Anti-CD40 monoclonal antibody, clone JM-B267 [FITC]

Objective Primary Sjogren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes. Methods Patients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed. Results Gene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors. Conclusions SGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 mu g of CpG-ODN or saline (n=40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n=3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4(+) and CD8(+) T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 mu g and 50 mu g of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1x104 or 1x105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 mu g and 50 mu g of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.