H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones.

We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

Characterization of a Synovial B Cell-Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints.

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast-like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA- RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti-cit-CRT Abs were preferentially detected in ACPA+ versus ACPA- RA sera. We identified a synovial B cell-derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Efficient generation of a stable CHO-K1 cell line overexpressing the human water channel aquaporin-5 as tool to generate therapeutic antibodies.

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren's syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Adaptor protein LNK is a negative regulator of brain neural stem cell proliferation after stroke.

Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain.

Generation of Recombinant Monoclonal Antibodies from Single B Cells Isolated from Synovial Tissue of Rheumatoid Arthritis Patients.

Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS+ synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.

The putative tumor suppressor gene EphA7 is a novel BMI-1 target.

Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation.

The functional interaction of 14-3-3 proteins with the ERK1/2 scaffold KSR1 occurs in an isoform-specific manner.

Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3gamma with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3gamma. Specific binding to 14-3-3gamma protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3gamma resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3gamma being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3gamma heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3gamma. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation.