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Despite the unique advantages of IgG3 over other IgG subclasses, such as mediating enhanced effector functions and increased flexibility in antigen binding due to a long hinge region, the therapeutic potential of IgG3 remains largely unexplored. This may be attributed to difficulties in recombinant expression and the reduced plasma half-life of most IgG3 allotypes. Here, we report plant expression of two SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) that exhibit high (P5C3) and low (H4) antigen binding. P5C3 and H4-IgG1 mAbs were subclass-switched to IgG3 formats, designed for efficient production and increased PK values, carrying three allotypic variations, referred to as -WT, -H, and -KVH. A total of eight mAbs were produced in glycoengineered plants that synthesize fucose-free complex N-glycans with great homogeneity. Antigen, IgG-FcγR immune complex and complement binding studies demonstrated similar activities of all mAbs. In accordance, P5C3 Abs showed minor alterations in SARS-CoV-2 neutralization (NT) and antibody-dependent cell-mediated virus inhibition (ADCVI). Clear functional differences were observed between H4 variants with superior ADCVI and NT potencies of H4 IgG3 H. Our comparative study demonstrates the production of an IgG3 variant carrying an Fc domain with equivalent or enhanced functions compared to IgG3-WT, but with the stability and PK values ​​of IgG1. Our data also demonstrate that both allotypic variability and antibody specificity are important for fine-tuning of activities, an important information for the development of future therapeutics.

ABSTRACTBreast cancer is the most common malignancy in women worldwide and a highly heterogeneous disease. Four different subtypes are described that differ in the expression of hormone receptors as well as the growth factor receptor HER2. Treatment modalities and survival rate depend on the subtype of breast cancer. However, it is still not clear which patients benefit from immunotherapeutic approaches such as checkpoint blockade. Thus, we aimed to decipher the immune cell signature of the different breast cancer subtypes based on high-dimensional flow cytometry followed by unbiased approaches. Here, we show that the frequency of NK cells is reduced in Luminal A and B as well as triple negative breast cancer and that the phenotype of residual NK cells is changed toward regulatory CD11b−CD16− NK cells. Further, we found higher frequencies of PD-1+ CD4+ and CD8+ T cells in triple negative breast cancer. Moreover, while Luminal A-type breast cancer was enriched for CD14+ cDC2 (named type 3 DC (DC3)), CD14− cDC2 (named DC2) were more frequent in triple negative breast cancer. In contrast, HER2-enriched breast cancer did not show major alterations in the composition of the immune cell compartment in the tumor microenvironment. These findings suggest that patients with Luminal A- and B-type as well as triple negative breast cancer might benefit from immunotherapeutic approaches targeting NK cells.

Analysing the take-off forces on the vaulting table can help to inform the athlete about the vaulting technique. A measuring device was used to measure the forces during the Artistic World Championships 2019. The greatest forces were measured during Forward handspring. The mean maximum values were 5.4 times body weight (BW). The highest peak height was calculated for Forward handspring (tucked) at 2.9 m. The longest flight distance was 3.62 m for forward handspring and 3.59 m for tsukahara/kasamatsu. The statistical correlations between the forces and the peak height and flight distance could only be verified for Forward handspring. For the Tsukahara/Kasamatsu and the Yurchenko vaults, no correlation could be proven.

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

Summary: Over the last decade, multiple studies have investigated the heterogeneity of murine conventional dendritic cells type 2 (cDC2s). However, their phenotypic similarity with monocytes and macrophages renders their clear identification challenging. By creating a protein atlas utilizing multiparameter flow cytometry, we show that ESAM+ cDC2s are a specialized feature of the spleen strongly differing in their proteome from other cDC2s. In contrast, all other tissues are populated by Clec12A+ cDC2s or Clec12A− cDC2s (high or low for Fcγ receptors, C-type lectin receptors, and CD11b, respectively), rendering Clec12A+ cDC2s classical sentinels. Further, expression analysis of CD301b, Clec12A, and FcγRIIB/III provides a conserved definition of cDC2 heterogeneity, including the discovery of putative FcγRIIB/III+ DC3s across tissues. Finally, our data reveal that cell identity (ontogeny) dictates the proteome that is further fine-tuned by the tissue environment on macrophages and dendritic cells (DCs), while monocytes and plasmacytoid DCs (pDCs) display subset intrinsic default settings.

Abstract Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

Summary: Understanding the mechanisms underlying cytotoxic immunoglobulin G (IgG) activity is critical for improving therapeutic antibody activity and inhibiting autoantibody-mediated tissue pathology. While prior research highlights the important role of the mononuclear phagocytic system for removing opsonized target cells, it remains unclear which monocyte or macrophage subsets stemming from fetal or post-natal bone-marrow (BM)-associated definitive hematopoiesis are involved in target cell depletion. By using a titrated irradiation approach as well as Kupffer-cell-specific deletion of activated Fcγ receptor signaling, we establish conditions under which the contribution of BM-derived monocytes versus yolk-sac-derived liver-resident macrophages to cytotoxic IgG activity can be studied. Our results demonstrate that liver-resident macrophages originating from either fetal or adult hematopoiesis play a central role in IgG-mediated depletion of opsonized target cells from the peripheral blood under steady-state conditions, highlighting the impact of the tissue niche and not macrophage origin for cytotoxic antibody activity.

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients.

Summary: Immunoglobulin G (IgG) antibodies coordinate immune effector responses by interacting with effector cells via fragment crystallizable γ (Fcγ) receptors. The IgG Fc domain directs effector responses through subclass and glycosylation variation. Although each Fc variant has been extensively characterized in isolation, during immune responses, IgG is almost always produced in Fc mixtures. How this influences effector responses has not been examined. Here, we measure Fcγ receptor binding to mixed Fc immune complexes. Binding of these mixtures falls along a continuum between pure cases and quantitatively matches a mechanistic model, except for several low-affinity interactions mostly involving IgG2. We find that the binding model provides refined estimates of their affinities. Finally, we demonstrate that the model predicts effector cell-elicited platelet depletion in humanized mice. Contrary to previous views, IgG2 exhibits appreciable binding through avidity, though it is insufficient to induce effector responses. Overall, this work demonstrates a quantitative framework for modeling mixed IgG Fc-effector cell regulation.

Sample preparation is a crucial step in single-molecule experiments and involves passivating the microfluidic sample chamber, immobilizing the molecules, and setting experimental buffer conditions. The efficiency of the experiment depends on the quality and speed of sample preparation, which is often performed manually and relies on the experience of the experimenter. This can result in inefficient use of single-molecule samples and time, especially for high-throughput applications. To address this, a pressure-controlled microfluidic system is proposed to automate single-molecule sample preparation. The hardware is based on microfluidic components from ElveFlow and is designed to be cost-effective and adaptable to various microscopy applications. The system includes a reservoir pressure adapter and a reservoir holder designed for additive manufacturing. Two flow chamber designs Ibidi µ-slide and Grace Bio-Labs HybriWell chamber are characterized, and the flow characteristics of the liquid at different volume flow rates V̇ are simulated using CFD-simulations and compared to experimental and theoretical values. The goal of this work is to establish a straightforward and robust system for single-molecule sample preparation that can increase the efficiency of experiments and reduce the bottleneck of manual sample preparation, particularly for high-throughput applications.

This study aimed (1) to assess the validity of a modified method (Mmod) based on heart rate (HR)—oxygen uptake (VO2) regression functions to calculate total energy costs (Wtotal) and aerobic (Waer) and anaerobic alactic energy contribution (Wpcr) and (2) to analyse the physiological and energetic demands of high-level pommel horse routines (PH routines). The Mmod was developed because VO2 measurements are limited during high-level PH routines. Answering Part 1, nine male artistic gymnasts performed a PH routine where energy costs were calculated from VO2 measurements and then compared with energy costs determined from the HR- VO2 regressions of Mmod’s two additional tests. Using the concordance correlation coefficient (CCC) and Deming regression, Waer (CCC = 0.955), Wpcr (CCC = 0.999), and Wtotal (CCC = 0.990) show substantial to almost perfect validity without constant or proportional bias. Data from eight further gymnasts performing a high-level PH routine and a graded exercise test (GXT), as well as four data sets from Part 1, were used to determine physiological and energetic demands using Mmod. VO2 and HR during PH routines reached 86.1% and 90.4% of the maximal values during GXT. Wpcr was 47.0%, anaerobic lactic energy contribution (Wblc) was 29.7%, and Waer was 23.3% of Wtotal required during PH routines. Summarising the energetic demands of high-level PH routines, they are mainly anaerobic, where Wpcr provides the largest energy share. Waer provides a substantial part of Wtotal and should therefore also be specifically trained.

The nanoscale morphology of bulk heterojunctions is highly important for the charge dissociation and transport in organic solar cells and ultimately defines the performance of the cell. The visualization of this nano-morphology in terms of domain size and polymer orientation in a fast and straightforward way is therefore of great interest to evaluate the suitability of a film for efficient solar cells. Here, we demonstrate that the morphology of different blends of poly(3-hexylthiophene-2,5-diyl) (P3HT) and phenyl-C61-butyric acid methyl ester (PCBM) can be imaged and analyzed by employing photoemission electron microscopy.

Epidermolysis bullosa acquisita (EBA) is a subepidermal autoimmune bullous disease caused by autoantibodies targeting type VII collagen (COL7). It is characterized by inflammation and subepidermal blistering mainly through immune complex (IC)-mediated activation of neutrophils. In experimental EBA, binding of neutrophils to ICs in the skin and induction of clinical disease depends on the expression of the Fc gamma receptor (FcγR) IV. As activating FcγR mediate both neutrophil extravasation and activation, we used multiphoton imaging to obtain further insights into the mechanistic contribution of FcγRIV in the pathogenesis of EBA. First, we demonstrated that blocking FcγRIV function completely protects LysM-eGFP mice against induction of antibody transfer-induced EBA. To visualize the interactions of anti-COL7 IgG and neutrophils in vivo, fluorescently labeled anti-COL7 IgG was injected into LysM-eGFP mice. Multiphoton microscopy was sequentially performed over a period of 8 days. At all time points, we observed a significantly higher extravasation of neutrophils into the skin of mice treated with anti-FcγRIV antibody compared to controls. However, the percentage of detected neutrophils localized to the target antigen along the dermal-epidermal junction was comparable between both groups. Additionally, reactive oxygen release and migration in vitro assay data demonstrate that FcγRIV antibody treatment inhibits the activation, but not the migration, of neutrophils. Our findings underscore the importance of advanced in vivo imaging techniques to understand the complexity of IC-mediated neutrophil-dependent inflammation, and indicate that the therapeutic utility of FcγRIV blockade is achieved through impairment of IC-mediated neutrophil activation.

Checkpoint control and immunomodulatory antibodies have become important tools for modulating tumor or self-reactive immune responses. A major issue preventing to make full use of the potential of these immunomodulatory antibodies are the severe side-effects, ranging from systemic cytokine release syndrome to organ-specific toxicities. The IgG Fc-portion has been demonstrated to contribute to both, the desired as well as the undesired antibody activities of checkpoint control and immunomodulatory antibodies via binding to cellular Fcγ-receptors (FcγR). Thus, choosing IgG subclasses, such as human IgG4, with a low ability to interact with FcγRs has been identified as a potential strategy to limit FcγR or complement pathway dependent side-effects. However, even immunomodulatory antibodies on the human IgG4 background may interact with cellular FcγRs and show dose limiting toxicities. By using a humanized mouse model allowing to study the immunomodulatory activity of human checkpoint control antibodies in vivo, we demonstrate that deglycosylation of the CD137-specific IgG4 antibody urelumab results in an amelioration of liver toxicity, while maintaining T cell stimulatory activity. In addition, our results emphasize that antibody dosing impacts the separation of side-effects of urelumab from its therapeutic activity via IgG deglycosylation. Thus, glycoengineering of human IgG4 antibodies may be a possible approach to limit collateral damage by immunomodulatory antibodies and allow for a greater therapeutic window of opportunity.

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies’ effector functions.

Summary: High-throughput antibody repertoire analysis via next-generation sequencing is a key method in understanding humoral immunity. Errors introduced during library preparation and sequencing can be corrected with molecular amplification fingerprinting using unique molecular identifiers. Here, we provide a step-by-step protocol for laboratory and bioinformatic workflows to perform sequencing in murine cells with isotype-specific primers, obtaining total and isotype-specific B cell receptor repertoires. This enables the examination of isotype-dependent immune responses and improves the understanding of underlying mechanisms in humoral immunity.For complete details on the use and execution of this protocol, please refer to Khan et al. (2016).

Moderate consumption of alcohol is associated with protection from some autoimmune diseases. Here the authors show that ethanol and its metabolite acetate can protect mice from collagen-induced arthritis and provide evidence that the mechanism of this effect might be via inhibition of the effector function of T follicular helper cells.

Three-dimensional tracking of cells is one of the most powerful methods to investigate multicellular phenomena, such as ontogenesis, tumor formation or wound healing. However, 3D tracking in a biological environment usually requires fluorescent labeling of the cells and elaborate equipment, such as automated light sheet or confocal microscopy. Here we present a simple method for 3D tracking large numbers of unlabeled cells in a collagen matrix. Using a small lensless imaging setup, consisting of an LED and a photo sensor only, we were able to simultaneously track ~3000 human neutrophil granulocytes in a collagen droplet within an unusually large field of view (>50 mm2) at a time resolution of 4 seconds and a spatial resolution of ~1.5 μm in xy- and ~30 μm in z-direction. The setup, which is small enough to fit into any conventional incubator, was used to investigate chemotaxis towards interleukin-8 (IL-8 or CXCL8) and N-formylmethionyl-leucyl-phenylalanine (fMLP). The influence of varying stiffness and pore size of the embedding collagen matrix could also be quantified. Furthermore, we demonstrate our setup to be capable of telling apart healthy neutrophils from those where a condition of inflammation was (I) induced by exposure to lipopolysaccharide (LPS) and (II) caused by a pre-existing asthma condition. Over the course of our experiments we have tracked more than 420.000 cells. The large cell numbers increase statistical relevance to not only quantify cellular behavior in research, but to make it suitable for future diagnostic applications, too.

Osteoarthritis (OA) is the leading degenerative joint disease in the western world and leads, if left untreated, to a progressive deterioration of joint functionality, ultimately reducing quality of life. Recent data has shown, that especially OA of the ankle and foot are among the most frequently affected regions. Current research in OA points towards a complex involvement of various cell and tissue types, often accompanied by inflammation. Low-dose radiotherapy (LDRT) is widely used for the treatment of degenerative and inflammatory diseases. While the reported analgesic effects are well known, the underlying molecular mechanisms are only poorly understood. We therefore correlated a clinical approach, looking at pain reduction in 196 patients treated with LDRT with a pre-clinical approach, utilizing the K/BxN serum transfer mouse model using flow cytometry and multiplex ELISA for analysis. While an improvement of symptoms in the majority of patients was found, patients suffering from symptoms within the tarsi transversa show a significantly lower level of improvement. Further, a significant impact of therapy success was detected depending on whether only one or both feet were affected. Further, patients of younger age showed a significantly better outcome than older ones while needing fewer treatment series. When looking on a cellular level within the mouse model, a systemic alteration of immune cells namely a shift from CD8+ to CD4+ T cells and reduced numbers of DCs was observed. A general reduction of inflammatory cytokines was detected, with significant alterations in IL-4 and IL-17 levels, all of which could potentially be responsible for the highly effective clinical improvement in patients. Taken together our data indicate that LDRT can be regarded as a highly effective treatment option for patients suffering from OA of the foot and ankle, in terms of analgesic effects, especially in younger patients. Furthermore, the observed effects are mediated by an interplay of cellular and soluble immune factors, as observed in the K/BxN serum transfer model. With this interdisciplinary approach we aim to encourage the usage of LDRT as an additive treatment strategy not only as a last resort, but also earlier in the course of disease.

Abstract Allelic variants of genes encoding for the Fc gamma receptors IIIA and IIA have been associated with the clinical response to cell‐depleting antibodies in lymphoma patients. Here, we tested the hypothesis that FCGR3A and FCGR2A high‐affinity polymorphisms predict clinical outcomes to alemtuzumab therapy in 85 patients with relapsing‐remitting multiple sclerosis. No differences in clinical and MRI‐based efficacy parameters, the development of severe infusion‐associated reactions and secondary autoimmune diseases during a 2 year follow‐up was observed based on FCGR3A or FCGR2A polymorphisms. This study does not support the use of FCGR genetic variants to predict clinical outcomes to alemtuzumab.

A link between high sodium chloride (salt) intake and the development of autoimmune diseases was previously reported. These earlier studies demonstrated exacerbation of experimental autoimmune encephalomyelitis and colitis by excess salt intake associated with Th17- and macrophage-mediated mechanisms. Little is known about the impact of dietary salt intake on experimental arthritides. Here, we investigated if salt restriction can exert beneficial effects on collagen-induced arthritis (CIA) and K/BxN serum transfer-induced arthritis (STIA). CIA depends on both adaptive and innate immunity, while STIA predominantly mimics the innate immune cell-driven effector phase of arthritis. In both models, low salt (LS) diet significantly decreased arthritis severity compared to regular salt (RS) and high salt (HS) diet. We did not observe an aggravation of arthritis with HS diet compared to RS diet. Remarkably, in STIA, LS diet was as effective as IL-1 receptor blocking treatment. Complement-fixing anti-CII IgG2a antibodies are associated with inflammatory cell infiltration and cartilage destruction. LS diet reduced anti-CII IgG2a levels in CIA and decreased the anti-CII IgG2a/IgG1 ratios pointing toward a more Th2-like response. Significantly less inflammatory joint infiltrates and cartilage breakdown associated with reduced protein concentrations of IL-1 beta (CIA and STIA), IL-17 (CIA), and the monocyte chemoattractant protein-1 (MCP-1) (CIA) were detected in mice receiving LS diet compared to HS diet. However, we did not find a reduced IL-17A expression in CD4+ T cells upon salt restriction in CIA. Analysis of mRNA transcripts and immunoblots revealed a link between LS diet and inhibition of the p38 MAPK (mitogen-activated protein kinase)/NFAT5 (nuclear factor of activated T-cells 5) signaling axis in STIA. Further experiments indicated a decreased leukodiapedesis under LS conditions. In conclusion, dietary salt restriction ameliorates CIA and STIA, indicating a beneficial role of LS diet during both the immunization and effector phase of immune-mediated arthritides by predominantly modulating the humoral immunity and the activation status of myeloid lineage cells. Hence, salt restriction might represent a supportive dietary intervention not only to reduce cardiovascular risk, but also to improve human inflammatory joint diseases like rheumatoid arthritis.

Summary: Systemic lupus erythematosus (SLE) is characterized by a loss of self-tolerance, systemic inflammation, and multi-organ damage. While a variety of therapeutic interventions are available, it has become clear that an early diagnosis and treatment may be key to achieve long lasting therapeutic responses and to limit irreversible organ damage. Loss of humoral tolerance including the appearance of self-reactive antibodies can be detected years before the actual onset of the clinical autoimmune disease, representing a potential early point of intervention. Not much is known, however, about how and to what extent this pre-phase of disease impacts the onset and development of subsequent autoimmunity. By targeting the B cell compartment in the pre-disease phase of a spontaneous mouse model of SLE we now show, that resetting the humoral immune system during the clinically unapparent phase of the disease globally alters immune homeostasis delaying the downstream development of systemic autoimmunity.

Novel molecules that directly target the neonatal Fc receptor (FcRn) and/or Fc gamma receptors (FcγRs) are emerging as promising treatments for immunoglobulin G (IgG)-dependent autoimmune pathologies. Mutated Fc regions and monoclonal antibodies that target FcRn are currently in clinical development and hold promise for reducing the levels of circulating IgG. Additionally, engineered structures containing multimeric Fc regions allow the dual targeting of FcRn and FcγRs; however, their tolerance needs to first be validated in phase I clinical studies. Here, for the first time, we have developed a modified monomeric recombinant Fc optimized for binding to all FcRns and FcγRs without the drawback of possible tolerance associated with FcγR cross-linking. A rational approach using Fc engineering allowed the selection of LFBD192, an Fc with a combination of six mutations that exhibits improved binding to human FcRn and FcγR as well as mouse FcRn and FcγRIV. The potency of LFBD192 was compared with that of intravenous immunoglobulin (IVIg), an FcRn blocker (Fc-MST-HN), and a trimeric Fc that blocks FcRn and/or immune complex-mediated cell activation through FcγR without triggering an immune reaction in several in vitro tests and validated in three mouse models of autoimmune disease.

The duration of holding elements represents a critical factor for judging routines on the still rings in artistic gymnastics. Athletes can be penalized with non-recognition of an element if the hold time is too short. Dynamometric and kinematic measuring methods offer the possibility to provide support to judges in evaluating the duration of the hold time. In this study a dynamometric method with two different variants (dms10 and dms5) as well as a kinematic method (kms) based on a trained neural network were presented and examined with regard to their agreement with judges’ evaluations when determining the hold time. To check the agreement, a) the percentage agreement and b) the interrater reliability were calculated using Cohen's kappa (k). The two dynamometric methods showed a percentage agreement of 83.5% (dms10) and 51.7% (dms5) with the hold time evaluation by judges. The percentage agreement of the kms was 38.8%. The interrater reliability showed for the dms10 a moderate (k = 0.58) and for the dms5 a fair (k = 0.23) agreement, while the kms showed a poor (k = 0.02) match. The results supported dms10 for its possible use as a practicable and reliable method to assist judges in evaluating hold times on the still rings. Dms5 and kms (in the current development stage) were not suitable as means of judges’ support.

Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.

In order to develop and optimize movements in gymnastics vault, knowledge of take-off velocity and angular momentum is important. Due to the short times of contact on the springboard, high-frequency kinemetric methods are very time-consuming for the determination of the take-off parameters. A multi-body model of a springboard was developed to determine the take-off forces to calculate specific take-off parameters. The Gymnova springboard was modeled using the simulation environment software alaska. The evaluation under dynamic conditions was carried out with a falling mass test, drop-jumps and forward handspring. The evaluation was done on the parameters of the ground reaction forces (GRF): force impact (p) and maximum vertical force (Fmax). For the drop-jump and forward handspring simulation the real measured acceleration of the upper board was given as input parameter in the model. When comparing the vertical displacement of the real and the modelled upper board, a discrepancy of 6.1 % can be observed. For the falling mass test differences for p=0.4 % and Fmax=28.2 % were achieved between the real board and the model. For typical loads for the gymnastics sport, drop-jumps have been used. There were realized differences of up to 8.4 % for Fmax and 6.8 % for p. For the final stage of the review, forward handstand vaults were examined. Horizontal and vertical forces were investigated. Through thorough evaluation on several stages, it was possible to develop a springboard model that is suitable to calculate the GRF under dynamic conditions successfully in 2-d. Therefore, the forces acting on the take-off position can be calculated. Take-off parameters can be determined from these forces. This evaluation also shows that the horizontal forces in especial have to be observed.

Particular interest to harness the innate immune system for cancer immunotherapy is fueled by limitations of immune checkpoint blockade. Plasmacytoid dendritic cells (pDC) are detected in a variety of solid tumors and correlate with poor clinical outcome. Release of type I interferons in response to toll-like-receptor (TLR)7 and TLR9 activation is the pDC hallmark. Mouse and human pDC differ substantially in their biology concerning surface marker expression and cytokine production. Here, we employed humanized mouse models (HIS) to study pDC function. We performed a comprehensive characterization of transgenic, myeloid-enhanced mouse strains (NOG-EXL and NSG-SGM3) expressing human interleukin-3 (hIL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) using identical humanization protocols. Only in HIS-NOG-EXL mice sufficient pDC infiltration was detectable. Therefore, we selected this strain for subsequent tumor studies. We analyzed pDC frequency in peripheral blood and tumors by comparing HIS-NOG-EXL with HIS-NOG mice bearing three different ovarian and breast tumors. Despite the substantially increased pDC numbers in peripheral blood of HIS-NOG-EXL mice, we detected TLR7/8 agonist responsive and thus functional pDCs only in certain tumor models independent of the mouse strain employed. However, HIS-NOG-EXL mice showed in general a superior humanization phenotype characterized by reconstitution of different myeloid subsets, NK cells and B cells producing physiologic IgG levels. Hence, we provide first evidence that the tumor milieu but not genetically introduced cytokines defines intratumoral (i.t.) frequencies of the rare pDC subset. This study provides model systems to investigate in vivo pro- and anti-tumoral human pDC functions.

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation.

Pathogen-specific antibody responses need to be tightly regulated to generate protective but limit self-reactive immune responses. While loss of humoral tolerance has been associated with microbial infections, the pathways involved in balancing protective versus autoreactive antibody responses in humans are incompletely understood. Studies in classical mouse model systems have provided evidence that balancing of immune responses through inhibitory receptors is an important quality control checkpoint. Genetic differences between inbred mouse models and the outbred human population and allelic receptor variants not present in mice; however, argue for caution when directly translating these findings to the human system. By studying Borrelia burgdorferi infection in humanized mice reconstituted with human hematopoietic stem cells from donors homozygous for a functional or a non-functional FcγRIIb allele, we show that the human inhibitory FcγRIIb is a critical checkpoint balancing protective and autoreactive immune responses, linking infection with induction of autoimmunity in the human immune system.

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.

Lipid cell membranes not only represent the physical boundaries of cells. They also actively participate in many cellular processes. This contribution is facilitated by highly complex mixtures of different lipids and incorporation of various membrane proteins. One group of membrane-associated receptors are Fc receptors (FcRs). These cell-surface receptors are crucial for the activity of most immune cells as they bind immunoglobulins such as immunoglobulin G (IgG). Based on distinct mechanisms of IgG binding, two classes of Fc receptors are now recognized: the canonical type I FcγRs and select C-type lectin receptors newly referred to as type II FcRs. Upon IgG immune complex induced cross-linking, these receptors are known to induce a multitude of cellular effector responses in a cell-type dependent manner, including internalization, antigen processing, and presentation as well as production of cytokines. The response is also determined by specific intracellular signaling domains, allowing FcRs to either positively or negatively modulate immune cell activity. Expression of cell-type specific combinations and numbers of receptors therefore ultimately sets a threshold for induction of effector responses. Mechanistically, receptor cross-linking and localization to lipid rafts, i.e., organized membrane microdomains enriched in intracellular signaling proteins, were proposed as major determinants of initial FcR activation. Given that immune cell membranes might also vary in their lipid compositions, it is reasonable to speculate, that the cell membrane and especially lipid rafts serve as an additional regulator of FcR activity. In this article, we aim to summarize the current knowledge on the interplay of lipid rafts and IgG binding FcRs with a focus on the plasma membrane composition and receptor localization in immune cells, the proposed mechanisms underlying this localization and consequences for FcR function with respect to their immunoregulatory capacity.

Immunoglobulin G (IgG) is the most abundant immunoglobulin isotype in the blood and is involved in the pathogenesis and progression of various diseases. Glycosylation of the IgG fragment crystallizable (Fc) region is shown to vary in different physiological and pathological states. Fc N-glycan composition can alter the effector functions of IgG by modulating its affinity for ligands, such as Fcγ receptors (FcγRs). However, it is not known whether IgG glycosylation is affected by the available repertoire of FcγRs, and if the Fc-linked N-glycome can compensate for modulation of the IgG–FcγR interaction. To explore this, we examined the subclass-specific Fc IgG glycoprofiles of healthy male and female FcγR knock-out mice on C57BL/6 and BALB/c backgrounds. We observed slight changes in IgG Fc N-glycan profiles in different knock-outs; however, it seems that the strain background and sex have a stronger effect on N-glycosylation of IgG Fc regions than the FcγR repertoire.