Essential role for CD103 in the T cell–mediated regulation of experimental colitis

The integrin CD103 is highly expressed at mucosal sites, but its role in mucosal immune regulation remains poorly understood. We have analyzed the functional role of CD103 in intestinal immune regulation using the T cell transfer model of colitis. Our results show no mandatory role for CD103 expression on T cells for either the development or CD4+CD25+ regulatory T (T reg) cell–mediated control of colitis. However, wild-type CD4+CD25+ T cells were unable to prevent colitis in immune-deficient recipients lacking CD103, demonstrating a nonredundant functional role for CD103 on host cells in T reg cell–mediated intestinal immune regulation. Non–T cell expression of CD103 is restricted primarily to CD11chighMHC class IIhigh dendritic cells (DCs). This DC population is present at a high frequency in the gut-associated lymphoid tissue and appears to mediate a distinct functional role. Thus, CD103+ DCs, but not their CD103− counterparts, promoted expression of the gut-homing receptor CCR9 on T cells. Conversely, CD103− DCs promoted the differentiation of IFN-γ–producing T cells. Collectively, these data suggest that CD103+ and CD103− DCs represent functionally distinct subsets and that CD103 expression on DCs influences the balance between effector and regulatory T cell activity in the intestine

Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms

Autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA are associated with inflammatory conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications. The mAbs recognized MDA-adducts in a variety of proteins. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. These anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses

IL-17 and IL-23 in lupus nephritis - association to histopathology and response to treatment

Background: Recent studies indicate a central role for the IL-23/IL-17 axis in the pathogenesis of lupus nephritis (LN) but the importance in the context of treatment outcome is unknown. We studied various cytokines, including the IL-23/IL-17 axis, in association to histopathology and response to therapy. Methods: Fifty-two patients with active LN were included. Renal biopsies were performed at baseline and after immunosuppressive treatment. Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-10, IL-17, IL-23 and TGF-beta were analysed at both biopsy occasions and in 13 healthy controls. IL-17 expression in renal tissue was assessed by immunohistochemistry. Biopsies were evaluated regarding WHO-classification and renal disease activity was estimated using the BILAG-index. Improvement of 2 grades in renal BILAG was regarded complete response, and 1 grade partial response. Results: At baseline, all patients had high disease activity (BILAG A/B). Baseline levels of IL-6, IL-10, IL-17, IL-23 (p < 0.001) and IFN-gamma (p = 0.03) were increased in patients vs. controls. In contrast, TGF-beta was lower in patients compared to controls (p < 0.001). Baseline levels of IL-17 were higher in patients with persisting active nephritis (WHO III, IV, V) after treatment, i.e. a poor histological response, vs. WHO I-II (p < 0.03). At follow-up, IL-23 were higher in BILAG-non-responders vs. responders (p < 0.05). Immunostaining of renal tissue revealed IL-17 expression in inflammatory infiltrates. Conclusions: High baseline IL-17 predicted an unfavourable histopathological response, and BILAG-non-responders had high IL-23, indicating that that a subset of LN-patients has a Th-17 phenotype that may influence response to treatment and could be evaluated as a biomarker for poor therapeutic response

IL-17 and IL-23 in lupus nephritis - association to histopathology and response to treatment

Background: Recent studies indicate a central role for the IL-23/IL-17 axis in the pathogenesis of lupus nephritis (LN) but the importance in the context of treatment outcome is unknown. We studied various cytokines, including the IL-23/IL-17 axis, in association to histopathology and response to therapy. Methods: Fifty-two patients with active LN were included. Renal biopsies were performed at baseline and after immunosuppressive treatment. Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-10, IL-17, IL-23 and TGF-beta were analysed at both biopsy occasions and in 13 healthy controls. IL-17 expression in renal tissue was assessed by immunohistochemistry. Biopsies were evaluated regarding WHO-classification and renal disease activity was estimated using the BILAG-index. Improvement of 2 grades in renal BILAG was regarded complete response, and 1 grade partial response. Results: At baseline, all patients had high disease activity (BILAG A/B). Baseline levels of IL-6, IL-10, IL-17, IL-23 (p < 0.001) and IFN-gamma (p = 0.03) were increased in patients vs. controls. In contrast, TGF-beta was lower in patients compared to controls (p < 0.001). Baseline levels of IL-17 were higher in patients with persisting active nephritis (WHO III, IV, V) after treatment, i.e. a poor histological response, vs. WHO I-II (p < 0.03). At follow-up, IL-23 were higher in BILAG-non-responders vs. responders (p < 0.05). Immunostaining of renal tissue revealed IL-17 expression in inflammatory infiltrates. Conclusions: High baseline IL-17 predicted an unfavourable histopathological response, and BILAG-non-responders had high IL-23, indicating that that a subset of LN-patients has a Th-17 phenotype that may influence response to treatment and could be evaluated as a biomarker for poor therapeutic response

Haplotype-Specific Expression Analysis of MHC Class II Genes in Healthy Individuals and Rheumatoid Arthritis Patients

HLA-DRB1 alleles have been associated with several autoimmune diseases. For anti-citrullinated protein antibody positive rheumatoid arthritis (RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to study the genetic regulation of major histocompatibility complex (MHC) Class II gene expression in immune cells, we investigated transcriptomic profiles of a variety of immune cells from healthy individuals carrying different HLA-DRB1 alleles. Sequencing libraries from peripheral blood mononuclear cells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes of 32 genetically pre-selected healthy female individuals were generated, sequenced and reads were aligned to the standard reference. For the MHC region, reads were mapped to available MHC reference haplotypes and AltHapAlignR was used to estimate gene expression. Using this method, HLA-DRB and HLA-DQ were found to be differentially expressed in different immune cells of healthy individuals as well as in whole blood samples of RA patients carrying HLA-DRB1 SE-positive versus SE-negative alleles. In contrast, no genes outside the MHC region were differentially expressed between individuals carrying HLA-DRB1 SE-positive and SE-negative alleles, thus HLA-DRB1 SE alleles have a strong cis effect on gene expression. Altogether, our findings suggest that immune effects associated with different allelic forms of HLA-DR and HLA-DQ may be associated not only with differences in the structure of these proteins, but also with differences in their expression levels.</p&gt

Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain

Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population