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Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Siglec-15 is a conserved sialic acid–binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15−/− mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti–Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.

A hallmark of many chronic inflammatory and autoimmune diseases is that there is an impaired resolution of inflammation and return to the steady state. The infusion of high doses of pooled serum IgG preparations from thousands of donors [intravenous immunoglobulin (IVIG) therapy] has been shown to induce resolution of inflammation in a variety of chronic inflammatory and autoimmune diseases, suggesting that IgG molecules can instruct the immune system to stop inflammatory processes and initiate the return to the steady state. The aim of this review is to discuss how insights into the mechanism of IVIG activity may help to understand the molecular and cellular pathways underlying resolution of inflammation. We will put a special emphasis on pathways dependent on the IgG FC domain and IgG sialylation, as several recent studies have provided new insights into how this glycosylation-dependent pathway modulates innate and adaptive immune responses through different sets of C-type or I-type lectins.

Serum autoantibodies have a wide range of glycosylation patterns. In this Review, the authors describe the role of autoantibody glycosylation in the pathology of autoimmune disease, and how understanding the role of specific antibody glycoforms might optimize current treatment approaches. A loss of humoral tolerance is a hallmark of many autoimmune diseases and the detection of self-reactive antibodies (autoantibodies) of the immunoglobulin G (IgG) isotype is widely used as a biomarker and diagnostic tool. However, autoantibodies might also be present in individuals without autoimmune disease, thus limiting their usefulness as a sole indicator of disease development. Moreover, while clear evidence exists of the pathogenic effects of autoantibodies in mouse model systems, the contribution of autoantibodies to the pathology of many autoimmune diseases has yet to be established. In this Review, the authors discuss the changes in total serum IgG and autoantibody glycosylation that occur during autoimmune disease and how these changes might help to predict disease development in the future. Furthermore, current knowledge of the signals regulating antibody glycosylation and how individual antibody glycoforms could be used to optimize current treatment approaches will be discussed.

Despite recent advances in activating immune cells to target tumors, the presence of some immune cells, such as tumor-associated macrophages (TAMs) or tumor-associated neutrophils (TANs), may promote rather than inhibit tumor growth. However, it remains unclear how antibody-dependent tumor immunotherapies, such as cytotoxic or checkpoint control antibodies, affect different TAM or TAN populations, which abundantly express activating Fcγ receptors. In this study, we show that the tissue environment determines which cellular effector pathways are responsible for antibody-dependent tumor immunotherapy. Although TAMs derived from Ly6Chigh monocytes recruited by the CCL2-CCR2 axis were critical for tumor immunotherapy of skin tumors, the destruction of lung tumors was CCL2-independent and required the presence of colony-stimulating factor 2-dependent tissue-resident macrophages. Our findings suggest that TAMs may have a dual role not only in promoting tumor growth in certain tissue environments on the one hand but also in contributing to tumor cell destruction during antibody-mediated immunotherapy on the other hand.

Lehmann et al. targeted antigens to Fcγ receptors expressed on various antigen-presenting cells. Induced CD4+ and CD8+ T cell responses were solely dependent on CD11b+ and CD8+ DC subsets, respectively, but independent of receptor intrinsic ITAM or ITIM signaling domains., Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8− DCs were critical for priming CD4+ T cell responses.

Isomerization and enantiomerization of Asp in the tetrapeptide Ala-Phe-Asp-GlyOH are studied at pH 10 and 80°C as well as 25°C. CE-MS allowed the distinction between α-Asp and β-Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfey's reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order Asp > Ala > Phe. The CE assay was validated with respect to linearity, LOQ, LOD, and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β-Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half-life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.

The aim of the present study was the investigation of the isomerization and epimerization kinetics of the aspartyl tetrapeptide Gly-Phe-Asp-GlyOH at alkaline conditions. Incubations of the model tetrapeptide in sodium borate buffer, pH 10 and ionic strength 0.2 M, at 25 °C and 80 °C were analyzed by a validated CE–UV assay and fitted according to a pharmacokinetic model. CE–ESI-MS was used for peptide identification. Enantiomerization and isomerization of the aspartyl residue of the model tetrapeptide was observed under all experimental conditions applied. Differences in the velocity and the ratios of the rates of the degradation reactions indicated different effects of temperature on the individual reactions. At 80 °C, a rapid formation of β-Asp and d -Asp containing isomers from Gly- l -Phe-α- l -Asp-GlyOH was monitored. Rate constants of the hydrolysis of the succinimide (Asu) intermediate generally exceeded the formation of the intermediate from α/β-Asp peptides. A higher rate constant was observed for the enantiomerization from l -configured Asu compared to d -Asu. At 25 °C, epimerization and isomerization equilibrium was not reached within 5208 h. Compared to 80 °C different ratios of the individual reaction rates were noted. Moreover, inversion of the sequence of the first 2 amino acids was noted as a minor side reaction at 80 °C.

CE-MS was employed to identify two unknown degradation products of the model tripeptide Phe-α-Asp-Gly heated at 80 °C in aqueous solution at pH 7.4. Both compounds displayed essentially identical mass spectra indicating the presence of peptide diastereomers. The [M + H]+-ion at m/z 338 suggested a tripeptide composed of the amino acids Phe, Gly and Asp. The fragmentation pattern indicated that Phe was not located at the N-terminus. Subsequently, the linear peptide α-Asp-Phe-Gly and the branched peptide Asp(Gly)-Phe were synthesized and analyzed by CE-MS. The mass spectrum of synthetic α-Asp-Phe-Gly was identical to that of the unknown compounds confirming the structure of the degradation products. Asp(Gly)-Phe displayed a complex fragmentation pattern. In conclusion, amino acid sequence inversion represents another degradation pathway of Phe-α-Asp-Gly at pH 7.4 besides known reactions including isomerization, enantiomerization, cyclization to diketopiperazine derivatives and backbone hydrolysis. The mechanism of the rearrangement of the amino acid sequence is proposed to proceed via an aza-bridged intermediate.

Dendritic cells (DCs) are important antigen presenting cells (APCs) and responsible for the induction of immune responses and preserving peripheral tolerance. We showed that targeting of antigens via C-type lectin receptors to different specialized DC subpopulations induced either CD4+ or CD8+ T cell responses in vivo. Fc receptors are also highly active in endocytosis enabling APCs to take up antigens in form of immune complexes. As they are expressed on various APCs, we aimed to identify responsible APCs for primary and secondary immune responses. Therefore, we assessed their expression in various organs and delivered antigens by specific recombinant antibodies. The targeting of the Fc receptors induced CD4+ and CD8+ T cell responses in a transgenic as well as a naïve system. Moreover, especially antigen delivery to the activating FcγRIV was superior in inducing CD4+ and CD8+ T cell responses at the same time, which could not be observed by classical targeting of the C-type lectin receptors DEC205 or DCIR2. Additionally, targeting of antigens to FcγRIV induced a pronounced CD4 helper response in naïve mice, whereas targeting to DCIR2 was inefficient. As FcγRIV is expressed on both major splenic DC subsets, we used this receptor to verify the subset intrinsic preference of DCs for CD4+ or CD8+ T cell responses. This was clearly shown by the induction of CD4+ T cell responses by splenic CD8− DCs, whereas splenic CD8+ DCs induced a CD8+ T cell response. The induced naïve CD8+ T cell responses were functional relevant, as we could demonstrate efficient dose-dependent killing of target cells in vivo. Therefore, we suggest this strategy as useful tool for the induction of de novo as well as the modulation of immune responses for therapeutic applications.

Isomerization and enantiomerization of Asp in the tetrapeptide Ala-Phe-Asp-GlyOH are studied at pH 10 and 80°C as well as 25°C. CE-MS allowed the distinction between α-Asp and β-Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfey's reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order AspAlaPhe. The CE assay was validated with respect to linearity, LOQ, LOD, and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β-Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half-life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.

The aim of the present study was the determination of the stability of a diketopiperazine (DKP) derived from the aspartyl tripeptide Phe-Asp-GlyOH at pH 10 and 80°C and 25°C as well as at pH 7.4 and 80°C. The analysis was performed using a validated capillary electrophoresis assay. Rapid epimerization of the incubated cis-DKP to the trans-DKP was observed under all conditions. Linear diastereomeric α-l/d-Asp and β-l/d-Asp peptides were the primary reaction products at pH 10 and 80°C, indicating that a cyclic succinimide is formed in the process. In contrast, the DKPs were by far the major compounds in the incubation solutions at pH 10 and 25°C and at pH 7.4 and 80°C, whereas the linear Asp peptides were found only at low concentrations. A kinetic model was derived to fit the concentration versus time data, which consider the succinimide as central intermediate for DKP formation and for isomerization and enantiomerization of the linear Asp peptides. Besides the back reaction of the DKPs to the succinimides, an additional hydrolysis reaction of the DKP ring was considered to obtain the fit of the experimental data, indicating that additional degradation reactions have to be considered for Asp-derived DKPs.