Mimicry of Pre–B Cell Receptor Signaling by Activation of the Tyrosine Kinase Blk

During B lymphoid ontogeny, assembly of the pre–B cell receptor (BCR) is a principal developmental checkpoint at which several Src-related kinases may play redundant roles. Here the Src-related kinase Blk is shown to effect functions associated with the pre-BCR. B lymphoid expression of an active Blk mutant caused proliferation of B progenitor cells and enhanced responsiveness of these cells to interleukin 7. In mice lacking a functional pre-BCR, active Blk supported maturation beyond the pro–B cell stage, suppressed VH to DJH rearrangement, relieved selection for productive heavy chain rearrangement, and stimulated κ rearrangement. These alterations were accompanied by tyrosine phosphorylation of immunoglobulin β and Syk, as well as changes in gene expression consistent with developmental maturation. Thus, sustained activation of Blk induces responses normally associated with the pre-BCR

Peripheral blood natural killer cell percentages in granulomatosis with polyangiitis correlate with disease inactivity and stage

Introduction: The role of CD3−CD56+ natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. Recently, it has been shown that peripheral blood NK cells can kill renal microvascular endothelial cells, suggesting a pathogenic role of NK cells in this disease. So far, subset distribution, phenotype, and function of peripheral blood NK cells in relation to GPA disease activity have not been elucidated. Moreover, it is not known whether NK cells infiltrate GPA tissue lesions. Methods: Paraffin sections of GPA granulomas and controls were stained with anti-CD56 and anti-CD3 antibodies. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. NK cell degranulation was analyzed using cocultures of patient PBMCs with target cells and surface expression of CD107a. Clinical data were extracted from medical records. Statistical analysis was performed in an exploratory way. Results: CD56+ cells were not detectable in active granulomatous GPA lesions but were found frequently in granulomas from tuberculosis and sarcoidosis patients. In GPA, the proportion of NK cells among peripheral blood lymphocytes correlated negatively with the Birmingham Vasculitis Activity Score (BVAS) (n = 28). Accordingly, NK cell percentages correlated positively with the duration of remission (n = 28) and were significantly higher in inactive GPA (BVAS = 0, n = 17) than in active GPA, healthy controls (n = 29), and inactive control diseases (n = 12). The highest NK cell percentages were found in patients with long-term remission and tapered immunosuppressive therapy. NK cell percentages >18.5 % of peripheral blood lymphocytes (n = 12/28) determined GPA inactivity with a specificity of 100 %. The differentiation into CD56dim and CD56bright NK cell subsets was unchanged in GPA (n = 28), irrespective of disease activity. Similar surface expression of the activating NK cell-receptors (NKp30, NKp46, and NKG2D) was determined. Like in healthy controls, GPA NK cells degranulated in the presence of NK cell receptor ligand bearing epithelial and lymphatic target cells. Conclusions: NK cells were not detectable in GPA granulomas. Peripheral blood NK cell percentages positively correlate with the suppression of GPA activity and could serve as a biomarker for GPA activity. Peripheral blood NK cells in GPA patients are mature NK cells with preserved immune recognition

Targeting of Janus Kinases Limits Pro-Inflammatory but Also Immunosuppressive Circuits in the Crosstalk between Synovial Fibroblasts and Lymphocytes

Crosstalk between synovial fibroblasts (SF) and immune cells plays a central role in the development of rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi) have proven efficacy in the treatment of RA, although clinical responses are heterogeneous. Currently, little is known regarding how JAKi affect pro- and anti-inflammatory circuits in the bidirectional interplay between SF and immune cells. Here, we examined the effects of tofacitinib, baricitinib and upadacitinib on crosstalk between SF and T or B lymphocytes in vitro and compared them with those of biologic disease modifying anti-rheumatic drugs (bDMARDs). JAKi dose-dependently suppressed cytokine secretion of T helper (Th) cells and decreased interleukin (IL)-6 and matrix metalloproteinase (MMP)3 secretion of SF stimulated by Th cells. Importantly, JAK inhibition attenuated the enhanced memory response of chronically stimulated SF. Vice versa, JAKi reduced the indoleamine-2,3-dioxygenase (IDO)1-mediated suppression of T cell-proliferation by SF. Remarkably, certain bDMARDs were as efficient as JAKi in suppressing the IL-6 and MMP3 secretion of SF stimulated by Th (adalimumab, secukinumab) or B cells (canakinumab) and combining bDMARDs with JAKi had synergistic effects. In conclusion, JAKi limit pro-inflammatory circuits in the crosstalk between SF and lymphocytes; however, they also weaken the immunosuppressive functions of SF. Both effects were dose-dependent and may contribute to heterogeneity in clinical response to treatment

Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

Background!#!Investigating the pathophysiological mechanisms of early osteoarthritis (OA) is of utmost interest since this stage holds the strongest promise for therapeutic interventions. The aims of this study were to analyze if synovial inflammation is already present in early OA and to characterize the involved cell populations, by investigating synovial fluid (SF) and synovial membrane (SM) of early OA patients for the presence and polarization status of CD4 T cells.!##!Methods!#!A quantitative analysis of CD4!##!Results!#!SF and SM showed a distinct infiltration of CD4!##!Conclusions!#!Early OA joints show already significant inflammation through CD

Activation of natural killer cells by rituximab in granulomatosis with polyangiitis

OBJECTIVE: In the last few years, anti-CD20 antibody rituximab profoundly changed the therapeutic landscape of granulomatosis with polyangiitis (GPA). Here, we investigated whether natural killer (NK) cells may play a role in rituximab’s mechanism of action in GPA. METHODS: B cell depletion, NK cell degranulation, and the expression of CD69 and CD16 on NK cells were measured in a series of in vitro experiments using peripheral blood mononuclear cells (PBMCs). In vivo activation of NK cells was investigated in patients receiving rituximab infusions. Cells were analyzed by seven-color flow cytometry. RESULTS: NK cells from GPA patients were activated by immobilized rituximab. Also soluble rituximab activated NK cells, provided that B cells were present. NK cells degranulated and expressed the activation marker CD69 while CD16 expression was decreased. This activation of NK cells by soluble rituximab was accompanied by a reduction of B cells. The next-generation anti-CD20 antibody obinutuzumab showed stronger effects compared to rituximab on both the reduction of B cells and the activation of NK cells. Finally, we found that rituximab led to the activation of NK cells in vivo, provided that B cells were not depleted due to prior rituximab infusions. CONCLUSION: B cell-bound rituximab activates NK cells in GPA. While NK cells therefore participate in rituximab’s mechanism of action in humans, their potential may be more efficiently exploited, e.g., by Fc engineering of therapeutic antibodies

Increase of aerobic glycolysis mediated by activated T helper cells drives synovial fibroblasts towards an inflammatory phenotype: new targets for therapy?

A dysregulated glucose metabolism in synovial fibroblasts (SF) has been associated with their aggressive phenotype in rheumatoid arthritis (RA). Even though T helper (Th) cells are key effector cells in the propagation and exacerbation of synovitis in RA, little is known about their influence on the metabolism of SF. Thus, this study investigates the effect of Th cells on the glucose metabolism and phenotype of SF and how this is influenced by the blockade of cytokines, janus kinases (JAKs) and glycolysis. Methods: SF from patients with RA or osteoarthritis (OA) were cultured in the presence of a stable glucose isotopomer ([U-13C]-glucose) and stimulated with the conditioned media of activated Th cells (ThCM). Glucose consumption and lactate production were measured by proton nuclear magnetic resonance (1H NMR) spectroscopy. Cytokine secretion was quantified by ELISA. The expression of glycolytic enzymes was analysed by PCR, western blot and immunofluorescence. JAKs were blocked using either baricitinib or tofacitinib and glycolysis by using either 3-bromopyruvate or FX11. Results: Quiescent RASF produced significantly higher levels of lactate, interleukin (IL)-6 and matrix metalloproteinase (MMP) 3 than OASF. Stimulation by ThCM clearly changed the metabolic profile of both RASF and OASF by inducing a shift towards aerobic glycolysis with strongly increased lactate production together with a rise in IL-6 and MMP3 secretion. Interestingly, chronic stimulation of OASF by ThCM triggered an inflammatory phenotype with significantly increased glycolytic activity compared to unstimulated, singly stimulated or restimulated OASF. Finally, in contrast to cytokine-neutralizing biologics, inhibition of JAKs or glycolytic enzymes both significantly reduced lactate production and cytokine secretion by Th cell-stimulated SF. Conclusions: Soluble mediators released by Th cells drive SF towards a glycolytic and pro-inflammatory phenotype. Targeting of JAKs or glycolytic enzymes both potently modulate SF’s glucose metabolism and decrease the release of IL-6 and MMP3. Thus, manipulation of glycolytic pathways could represent a new therapeutic strategy to decrease the pro-inflammatory phenotype of SF

Effect of JAK Inhibition on the Induction of Proinflammatory HLA–DR+CD90+ Rheumatoid Arthritis Synovial Fibroblasts by Interferon-γ

OBJECTIVE: Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15-fold expanded HLA–DR+CD90+ synovial fibroblasts potentially act as key mediators of inflammation. The reasons for the expansion of HLA–DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon-γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA–DR+CD90+ synovial fibroblasts. METHODS: We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets. RESULTS: We detected enriched and activated Fcγ receptor type IIIa–positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell–derived IFNγ induced HLA–DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine-secreting HLA–DR+CD90+ phenotype. HLA–DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA–DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell–derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA–DR induction and blocked proinflammatory signals to T cells. CONCLUSION: The HLA–DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin-6–producing, and likely antigen-presenting synovial fibroblasts can be targeted by JAK inhibition