperfused in vivo for 12 h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot.ResultsOur data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9 h ischemia compared to the 1 h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9 h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs’ and NETs’ intracellular regulatory functions.

Introduction: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1β in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. Methods: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1β. Results: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1β was measured in mice treated with rFc-µTP-L309C. Conclusion: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1β production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Recombinant Fc-μTP-L309C is more efficacious than intravenous immunoglobulin (IVIg) at ameliorating antibody-mediated autoimmune diseases through its effects on Fcγ receptors (FcγRs). Fc-μTP-L309C inhibited in-vitro FcγR-mediated phagocytosis 104/105-fold better than IVIg. Fc-μTP-L309C, given subcutaneously, recovered platelet counts in an immune thrombocytopenia (ITP) mouse model to a higher degree than IVIg at a 10-fold lower dose. We show, using confocal microscopy, that Fc-μTP-L309C binds to monocyte-macrophages and is rapidly internalized, whereas, IVIg remains on the cell surface. Western blotting showed that internalized FcγRIII is degraded through a lysosomal pathway, and this reduction of cell surface FcγRIII is likely responsible for the increased efficacy to ameliorate ITP.

IgG products from human plasma are used to treat an increasing number of conditions including antibody deficiencies and autoimmune diseases.1Chaigne B. Mouthon L. Mechanisms of action of intravenous immunoglobulin.Transfus Apher Sci. 2017; 56: 45-49Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Although multiple mechanisms of action for intravenous IgG (IVIg) therapy have been reported, little attention has been given to the effects on natural killer (NK) cells. The Fc fragment of IgG binds to Fc-gamma receptors (FcγRs), which play an important role in the clearance of soluble immune complexes and presentation of complexed antigens on pathogens, thus providing proinflammatory or anti-inflammatory regulation of immune cell responses.2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar There are 3 classical FcγRs, high-affinity FcγRI (CD64), low-affinity FcγRII (CD32), and FcγRIII (CD16), in all together 5 isoforms. FcγRs trigger activating signaling pathways, except for FcγRIIB, which is inhibitory. The FGCR gene locus is found on chromosome 1.2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar Single nucleotide polymorphisms (SNPs) of the FCGR3A and FCGR2C genes influence the expression of FcγRs and are associated with diseases and/or drug response.2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar,3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci Rep. 2018; 8: 15983Crossref PubMed Scopus (31) Google Scholar FcγRIIIA/CD16a is the major activating Fc receptor expressed on NK cells,2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar which are a heterogeneous subpopulation of CD3–CD56+ lymphocytes recognizing infected, transformed, allo- and xenogeneic cells. CD16a is responsible for antibody-dependent cell cytotoxicity (ADCC) upon binding to antibody-coated target cells.2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar Beside exerting cytotoxicity, NK cells play an important role in regulating the function of other immune cells through the secretion of cytokines such as IFN-γ.4Zimmer J. Natural killer cells: at the forefront of modern immunology. Springer, Berlin2010Crossref Scopus (7) Google Scholar Previous reports collecting blood samples immediately after IVIg infusion indicated a reduction in circulating NK cells post-IVIg administration.5Bohn A.B. Nederby L. Harbo T. Skovbo A. Vorup-Jensen T. Krog J. et al.The effect of IgG levels on the number of natural killer cells and their Fc receptors in chronic inflammatory demyelinating polyradiculoneuropathy.Eur J Neurol. 2011; 18: 919-924Crossref PubMed Scopus (18) Google Scholar,6Jacobi C. Claus M. Wildemann B. Wingert S. Korporal M. Romisch J. et al.Exposure of NK cells to intravenous immunoglobulin induces IFN gamma release and degranulation but inhibits their cytotoxic activity.Clin Immunol. 2009; 133: 393-401Crossref PubMed Scopus (37) Google Scholar The question whether IVIg decreases blood cell counts in vivo, in particular circulating NK cells and their functions, is of high clinical relevance with respect to immunosurveillance and mAb therapies depleting targeted cell populations. Therefore, we studied NK-cell numbers and function in 3 patient groups treated with IVIg for immunodeficiencies, autoimmune myopathies, and neuropathies (see Table E1 in this article’s Online Repository at www.jacionline.org). In parallel we investigated FCGR polymorphisms, especially the most relevant SNPs and copy number variations. No clinically relevant reductions in the blood cell counts were observed 3 days post-IVIg treatment (Fig 1, A; see Fig E1 and Table E2 in this article’s Online Repository at www.jacionline.org); in particular, the percentages of NK cells and the expression of CD56 remained stable (Fig 1, B). In 4 patients, very low NK-cell numbers were found: in patient IVIG50, only 0.4% and 0.2% NK cells before and after infusion, respectively; NK cells were detected only post-IVIg in patient IVIG43; and only before IVIg in patients IVIG36 and IVIG41. The reasons for this observation are not clear; however, we found no correlation with a specific disease or treatment. To explain the discrepancy with previous studies reporting a reduction in NK-cell numbers induced by IVIg,5Bohn A.B. Nederby L. Harbo T. Skovbo A. Vorup-Jensen T. Krog J. et al.The effect of IgG levels on the number of natural killer cells and their Fc receptors in chronic inflammatory demyelinating polyradiculoneuropathy.Eur J Neurol. 2011; 18: 919-924Crossref PubMed Scopus (18) Google Scholar,6Jacobi C. Claus M. Wildemann B. Wingert S. Korporal M. Romisch J. et al.Exposure of NK cells to intravenous immunoglobulin induces IFN gamma release and degranulation but inhibits their cytotoxic activity.Clin Immunol. 2009; 133: 393-401Crossref PubMed Scopus (37) Google Scholar we speculated that shedding of CD56 might be a cause as shown for CD16a in activated NK cells.7Romee R. Foley B. Lenvik T. Wang Y. Zhang B. Ankarlo D. et al.NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17).Blood. 2013; 121: 3599-3608Crossref PubMed Scopus (314) Google Scholar Thus, we determined soluble CD56 and CD16 serum levels (sCD56 and sCD16) in IVIg-treated patients, and for the first time also in healthy controls. In general, there was a large heterogeneity in both sCD56 and sCD16 levels and we did not detect any significant differences as a result of IVIg administration (Fig 1, C, upper panel). Furthermore, no correlation was found, neither pre- nor post-IVIg, between NK-cell surface expression of CD56 and CD16 compared with sCD56 (Spearman r = .3922 and −.1860) and sCD16 (Spearman r = −.0444 and −.1042) (Fig 1, C, lower panel; see Table E3 in this article’s Online Repository at www.jacionline.org). Nevertheless, because the half-life of sCD56 and sCD16 in blood is unknown, the analysis 3 days post-IVIg might not have been the ideal time point to detect a rise. Moreover, sCD16 is shed from activated neutrophils and CD16+ monocytes in addition to NK cells, which could have concealed the effect of IVIg on NK cells. In summary, some patients presented low numbers of circulating NK cells, irrespective of IVIg therapy, and no effect of IVIg on circulating NK-cell numbers, CD56 surface expression, and soluble CD56/CD16 was observed. As expected, blood levels of total IgG and IgG subclasses increased post-IVIg infusion, whereas the levels of naturally bound IgG to NK cells did not change (Table E2). No differences were noticed between low- and high-dose IVIg, and there was no association between the levels of IgG bound to CD16a on NK cells and the high-affinity SNP rs396991 of FCGR3A (see below). The NK-cell activating markers (CD25, CD69), maturation marker CD57, and receptors CD94 and CD158a also remained unchanged (Table E3). In a recent study we have demonstrated that IVIg inhibits NK cytotoxicity in vitro, especially ADCC.8Pradier A. Papaserafeim M. Li N. Rietveld A. Kaestel C. Gruaz L. et al.Small-molecule immunosuppressive drugs and therapeutic immunoglobulins differentially inhibit NK cell effector functions in vitro.Front Immunol. 2019; 10: 556Crossref PubMed Scopus (10) Google Scholar In contrast, IVIg therapy had no effect on NK-cell–mediated ADCC ex vivo 3 days after administration in the present study (Fig 1, D). Overall, ADCC did not change (P = .3402) neither after stratification of the groups receiving low-dose (antibody deficiencies P = .9945) or high-dose IVIg (myopathies/neuropathies; P = .6670 and P = .7389, respectively). Direct ex vivo NK-cell cytotoxicity against porcine endothelial cells did not change after IVIg therapy in any of the 3 treatment groups (P = .9774) (Fig 1, E; see Table E4 in this article’s Online Repository at www.jacionline.org). These results are in agreement with the levels of the degranulation marker CD107a, which were also unaffected by IVIg therapy (Fig 1, F; see Table E4). Although limited by the small sample size, patients with neuropathy seem to exhibit lower ADCC levels and direct NK-cell cytotoxicity compared with patients treated for antibody deficiencies and myopathies. Finally, there were no changes in the ex vivo IFN-γ production by NK cells pre- and post-IVIg infusion (P = .7717 and .1966, for % and mean fluorescence intensity ratio, respectively), and no differences between the patients groups (Fig 1, G; see Table E4). At the genetic level, the most common ClinVar SNPs for FCGR3A, rs396991 and rs10127939, were assessed in our patients’ groups, resulting in similar frequencies to the world population records (see Table E5 in this article’s Online Repository at www.jacionline.org).3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci Rep. 2018; 8: 15983Crossref PubMed Scopus (31) Google Scholar Two additional SNPs, rs12071048 and rs4656317, located in an enhancer region of FCGR3A, were reported to affect ADCC and CD16a surface expression in NK cells.3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci Rep. 2018; 8: 15983Crossref PubMed Scopus (31) Google Scholar The current study population showed 7 heterozygous and 24 homozygous samples for rs12071048; 21 heterozygous and 1 homozygous for rs4656317, but no correlation of the SNPs to ADCC or CD16a expression levels was detected (Fig 2, A). All patients were carriers of the FCGR2C-Stop57 (CD32c), in agreement with the reported low frequency of glutamine carriers. The additional SNPs analyzed for the FCGR locus are also described in Table E5. In our cohort we found FCGR copy number variations in 2 of 31 (nearly 6.5%) patients, IVIG08 and IVIG16 (Fig 2, B). Four different copy number regions (CNRs) have been described for the FCGR locus2Nagelkerke S.Q. Schmidt D.E. de Haas M. Kuijpers T.W. Genetic variation in low-to-medium-affinity Fcgamma receptors: functional consequences, disease associations, and opportunities for personalized medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (43) Google Scholar; NA18555 and IVIG16 showed increased reads for FCGR2C/3B, suggesting a duplication of CNR4; IVIG08 a deletion in FCGR3A/2C, suggesting nonallelic homologous recombination of CNR2 (Fig 2, C). ADCC and CD16a were not affected by the SNPs of FCGR3A, and only an association between pre- and post-IVIg was observed (Fig 2, D). Interestingly, inverse correlation was found between rs396991 (V178F) and rs10919543 (associated with arteritis3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci Rep. 2018; 8: 15983Crossref PubMed Scopus (31) Google Scholar); all 11 homozygotes for ancestral nt for rs10919543 were also homozygotes for the changed nt in rs396991. A direct moderate association was found between rs148181339 (I106V) and rs771144485 (S65N); and between rs10127939 (L66H/R) and the 2KB upstream variant rs56199187 (Fig 2, D). Finally, no correlations were found between ADCC and CD16a expression on NK cells, patient characteristics, and as expected, IVIg dose and duration of IVIg administartion; however, minor correlations were found for the clinical parameters (Fig 2, E). Other investigators previously reported that IVIg resulted in a reduction in the number of NK cells as well as suppression of ADCC activity of NK cells (for details, see Table E7 in this article’s Online Repository at www.jacionline.org). This discrepancy might be explained by the use of either diluted blood, peripheral blood lymphocytes, or mononuclear cells instead of purified NK cells in the functional assays. Second, the time point chosen for blood collection is of relevance. Suppression of NK-cell cytotoxicity was observed, when the blood samples were obtained immediately after IVIg administration, matching the in vitro results reported by us and others. Furthermore, the disease group and additional pharmacologic agents such as corticosteroids could have had an impact on NK-cell counts and function. The current study populations received 4 different IVIg products (mainly Kiovig and Privigen) supporting the notion that findings were not product-related. Finally, we included study groups receiving high- and low-dose IVIg with similar results, suggesting that the dose of IVIg had no impact on NK cells. In summary, we report the important finding that NK-cell functions, antibody-dependent and -independent cytotoxicity as well as IFN-γ production were not compromised in monthly treated patients 3 days after IVIg administration, irrespective of the dose. Therefore, we conclude that NK-cell–mediated immunosurveillance and mAb therapies depleting targeted cell populations remain efficient after IVIg treatment. Download .docx (.05 MB) Help with docx files Tables E1-E4 and Tables E6-E7 Download .docx (.31 MB) Help with docx files Fig E1 Download .docx (.03 MB) Help with docx files Supplemental Materials and Methods Download .docx (.02 MB) Help with docx files Supplemental References Download .docx (.02 MB) Help with docx files Supplemental Results Download .docx (.02 MB) Help with docx files Table E5

High-dose intravenous immunoglobulin (IVIg), and more recently, subcutaneously-delivered Ig (SCIg), are used to treat a variety of autoimmune diseases; however, there are challenges associated with product production, availability, access and efficacy. These challenges have provided incentives to develop a human recombinant Fc as a more potent alternative to IVIg and SCIg for the treatment of autoimmune diseases. Recently, a recombinant human IgG1 Fc hexamer (Fc-μTP-L309C) was shown to be more efficacious than IVIg in a variety of autoimmune mouse models. We have now examined its efficacy compared to IVIg and SCIg in the K/BxN mouse model of endogenous, chronic rheumatoid arthritis (RA). Using the serum-transfer K/BxN model and the endogenous autoimmune model, amelioration of the arthritis was achieved. Effective treatment required high and frequent doses of IVIg, SCIg and Fc-μTP-L309C. However, Fc-μTP-L309C was efficacious at 10-fold lower doses that IVIg/SCIg. Also, arthritis could be prevented when Fc-μTP-L309C was given prior to onset of the arthritis in both the endogenous model and in the serum transfer model. Our results show that Fc-μTP-L309C is a powerful treatment for the prevention and amelioration of severe, chronic arthritis in a true autoimmune mouse model of RA. Thus, the K/BxN endogenous arthritis model should be useful for testing potential therapeutics for RA. Our findings provide rationale for further examination of the treatment efficacy of immunoglobulin-based therapeutics in rheumatoid arthritis.

Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection (AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat controversial in the current literature, the aim of this comparative study was to investigate healthy donor's human NK cell functions after exposure to ISD and IVIg, and to comprehensively review the current literature. NK cells were incubated overnight with IL2/IL12 and different doses and combinations of ISD and IVIg. Proliferation was evaluated by 3[H]-thymidine incorporation; phenotype, degranulation and interferon gamma (IFNγ) production by flow cytometry and ELISA; direct NK cytotoxicity by standard 51[Cr]-release and non-radioactive DELFIA assays using K562 as stimulator and target cells; porcine endothelial cells coated with human anti-pig antibodies were used as targets in antibody-dependent cellular cytotoxicity (ADCC) assays. We found that CD69, CD25, CD54, and NKG2D were downregulated by ISD. Proliferation was inhibited by methylprednisolone (MePRD), mycophenolic acid (MPA), and everolimus (EVE). MePRD and MPA reduced degranulation, MPA only of CD56bright NK cells. MePRD and IVIg inhibited direct cytotoxicity and ADCC. Combinations of ISD demonstrated cumulative inhibitory effects. IFNγ production was inhibited by MePRD and ISD combinations, but not by IVIg. In conclusion, IVIg, ISD and combinations thereof differentially inhibit NK cell functions. The most potent drug with an effect on all NK functions was MePRD. The fact that MePRD and IVIg significantly block NK cytotoxicity, especially ADCC, has major implications for AbMR as well as therapeutic strategies targeting cancer and immune cells with monoclonal antibodies.

In recent years, there has been a surge in the research and development of novel molecules as potential therapeutic alternatives to traditional treatments (such as intravenous immunoglobulins) for autoimmune disorders. The aim of this review is to describe different drug development strategies and evaluate how various molecules have performed in clinical trials to date. Broadly, three main approaches have been pursued. Recombinant fragment crystallisable (rFc) multimers primarily target Fcγ receptors (FcγRs) but may also affect the complement system. These include PF-06755347 (GL-2045), CSL730 (M230), CSL777 and Pan Fc Receptor Interacting Molecule (PRIM). Neonatal Fc receptor (FcRn)-targeting therapeutics block the FcRn receptor and are represented by candidate drugs such as the Fc fragment efgartigimod and the monoclonal antibodies rozanolixizumab (UCB7665), M281 and SYNT001. Finally, Fc and FcγR-targeting therapeutics, comprise molecules that target the Fc of IgG, such as the recombinant soluble FcγIIb receptor valziflocept (SM101/SHP652) and various monoclonal antibodies directed against the receptors. The developmental status of these three classes of molecules ranges from preclinical to ongoing phase 3 clinical studies. Efgartigimod and rozanolixizumab are the most advanced and have demonstrated encouraging results from phase 2 trials in immune thrombocytopenia and myasthenia gravis. Although initial results are promising, further long-term data and a better understanding of the unique mechanisms of action of the different molecules are needed. The efficacy, safety, convenience of administration, duration of effects, and cost will all contribute to determining which of the molecules will be successful in the clinic.

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO.

Objective: Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. Methods: Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. Results: Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. Conclusions: Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities. (J Vasc Surg 2018;68:209S-21S.) Clinical Relevance: Our animal model reveals a range of pathologic changes seen in patients with elective surgery on extremities requiring prolonged arterial occlusion. Pretreatment with C1 inhibitor shows the potential to reduce the extent of reperfusion injury by preventing endothelial cell damage. Our results may lead to novel, targeted therapies that could be used alone or in combination with other reperfusion strategies to improve clinical care.