Extensive research into ankylosing spondylitis (AS) has suggested the major role of genetics, immune reactions, and the joint–gut axis in its etiology, although an ultimate consensus does not yet exist. The available evidence indicates that both autoinflammation and T-cell-mediated autoimmune processes are actively involved in the disease process of AS. So far, B cells have received relatively little attention in AS pathogenesis; this is largely due to a lack of conventional disease-defining autoantibodies. However, against prevailing dogma, there is a growing body of evidence suggestive of B cell involvement. This is illustrated by disturbances in circulating B cell populations and the formation of auto-reactive and non-autoreactive antibodies, along with B cell infiltrates within the axial skeleton of AS patients. Furthermore, the depletion of B cells, using rituximab, displayed beneficial results in a subgroup of patients with AS. This review provides an overview of our current knowledge of B cells in AS, and discusses their potential role in its pathogenesis. An overarching picture portrays increased B cell activation in AS, although it is unclear whether B cells directly affect pathogenesis, or are merely bystanders in the disease process.

B-cells have received little attention in axial spondyloarthritis (axSpA) and for this reason their role in pathogenesis remains unclear. However, there are indications that B-cells may be involved in the disease process. Our objective was to obtain insights into the composition of the peripheral B-cell compartment of axSpA patients compared to healthy donors (HD) and patients with primary Sjögren’s syndrome (pSS), a typical B-cell-associated autoimmune disease. Special emphasis was given to CD27-negative B-cells expressing low levels of CD21 (CD21^low B-cells), since this subset is implicated in autoimmune diseases with strong involvement of B-cells. Transitional B-cells (CD38^hi) were excluded from the analysis of the CD27^-CD21^low B-cell compartment. This study included 45 axSpA patients, 20 pSS patients and 30 HDs. Intriguingly, compared to HDs the frequency of CD27^-CD38^lowCD21^low B-cells was significantly elevated in both axSpA and pSS patients (P<0.0001 for both comparisons). The frequency of CD27^-CD38^lowCD21^low B-cells expressing the activation-induced immune markers T-bet and CD11c was decreased in axSpA patients compared to HDs. A higher proportion of CD27^-CD38^lowCD21^low B-cells expressed the chemokine receptor CXCR3 in axSpA compared to HDs, suggestive for active involvement of these cells in an inflammatory process. The frequency of CD27^-CD38^lowCD21^low B-cells in axSpA patients correlated positively with age and erythrocyte sedimentation rate. Furthermore, axSpA patients with extra-skeletal manifestations (ESM) showed increased frequencies of CD27^-CD38^lowCD21^low B-cells compared to patients without ESM. In conclusion, our findings are suggestive of active B-cell involvement in the pathogenesis of axSpA, against prevailing dogma.

Objective: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS.Methods: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]).Results: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P< 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P< 0.01 for both pathways). Conversely, transforming growth factor-β signaling and angiogenesis pathway scores were lower in pSS (P= 0.02 andP= 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity.Conclusion: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Radiographic axial spondyloarthritis (r-axSpA) is a chronic rheumatic disease in which innate immune cells and T cells are thought to play a major role. However, recent studies also hint at B cell involvement. Here, we performed an in-depth analysis on alterations within the B-cell compartment from r-axSpA patients. We performed immune gene expression profiling on total peripheral blood B cells from 8 r-axSpA patients and 8 healthy controls (HCs). Next, we explored B cell subset distribution and B-cell receptor (BCR) signaling responses in circulating B cells from 28 r-axSpA patients and 15 HCs, by measuring spleen tyrosine kinase, phosphoinositide 3-kinase and extracellular signal regulated kinase 1/2 phosphorylation upon α-Ig stimulation using phosphoflow cytometry. Immune gene expression profiling indicated an elevated pathway score for BCR signaling in total B cells from r-axSpA patients compared with HCs. Flow cytometric analysis revealed an increase in frequency of both total and double-negative 2 (DN2) B cells in r-axSpA patients compared with HCs. In r-axSpA patients, DN2 B cells displayed an isotype shift towards IgA. Remarkably, where DN2 B cells from HCs were hyporesponsive, these cells displayed significant proximal BCR signaling responses in r-axSpA patients. Enhanced BCR signaling responses were also observed in the transitional and naïve B cell population from r-axSpA patients compared with HCs. The enhanced BCR signaling responses in DN2 B cells correlated with clinical disease parameters. In r-axSpA patients, circulating DN2 B cells are expanded and, together with transitional and naïve B cells, display significantly enhanced BCR signaling responses upon stimulation. Together, our data suggest B cell involvement in the pathogenesis of r-axSpA.

Background The role of B cells in the pathogenesis of ankylosing spondylitis (AS) remains relatively understudied. Nevertheless, available evidence shows presence of B cells at sites of inflammation, higher frequencies of circulating plasmablasts, presence of autoantibodies and beneficial results of B cell depleting therapy with rituximab [1]. We observed previously that a subpopulation of CD27^– B cells, characterized by low expression of the complement receptor CD21 (CD21^low), is increased in patients with AS and patients with primary Sjogren’s syndrome (pSS), a typical B cell mediated systemic autoimmune disease [2]. These CD21^lowCD38^lowCD27^– B cells display a pro-inflammatory phenotype, suggestive for a pathogenic role. Objectives To gain insight into the origin of these CD21^lowCD38^lowCD27^– B cells we analysed the mutation status of the immunoglobulin heavy chain variable (IGHV) gene regions in the total B cell compartment, CD21^low B cell subpopulations and plasmablasts in AS patients in comparison to pSS patients and healthy donors (HD). In addition, we investigated clonal relations between CD21^low B cell subpopulations and plasmablasts in AS. Methods RNA was isolated from sorted total B cells (CD19⁺), two CD21^low B cell populations (CD21^lowCD38^lowCD27^– and CD21^lowCD38^lowCD27⁺) and plasmablasts (CD19⁺CD38⁺⁺CD27⁺) from peripheral mononuclear cells of 10 AS patients fulfilling the mNY criteria (mean age 46 ± 9 years, 60% male, mean ASDAS 2.3 ± 0.8, all HLA-B27+), 10 sex- and age-matched HDs (mean age 48 ± 21 years, 60% male) and 10 age-matched pSS patients (mean age 46 ± 9 years, 20% male). Repertoire analysis of the B cell receptor heavy chain was performed using high throughput sequencing on the Illumina MiSeq Platform. Sequences were analyzed for degree of somatic hypermutation, usage of immunoglobulin heavy chain variable (IGHV) and joining (IGHJ) regions and clonal relationships. Results IGHV sequences with identical heavy chain CDR3 regions were grouped together and were defined as a clone. Analysis of the IGHV mutation status in CD21^low B cells revealed that the majority of clones (71% SD ± 14.1) in the CD21^lowCD38^lowCD27^– subset from patients with AS consisted of clones with a low mutation rate (0-2 mutations in the IGHV region) (Figure 1b). On the contrary, most of the clones in the CD21^lowCD38^lowCD27⁺ (49% SD ± 12.5) and plasmablasts (59% SD ± 12.7) subsets were clones with a higher mutation load (>10 mutations in the IGHV region). A similar mutational pattern was seen in pSS patients and HDs. In AS, IGHV3 and IGHJ4 family genes were predominantly used (Figure 1c). However, we observed no differences in family gene usage among B cell subsets nor between groups. To investigate clonal relationships we performed a CDR3 amino acid network analysis on the top 100 most expanded clones per subset in AS patients and found that the CD21^lowCD38^lowCD27^– subset shared more clones with the CD21^lowCD38^lowCD27⁺ subset (median 3.0 (0.8-7.0 IQR), p< 0.05) compared to plasmablasts (median 0.0 (0-1.3 IQR). Conclusion This study shows that the expanded circulating CD21^lowCD38^lowCD27^– B cell population in AS consists mainly of antigen-inexperienced cells. The presence of shared clones with their CD27⁺ counterpart and plasmablasts suggest that the three subsets are to some extent related. Hence, CD21^lowCD38^lowCD27^– B cells may differentiate towards CD21^lowCD38^lowCD27⁺ B cells and plasmablasts. The relative roles of these CD21^low B cell subsets and plasmablasts in AS pathogenesis remain to be established. References [1]Wilbrink, R et al. (2021). B Cell Involvement in the Pathogenesis of Ankylosing Spondylitis. International journal of molecular sciences. [2]Wilbrink, R et al. (2021). CD27^-CD38^lowCD21^low B-Cells Are Increased in Axial Spondyloarthritis. Frontiers in immunology. Acknowledgements: NIL. Disclosure of Interests Rick Wilbrink: None declared, Linda Van der weele: None declared, Anneke Spoorenberg Consultant of: Abbvie, Pfizer, MSD, UCB, Lilly and Novartis, Grant/research support from: Abbvie, Pfizer, Union Chimique Belge (UCB), Novartis, Niek de Vries: None declared, Frans G.M. Kroese Speakers bureau: BMS Roche and Jannsen-Cilag, Consultant of: BMS, Grant/research support from: BMS, Gwenny M. Verstappen: None declared.

Background In spite of advancements in treatment of ankylosing spondylitis (AS), its pathogenesis remains incompletely understood. The current evidence suggests that AS is both an autoinflammatory and autoimmune disease involving innate immune cells along with autoreactive lymphocytes. B cells have received little attention regarding their role in the pathogenesis of ankylosing spondylitis (AS), although the available evidence shows presence of B cells at inflammatory sites, imbalances in B cell subsets, presence of autoantibodies (anti-CD74/CLIP), and beneficial effects of B cell depletion (anti-CD20) with rituximab [1]. In AS, the overarching picture portrays increased B cell activation, although it is unclear whether B cells directly affect pathogenesis, or are merely bystanders in the disease process. Objectives Our main objective was to study gene expression and gene signalling pathways involved in B cell activation in AS. To this end, we analysed the transcriptomic profile of isolated peripheral B cells from AS patients, healthy donors (HD) and patients with primary Sjögren syndrome (pSS), a typical B-cell-associated autoimmune disease. Methods RNA was isolated from CD19⁺ B cells sorted from peripheral blood mononuclear cells of 8 AS patients (mean age 48.0 ± 9.7 years, 63% male, mean ASDAS 2.5 ± 0.7), 8 sex and age-matched HDs (42.3 ± 17.8 years, 63% male) and 8 age-matched pSS patients (48.4 ± 17.8 years, 13% male). Next, characterization of differentially expressed genes was performed using transcriptomic analysis by the NanoString nCounter Autoimmune Profiling Panel, which utilizes a direct hybridization technique without PCR amplification. After normalization and z-score scaling of pathway genes, pathway scores were calculated based on the first principle component using nCounter advanced analysis software. P-values <0.05 were considered significant. Results All samples passed quality control, except for one pSS patient sample that was being flagged as an outlier by the nCounter advanced analyses software. Analysis of differential gene expression between AS patients and HDs revealed upregulation of 8 genes, including MAPK14, KMT2A and PKM, in B cells from AS patients, and downregulation of two genes, DDIT4 and ATG5. In AS patients, the relative expression of B cell receptor (BCR) signalling (n=60 genes) and Fc receptors and phagocytosis (n=55 genes) pathway genes were both significantly enriched compared to HDs (Figure 1). Although not significant, these pathways also displayed higher pathway scores in pSS patients compared to HDs (P=0.30 and P=0.37, respectively). Other gene pathways involved in B cell activation, such as toll-like receptor signalling were not significantly enriched. Conclusion In this study, we observed several differentially expressed genes involved in signal transduction pathways in B cells from AS patients. Furthermore, we showed increased expression of gene pathways involved in B cell activation in patients with AS compared to HDs. The BCR-mediated activation in AS displays similarities to a bona fide autoimmune disease pSS. These results suggest active involvement of B cells in the disease process of AS. Reference [1]Wilbrink, R et al. (2021). B Cell Involvement in the Pathogenesis of Ankylosing Spondylitis. International journal of molecular sciences. Gene pathway scores of (A) B cell receptor signalling and (B) Fc receptors and phagocytosis between AS, pSS patients and HDs. P-values <0.05 were considered significant. Acknowledgements: NIL. Disclosure of Interests Rick Wilbrink: None declared, Anneke Spoorenberg Consultant of: Abbvie, Pfizer, MSD, UCB, Lilly and Novartis, Grant/research support from: Abbvie, Pfizer, Union Chimique Belge (UCB), Novartis, Frans G.M. Kroese Speakers bureau: BMS Roche and Jannsen-Cilag, Consultant of: BMS, Grant/research support from: unrestricted grants from BMS, Gwenny M. Verstappen: None declared.