Anil K. Chauhan

Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC)

The complement regulatory (CR) proteins clusterin and vitronectin bind to the membrane attack complex (MAC) and thus prevent cytolysis. In this report, we demonstrate the presence of both of these CR proteins on MAC bound to circulating immune complexes (CIC). We measured the amount of clusterin and vitronectin on MAC in plasma, also referred to as soluble MAC (SMAC), as well as on MAC bound to CIC (MAC–CIC), using antibody directed to polymerized C9 in systemic lupus erythematosus (SLE) patients. We observed a strong correlation among the quantities of SMAC and MAC–CIC. The amount of both clusterin and vitronectin associated with MAC–CIC was two‐ to threefold higher in comparison to the SMAC. Patients with high levels of clusterin and vitronectin demonstrated renal involvement. We hypothesize that these complement regulatory proteins besides regulating the insertion of MAC play other critical roles, in disease pathogenesis.

Evidence of fibrinogen as a target of citrullination in IgM rheumatoid factor-positive polyarticular juvenile idiopathic arthritis

Background Several studies have noted the significance of measuring anti-cyclic citrullinated peptide (CCP) antibodies in juvenile idiopathic arthritis (JIA) as an important indicator for destructive disease, as is the case in rheumatoid arthritis (RA). While the role of anti-CCP antibodies in RA and JIA has become better understood, the identity of the target proteins of this modification has remained elusive. In this study, we evaluated serum from patients with various subtypes of JIA to investigate the presence of anti-deiminated (citrullinated) fibrinogen and anti-citrullinated α-enolase antibodies, and their association with RF and anti-CCP antibody isotypes.

Proteomic analysis of circulating immune complexes in juvenile idiopathic arthritis reveals disease-associated proteins

Juvenile idiopathic arthritis reflects a group of clinically heterogeneous arthritides hallmarked by elevated concentrations of circulating immune complexes. In this study, the circulating immune complex proteome was examined to elucidate disease‐associated proteins that are overexpressed in patients with an aggressive, and at times destructive, disease phenotype. To solve this proteome, circulating immune complexes were isolated from the sera of patients with chronic, erosive or early‐onset, aggressive disease and from patients in medical remission or healthy controls subsequent to protein separation by 2‐DE. Thirty‐seven protein spots were overexpressed in the circulating immune complexes of the aggressive disease groups as compared to controls, 28 of which have been confidently identified to date. Proteolytic fragments of glyceraldehyde‐3‐phosphate dehydrogenase, serotransferrin, and α‐1‐antitrypsin have been identified among others. In total, these 28 putative disease‐associated proteins most definitely contribute to immune complex formation and likely have a significant role in disease etiology and pathogenesis. Moreover, these proteins represent markers of aggressive disease, which could aid in diagnosis and management strategies, and potential therapeutic targets to prevent or control disease outcome. This is the first in‐depth analysis of the circulating immune complex proteome in juvenile idiopathic arthritis.

Immune complexes and late complement proteins trigger activation of Syk tyrosine kinase in human CD4+ T cells

In systemic lupus erythematosus (SLE), the autoantibodies that form immune complexes (ICs) trigger activation of the complement system. This results in the formation of membrane attack complex (MAC) on cell membrane and the soluble terminal complement complex (TCC). Hyperactive T cell responses are hallmark of SLE pathogenesis. How complement activation influences the T cell responses in SLE is not fully understood. We observed that aggregated human γ‐globulin (AHG) bound to a subset of CD4+ T cells in peripheral blood mononuclear cells and this population increased in the SLE patients. Human naive CD4+ T cells, when treated with purified ICs and TCC, triggered recruitment of the FcRγ chain with the membrane receptor and co‐localized with phosphorylated Syk. These events were also associated with aggregation of membrane rafts. Thus, results presented suggest a role for ICs and complement in the activation of Syk in CD4+ T cells. Thus, we propose that the shift in signalling from ζ‐chain‐ZAP70 to FcRγ chain‐Syk observed in T cells of SLE patients is triggered by ICs and complement. These results demonstrate a link among ICs, complement activation and phosphorylation of Syk in CD4+ T cells.

Evaluation of anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies in patients with juvenile idiopathic arthritis

BackgroundTo determine the prevalence and significance of anti-citrullinated vimentin and anti-citrullinated type II collagen antibodies and elucidate their role in the disease process of juvenile idiopathic arthritis (JIA).MethodsSera were obtained from 95 patients with various subtypes of JIA, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. Antibodies were measured in the sera against citrullinated and native type II collagen and vimentin (vim1-16 and vim 59-74) by enzyme-linked immunosorbent assay. Samples were compared to anti-cyclic citrullinated peptide (anti-CCP) antibody and rheumatoid factor (RF) isotypes, and our previously measured anti-citrullinated fibrinogen and α-enolase antibodies on the same patient population, in addition to erythrocyte sedimentation rate and C-reactive protein. The relationship between the anti-citrullinated antibody profile and disease activity and joint damage were also investigated.ResultsTwenty-three JIA patients (24%) demonstrated reactivity to anti-citrullinated type II collagen. Ten JIA patients (10.5%) demonstrated reactivity to anti-citrullinated vimentin 1–16 antibodies and 7 (7.4%) to anti-citrullinated vimentin 59–74 antibodies. One IgM RF-positive polyarticular patient was positive for all 5 of the citrullinated autoantibodies tested. Thirty-seven different subsets of patients were identified based on their anti-citrullinated autoantibody and RF isotype profile. No significant associations were noted with anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies with joint damage or disease activity. Anti-citrullinated vimentin 59–74 antibodies demonstrated the highest overall specificity at 89.7%, with anti-citrullinated vimentin 1–16 and anti-citrullinated type II collagen antibodies at 86.2%.ConclusionThis study demonstrates that antibodies to multiple citrullinated epitopes are present in the sera of patients with various subtypes of JIA. It also demonstrates the frequent occurrence of anti-citrullinated type II collagen and anti-citrullinated fibrinogen antibodies. The presence of autoantibodies to citrullinated antigens in JIA patients is highly diverse.

Abnormal κ:λ Light Chain Ratio in Circulating Immune Complexes as a Marker for B Cell Activity in Juvenile Idiopathic Arthritis

Patients with juvenile idiopathic arthritis (JIA) have been shown to have elevated levels of circulating immune complexes (CICs) which correlated with disease activity. Our aim was to assess B cell activity by measuring the amount of and the κ:λ chain immunoglobulin light (L) chain ratio in CICs from JIA patients and to determine potential evidence for either an antigen‐driven response or B‐cell receptor editing. We used an enzyme‐linked immunosorbent assay to measure κ and λ chains present in the CICs from the sera of patients with JIA. Statistical analysis was performed using Pearsons correlation, one‐way ANOVA and Bonferroni post hoc analysis. Sera from 44 JIA patients were examined for the concentration of L chains in CICs. Healthy controls had a κ:λ chain ratio of 1.2:1, whereas this ratio was reversed among JIA subgroups with RF‐positive polyarthritis (1:1.2), RF‐negative polyarthritis (1:1.3), oligoarthritis (1:2.3) and systemic‐onset arthritis (1:2.5). In addition, overall λ chain selection was not significantly associated with a particular immunoglobulin heavy (H) chain and occurred with all immunoglobulin isotypes. We showed preferential selection of λ chains contributing to the formation of potentially pathogenic CICs from JIA patients, of all onset types compared to healthy controls, in an H chain‐independent manner. The reversal of κ:λ chain ratio within the JIA CICs and association with all immunoglobulin isotypes demonstrated the potential for L chain editing. Furthermore, we conclude that a reversal of the normal κ:λ chain ratio in JIA CICs may be used as a marker for increased B‐cell activity.

FcγRIIIa-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulates Toll-like Receptor (TLR) Expression.

CD4+ T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4+ T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4+ T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4+ T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A+ and IFN-γhigh CD4+ T-cells in vitro. Cytokines IL-1β, IL-6, TGF-β1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4+ T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk up-regulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these toll-like receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses.

Detection of drug specific circulating immune complexes from in vivo cynomolgus monkey serum samples

BACKGROUND Administration of a biotherapeutic can result in the formation of anti-drug antibodies (ADAs). The resulting ADA can potentially form immune complexes (ICs) with the drug leading to altered pharmacokinetic (PK) profiles and/or adverse events. Furthermore the presence of such complexes may interfere with accurate PK assessment, and/or detection of ADA in immunogenicity assays. Here, we present two assays to detect the presence of drug-ADA immune complexes in cynomolgus monkeys. RESULTS Serum samples were analyzed for IC formation in vivo. 8/8 tested animals were positive for drug specific IC. Depending on the time point tested 4/8 or 7/8 animals tested positive for ADA during drug dosing. All 8 animals were confirmed positive for ADA during the washout phase, indicating drug interference in the bridging assay. Relative amount of IC over time was determined and its correlation with PK and ADA was then assessed. Multivariate data analysis demonstrates good correlation between signals obtained from the anti-drug and FcγRIIIa based capture assays, although due to its biological characteristic FcγRIIIa based assay captured only a subset of drug specific IC. In one animal IC remained in circulation even when the drug levels decreased below detection limit. CONCLUSION Results from this study indicate the presence of IC during administration of an immunogenic biotherapeutic. Potential application of these assays includes detection of ADA in an IC during high drug levels. The results on the kinetics of IC formation during ADA response can complement the understanding of PK and ADA profiles. Moreover, the presence of IC indicates possible ADA interference in standard PK assays and potential underestimation of total drug exposure in toxicology studies. In addition this study also highlights the need to understand downstream in vivo consequences of drug-ADA IC as no animals under investigation developed adverse events.

Induced Expression of FcγRIIIa (CD16a) on CD4+ T Cells Triggers Generation of IFN-γhigh Subset

Background: Fcγ-receptors play an important role in the immune responses. Results: We show that upon activation by immune complexes peripheral CD4+ T-lymphocytes express FcγRIIIa and produce a subset that express high levels of IFN-γ. Conclusion: We describe for the first time a role for FcγRIIIa in CD4+ T-cell differentiation. Significance: This CD4+FcγRIIIa+IFN-γhigh subset will be critical in understanding the underlying autoimmune pathologies. Whether or not CD4+ T-cells express low affinity receptor FcγRIIIa (CD16a) in disease pathology has not been examined in great detail. In this study, we show that a subset of activated CD4+ T-cells in humans express FcγRIIIa. The ligation of FcγRIIIa by immune complexes (ICs) in human CD4+ T-cells produced co-stimulatory signal like CD28 that triggered IFN-γ production. The induced expression of FcγRIIIa on CD4+ helper T-cells is an important finding since these receptors via ITAM contribute to intracellular signaling. The induced expression of FcγRIIIa on CD4+ T helper cells and their ability to co-stimulate T-cell activation are important and novel findings that may reveal new pathways to regulate adaptive immune responses during inflammation and in autoimmunity.

Immune complexome analysis reveals the specific and frequent presence of immune complex antigens in lung cancer patients: A pilot study.

Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor‐infiltrating lymphocyte (TIL) transfer, have been developed to boost the endogenous immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Also, the selection of tumor‐reactive T cells for TIL relies on information regarding mutated antigens in tumors and does not reflect other factors involved in protein antigenicity. It is therefore essential to engineer therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., “focused cancer immunotherapy”) based on tumor antigens that are specifically expressed in the tumor of a certain cancer and in many patients with this cancer. Immune complexes (ICs) are the direct and stable products of immunological recognition by humoral immunity. Here, we searched for tumor‐specific IC antigens in each of five cancers (lung (n = 28), colon (n = 20), bladder (n = 20), renal cell (n = 15) and malignant lymphoma (n = 9)), by using immune complexome analysis that comprehensively identifies and profiles the constituent antigens in ICs. This analysis indicated that gelsolin and inter‐alpha‐trypsin inhibitor heavy chains were specifically and frequently detected (at a frequency higher than 80%), and that phosphoproteins (VENTX, VCIP135) were also specifically present in the ICs of lung cancer patients. Immune complexome analysis successfully identified several tumor‐specific IC antigens with high detection frequency in lung cancer patients. These specific antigens are required to validate the clinical benefit by further analysis using a large number of patients.