Bruton’s tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis. The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA). HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA. HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

Methotrexate (MTX) with its proven efficacy and safety profile remains as the anchor drug for the treatment of rheumatoid arthritis (RA).1 2 However, the impact of MTX alone or in conjunction with antitumour necrosis factor (anti-TNF) on humoral immune system and infection risk varies markedly among patients with RA, suggesting that other host factors influence the therapeutic response to MTX and/or anti-TNF treatment.3 A possible candidate is B-cell activating factor (BAFF), which promotes B-cell activation and differentiation for antibody production.4 When patients with RA received anti-TNF treatment, a high BAFF serum level prevented formation of antidrug antibody in patients taking MTX but not those who did not.5 Thus, in the presence of MTX, BAFF may exert a paradoxical anti-inflammatory effect. Here, we investigated whether high BAFF levels negatively impact vaccine response via the inhibitory BAFF–MTX interaction in patients with RA taking MTX. Patients with RA according to the revised 1987 American College of Rheumatology from the randomised controlled trial (ClinicalTrials.gov identifier: NCT02897011) that aimed to investigate the effects of a 2-week MTX discontinuation on vaccine response to seasonal influenza vaccination were included in this study.6 Patients with RA were randomised to continue MTX or to hold …

Since T cells are key mediators in the adaptive immune system, evaluating antigen-specific T cell immune responses is pivotal to understanding immune function. Commonly used methods for measuring T cell responses include Activation-Induced Marker (AIM) assays and Intracellular Cytokine Staining (ICS). However, combining these approaches has rarely been reported. This study describes a combined AIM + ICS assay and the effect of collecting the supernatant. Peripheral blood mononuclear cells (PBMCs) from seven healthy donors were stimulated with DMSO (negative control), Epstein-Barr virus (EBV) peptide pools, and PHA (positive control). The AIM markers OX40 + CD137+ were used for CD4+ T cells and CD69 + CD137+ and CD107a + CD137+ for CD8+ T cells. Cytokine-secreting cells were identified as CD40L+ cytokine+ for CD4+ and CD69+ or CD107 + cytokine+ for CD8+ T cells. Half of the supernatant was collected before adding the BFA/Monensin/CD137 antibody solution to assess the impact on T cell responses. The CD107a + CD137+ AIM markers combination had a lower background than CD69 + CD137+, making CD107a+ a more sensitive marker for CD8+ AIM markers. Collecting half of the supernatant did not significantly affect the immune responses. Our AIM + ICS combined protocol enables the simultaneous assessment of activation and cytokine release reducing the sample volume for testing T cell responses. We also show that collecting half of the supernatant does not significantly interfere with immune responses detection.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes acute febrile illness that can progress into chronic arthritis-like disease (CHIKVD) in humans. CD4⁺ T cells have important functions in CHIKV infection, yet the CHIKV target proteins for these CD4 + T cells are poorly characterized. Here, by stimulating PBMCs collected from individuals with chronic CHIKVD with peptides spanning the entire CHIKV proteome, we provide a comprehensive landscape of CHIKV CD4⁺ T cell epitopes. We identify three immunodominant regions and associated core motifs in CHIKV E1, nsP1 and CP proteins. In addition, by in silico assessment of the sequence conservation of CHIKV proteome with closely related alphaviruses, we define CHIKV epitopes conserved across arthritogenic and encephalitic viruses. Overall, our work describes CD4⁺ T cell targets of CHIKV in humans, thereby assisting in studying the functions of CD4⁺ T cells in CHIKV pathogenesis and vaccine design.

Background: To investigate the role of CD47 in inflammatory responses in systemic lupus erythematosus (SLE). Methods: Expression of CD47 and signal regulatory protein alpha (SIRPα) by peripheral blood mononuclear cells (PBMCs) and changes in CD47 expression after exposure to SLE serum, healthy control (HC) serum, recombinant interferon (IFN)-α, or tumor necrosis factor (TNF)-α were examined. Human monocytes and THP1 cells were incubated with lipopolysaccharide (LPS), an anti-CD47 antibody, or both. TNF-α production was examined. Sera from SLE patients and HCs were screened to detect autoantibodies specific for CD47. Results: Twenty-five SLE patients and sixteen HCs were enrolled. CD47 expression by monocytes from SLE patients was higher than those from HCs (mean fluorescence intensity ± SD: 815.9 ± 269.4 vs. 511.5 ± 199.4, respectively; p < 0.001). CD47 expression by monocytes correlated with SLE disease activity (Spearman’s rho = 0.467, p = 0.019). IFN-α but not TNF-α, increased CD47 expression. Exposing monocytes to an anti-CD47 antibody plus LPS increased TNF-α production by 21.0 ± 10.9-fold (compared with 7.3 ± 5.5-fold for LPS alone). Finally, levels of autoantibodies against CD47 were higher in SLE patients than in HCs (21.4 ± 7.1 ng/mL vs. 16.1 ± 3.1 ng/mL, respectively; p = 0.02). Anti-CD47 antibody levels did not correlate with disease activity (Spearman’s rho = −0.11, p = 0.759) or CD47 expression on CD14 monocytes (Spearman’s rho = 0.079, p = 0.838) in patients. Conclusions: CD47 expression by monocytes is upregulated in SLE and correlates with disease activity. CD47 contributes to augmented inflammatory responses in SLE. Targeting CD47 might be a novel treatment for SLE.