Resolution of Th/Tc17-driven inflammation during anti-TNFα treatment of rheumatoid arthritis reveals a unique immune biomarker profiling pattern.

Rheumatoid arthritis (RA) is a chronic multisystem disease with a complex immunopathology. Its inflammatory state is dominated by pro-inflammatory cytokines such as TNFα and activated Th1/Th17. Only proportion of patients achieve clinical remission despite potent biologics targeting these pathways. This study investigated the resolution of inflammation in RA patients (naïve for biologics) receiving TNFα inhibitors (TNFi) and evaluated the biological mechanisms behind treatment response and assessed them using clinical scoring systems. The majority showed a good clinical response after six months (6M) and a significant drop in DAS28-CRP (P ≤ .002), CDAI (P ≤ .0001) and RheumXpert (P ≤ .0001). Before treatment, the patients demonstrated a chronic innate and adaptive inflammatory state. The improved clinical condition was reflected with a decrease in Th17/Tc17 (P ≤ .05) and an increase in Tregs after 6M (P ≤ .05). Using a logistic regression model on serum data, IL-6, IL-18, IL-21, IL-22, IFNγ and TNFα were identified as the main contributing biomarkers in the chronic inflammatory state of RA. A specific test score (STS) was defined and converted to a single cytokine composite test score (CCTS), which showed the disease outcome on a scale 0-100, providing sensitivity and specificity of ≥90%. Thus, the immunological complexity in RA is driven by a complex interplay of pro-inflammatory cytokines and effector T-cell response dominated by Th17/Tc17. In addition, the resolution of inflammation could be linked to a partially Treg-driven homeostatic innate immune response. Therefore, a more complex therapeutic approach against the above markers might be of value to obtain full clinical remission in the future.

Mapping of Signaling Pathways Linked to sIgAD Reveals Impaired IL-21 Driven STAT3 B-Cell Activation.

Objectives: It has recently been shown that individuals with selective IgA deficiency (sIgAD) have defective B cell responses both to T cell dependent and independent mimicking stimulations. The complex intracellular signaling pathways from different stimuli leading to IgA isotype switching have not been fully elucidated. Thus, the main objective of this study was to delineate these pathways and their potential role in the immunopathology linked to sIgAD. Materials and Methods: PBMCs from 10 individuals with sIgAD and 10 healthy controls (HC) were activated in vitro via either a T cell dependent or independent mimicking stimulation. Intracellular phosphorylation of pSTAT3, pSTAT5, pSTAT6, and as pERK1/2 was evaluated in T and B cells using phosphoflow cytometry. Results: By evaluating T cell dependent cytokine driven pathways linked to IgA isotype induction we identified a defect involving an IL-21 driven STAT3 activation isolated to B cells in sIgAD individuals. However, all other signaling pathways studied were found to be normal compared to HC. In T cell dependent cytokine driven stimulations linked to IgA isotype induction the following patterns emerged: (i) IL-10 led to significant STAT3 activation in both T- and B cells; (ii) IL-4 stimulation was predominantly confined to STAT6 activation in both T- and B cells, with some effects on STAT3 activation in T-cells; (iii) as expected, of tested stimuli, IL-2 alone activated STAT5 and some STAT3 activation though in both cases only in T-cells; (iv) IL-21 induced significant activation of STAT3 in both T- and B cells, with some effects on STAT5 activation in T-cells; and finally (v) synergistic effects were noted of IL-4+IL-10 on STAT5 activation in T-cells, and possibly STAT6 in both T- and B cells. On the other hand, CPG induced T cell independent activation was confined to ERK1/2 activation in B cells. Conclusion: Our results indicate a diminished STAT3 phosphorylation following IL-21 stimulation solely in B cells from sIgAD individuals. This can represent aberrant germinal center reactions or developmental halt. Thus, our work provides further insight into the unraveling of the previously hypothesized role of IL-21 to reconstitute immunoglobulin production in primary antibody deficiencies.

Transitional B Cells and TLR9 Responses Are Defective in Selective IgA Deficiency.

Selective IgA deficiency (IgAD) is the most common primary antibody deficiency in the western world with affected individuals suffering from an increased burden of autoimmunity, atopic diseases and infections. It has been shown that IgAD B cells can be induced with germinal center mimicking reactions to produce IgA. However, IgA is the most prevalent antibody in mucosal sites, where antigen-independent responses are important. Much interest has recently focused on the role of TLR9 in both naïve and mature B cell differentiation into IgA secreting plasma cells. Here, we analyze the phenotype and function of T and B cells in individuals with IgAD following IgA-inducing CpG-TLR9 stimulations. The IgAD individuals had significantly lower numbers of transitional B cells (CD19+CD24hiCD38hi) and class-switched memory B cells (CD20+CD27+IgD-) ex vivo. However, proportions of T cell populations ex vivo as well as in vitro induced T effector cells and T regulatory cells were comparable to healthy controls. After CpG stimulation, the transitional B cell defect was further enhanced, especially within its B regulatory subset expressing IL-10. Finally, CpG stimulation failed to induce IgA production in IgAD individuals. Collectively, our results demonstrate a defect of the TLR9 responses in IgAD that leads to B cell dysregulation and decreased IgA production.

Human IgG3 with extended half-life does not improve Fc-gamma receptor-mediated cancer antibody therapies in mice.

BACKGROUND: Current anti-cancer therapeutic antibodies that are used in the clinic are predominantly humanized or fully human immunoglobulin G1 (IgG1). These antibodies bind with high affinity to the target antigen and are efficient in activating the immune system via IgG Fc receptors and/or complement. In addition to IgG1, three more isotypes are present in humans, of which IgG3 has been found to be superior compared to human IgG1 in inducing antibody dependent cell cytotoxicity (ADCC), phagocytosis or activation of complement in some models. Nonetheless, no therapeutic human IgG3 mAbs have been developed due to the short in vivo half-life of most known IgG3 allotypes. In this manuscript, we compared the efficacy of V-gene matched IgG1 and IgG3 anti-tumour mAb (TA99) in mice, using natural variants of human IgG3 with short- or long half-life, differing only at position 435 with an arginine or histidine, respectively. RESULTS: In vitro human IgG1 and IgG3 did not show any differences in opsonisation ability of B16F10-gp75 mouse melanoma cells. IgG1, however, was superior in inducing phagocytosis of tumour cells by mouse macrophages. Similarly, in a mouse peritoneal metastasis model we did not detect an improved effect of IgG3 in preventing tumour outgrowth. Moreover, replacing the arginine at position 435 for a histidine in IgG3 to enhance half-life did not result in better suppression of tumour outgrowth compared to wild type IgG3 when injected prior to tumour cell injection. CONCLUSION: In conclusion, human IgG3 does not have improved therapeutic efficacy compared to human IgG1 in a mouse tumour model.

The multiple facets of FcRn in immunity.

The neonatal Fc receptor, FcRn, is best known for its role in transporting IgG in various tissues, providing newborns with humoral immunity, and for prolonging the half-life of IgG. Recent findings implicate the involvement of FcRn in a far wider range of biological and immunological processes, as FcRn has been found to bind and extend the half-life of albumin; to be involved in IgG transport and antigen sampling at mucosal surfaces; and to be crucial for efficient IgG-mediated phagocytosis. Herein, the function of FcRn will be reviewed, with emphasis on its recently documented significance for IgG polymorphisms affecting the half-life and biodistribution of IgG3, on its role in phagocyte biology, and the subsequent role for the presentation of antigens to lymphocytes.

On the perplexingly low rate of transport of IgG2 across the human placenta.

The neonatal receptor, FcRn, mediates both serum half-life extension as well as active transport of maternal IgG to the fetus during pregnancy. Therefore, transport efficiency and half-life go hand-in-hand. However, while the half-life of the human IgG2 subclass is comparable to IgG1, the placental transport of IgG2 is not, with the neonatal IgG1 levels generally exceeding maternal levels at birth, but not for IgG2. We hypothesized that the unique short-hinged structure of IgG2, which enables its κ-, but not λ-isotype to form at least three different structural isoforms, might be a contributing factor to these differences. To investigate whether there was any preference for either light chain, we measured placental transport of IgG subclasses as well as κ/λ-light chain isotypes of IgG1 and IgG2 in 27 matched mother-child pairs. We also studied the half-life of IgG1 and IgG2 light chain isotypes in mice, as well as that of synthesized IgG2 structural isotypes κA and κB. In order to investigate serum clearance of IgG1 and IgG2 light-chain isotypes in humans, we quantified the relative proportions of IgG1 and IgG2 light chains in hypogammaglobulinemia patients four weeks after IVIg infusion and compared to the original IVIg isotype composition. None of our results indicate any light chain preference in either of the FcRn mediated mechanisms; half-life extension or maternal transport.

IgG-effector functions: "the good, the bad and the ugly".

IgG-antibodies are potent and versatile mediators of host protection. They elicit their biological effects through specific interaction of the Fc-part with complement, specific cellular receptors, or both. Several factors should be taken into consideration when analyzing the nature and intensity of the immunological response elicited via IgG-effector functions, especially for the family of IgG-Fc receptors (FcγRs) exclusively expressed on immune cells. These include the various classes of leukocyte FcγR, expressed variably on different immune cells, each with distinct affinity for every IgG subclass, as well as genetic FcγR-polymorphisms affecting expression and affinity for IgG. Furthermore, various aspects of the IgG itself are also crucial for the outcome of the biological response. These include endogenously encoded IgG-polymorphisms, such as IgG3 polymorphisms, and post-transcriptional IgG-modifications, in particular IgG-Fc-glycosylation, affecting IgG effector functions through modified binding affinity to FcγR. These latter aspects concerning the variability in IgG3 on its half-life and placental transport and the clinical consequences of altered IgG-quality through glycosylation, will be the focus of this review.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice.

Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn(-/-) mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4(+) T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8(+) T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.

Comparison of the Fc glycosylation of fetal and maternal immunoglobulin G.

Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylation-selective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fc-specific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.

Familial monoclonal gammopathy: hyper-responsive B cells in unaffected family members.

BACKGROUND: In Iceland, eight families have been identified with multiple cases of monoclonal gammopathies (MG) and other lymphoproliferative diseases. In one of these families with several cases of monoclonal gammopathy of undetermined significance (MGUS) and Waldenströms macroglobulinemia, in vitro stimulation with poke-weed mitogen revealed hyper-responsive B cells showing increased immunoglobulin production in one-third of disease-free family members. DESIGN AND METHODS: In this study, the families were further traced and the list of names produced was compared with The Icelandic Cancer Registry (ICR) to find all recent cases of lymphoproliferative diseases. First-degree relatives and descendants older than 20yrs of age (n=350) were selected for screening for paraprotein. Selected family members were tested for B-cell hyper-responsiveness and the lymphocyte phenotype was analysed by flow cytometry. RESULTS: Comparison of the total list of 4370 family members with the ICR revealed 22 new cases and screening for serum paraprotein identified nine new cases of MG, eight being first-degree relatives of known probands. Sixty cases of lymphoproliferative diseases are currently known within the eight families, five of them containing both IgG/A and IgM disorders. Twelve hyper-responders (HR) were identified in four families, eight from one family, of whom four were known already. Stimulated B cells from HR had a significantly higher proportion of CD27(+) memory/plasma cells than controls. CONCLUSION: Identification of new affected family members by screening confirms a hereditary predisposition to B-cell proliferative diseases. Contrary to most studies, IgG/A and IgM disorders occurred together in five families. In four families, enhanced B-cell responsiveness was found in healthy subjects clustered around cases.

Familial predisposition to monoclonal gammopathy of unknown significance, Waldenström's macroglobulinemia, and multiple myeloma.

The medical literature contains reports of around 130 families with two or more cases of MM, MGUS, or WM. An Icelandic family with multiple cases of MGUS, WM, and lymphoma was first described in 1978. In vitro testing of peripheral blood lymphocytes revealed increased production of immunoglobulins in response to poke-weed mitogen in 10 out of 35 family members, referred to as hyperresponders (HR). Enhanced B-cell survival after stimulation was associated with prolonged expression of Bcl-2. A population-based cancer registry study of 218 MM patients identified 7 additional families. Nine new cases of monoclonal gammopathy were detected by the screening of 350 family members. Further testing confirmed previously identified HR in the originally described family as well as detecting new cases. Only two HR were found in the recently identified families. The long-term aim is to identify the genetic background(s) and biology predisposing to the emergence of a persistent clone of immunoglobulin-producing cells.