Helicobacter-aktive B(HAKT-B) hücrelerinin CD4+ T hücreleri ile fonksiyonel etkileşimleri

Helicobacter pylori is a spiral-shaped, gram-negative bacterium that infects the gastric mucosa of more than half of the world's population. The infection initially occurs in childhood, becomes persistent and the chronic infection leads to gastric inflammation. A major virulence factor of H. pylori is the cytotoxin-associated gene A (CagA) protein and this CagA protein interacts with several intracellular components of signal transduction and activates some crucial signaling pathways. Helicobacter has developed a variety of mechanisms to persist in the gastric mucosa. Gastric epithelial cells (GECs) are primary target for H. pylori infection, therefore they are the first point of contact for H. pylori and activate an innate immune response through TLRs. Rather than being a strong TLR4 ligand, H. pylori LPS is thought to activate TLR2 on gastric epithelial cells. Animal and cell culture experiments suggested that ligands in Helicobacter species can bind to TLR2 and activate NF-κB in epithelial cells. In mouse studies, Helicobacter felis (H. felis) is mainly used because it is more immunogenic than H.pylori in mice. B cells and their cytokines have important roles in Helicobacter infections by balancing between the infection and T cell driven gastric immunopathology. The ability of B cells to interact with pathogenic T cells and to produce anti-inflammatory cytokines such as IL-10 is crucial to dampen harmful immune responses. It has been found that B-cells exposed to Helicobacter sonicate produced large amounts of the regulatory cytokine IL-10. Using mouse models of Helicobacter-induced gastric premalignant pathology, it is shown that IL-10 secretion by purified B cells absolutely required MyD88 signaling and TLR-2. The TLR-2- dependent Helicobacter activation of B cells differentiates them into IL-10 and TGF-β producing regulatory B cells. Both IL-10 and TGF-β have crucial effects on T cell differentiation. When co-cultured with Helicobacter-activated B cells, naive CD4+ T-cells are shown to produce IL-10 and differentiate into T regulatory 1 (Tr1)-like cells. In addition to that, it is suggested that Breg cells contribute to regulatory T-cell induction by producing TGF-β. At the same time, studies have shown convincingly that TGF-β is required for Th17 differentiation in vitro and in vivo. In previous studies in our laboratory, Helicobacter-activated total B cells were separated into two subgroups: IL-10+ B cells and IL-10- B cells. The experimental results showed that Helicobacter-activated IL-10+ B cells are the source of the IL-10 production while Helicobacter-activated IL-10-B cells are mostly TGF-β positive. However, it was not clear if the Helicobacter-acitvated-IL-10+ B cells or the Helicobacter-activated-IL-10- B were specifically causing the Tr1 differentiation. Taking account that Bregs are known for producing IL-10 and TGF-β which are key cytokines in T cell differentiation, the interaction between Helicobacter-activated B cell subgroups and CD4+ T cell differentiation was investigated. By magnetic isolation techniques, CD19+B cells and CD4+T cells were isolated from the spleens of C57BL/6 mice with high purities (with an average of 90% and 93%, respectively). Following the B cell isolation, cells were treated with Helicobacter felis sonicate (10 µg/ml) for 24 hours. For the last 5 hours of incubation, to induce an optimal IL-10 production and secretion, PMA (50 ng/ml) and ionomycin (500 ng/ml) were added. After the in vitro stimulation of B cells, IL-10 producing B cells were labeled and the IL-10+B and IL-10-B cells were separated. To observe the interaction between the Helicobacter-activated-B cell subgroups and CD4+ T cells, isolated CD4+ T cells were put on co-culture in 1:1 ratio with the Helicobacter-activated-IL-10+ B cells and Helicobacter-activated-IL-10- B cells, for 24 hours. The cell surface markers and intracellular cytokine productions were examined by flow cytometry. While the supernatants of the co-culture groups were subjected to ELISA tests, the cell pellets were used for gene expression analyses. The intracellular staining of IL-10 of T cells co-cultured with Helicobacter-activated B cell subgroups showed that about 15% of T cells co-cultured with HACT-IL-10+ B cells produced IL-10 while almost 20% of the T cell population was IL-10 positive when T cells were co-cultured with HACT-IL-10- B cells. In addition, according to IL-10 ELISA results, CD4+T cells co-cultured with IL-10+B cells and IL-10-B cells secreted twice IL-10 when compared to only T cells. For IL-10+B cell and T cell co-culture, a part of secreted IL-10 came from B cells while most of the IL-10 secreted from IL-10-B cell and T cell co-culture originated is suggested to be from T cells. CD25 has been used as a marker to identify activated T cells as well as some regulatory T cell subsets in mice; while CD62L has been known to rapidly shed from lymphocytes upon cellular activation. Both CD25 and CD62L levels in co-culture groups showed significant differences compared to T only groups. Furthermore, the differences in T cell CD25 and CD62L levels together might indicate that T cells co-cultured with Helicobacter-activated IL-10-B cells are more activated/differentiated compared to the T cells interacting with IL-10+B cells. In literature, it has been shown that B cells which were activated by Helicobacter induce IL-10–producing CD4+CD25+ Tr1–like cells in vitro. In addition, it has been identified that the co-expression of CD49b and LAG-3 distinguishes Tr1 cells from Th1, Th2 and Th17 cells. Flow cytometry results of CD4-CD49b-LAG3 stainings showed that almost 20% of T cells co-cultured with IL-10+B cells express CD4-CD49b-LAG3 surface markers while only around 10% of CD4+ T cells co-cultured with IL-10-B cells express CD49b and LAG3 together. CD4+ T cells obtain distinct functional properties in response to signals sent by commensal and pathogenic microbe-activated cells of the innate immune system. Th17 cells secrete interleukin-17 (IL-17), IL-17F, and IL-22 and have significant roles in protecting the host from bacterial and fungal infections, particularly at mucosal surfaces. Data of four independent experiments showed that the approximately 15% of CD4+ T cells co-cultured with IL10-B cells turned into IL17 producing CD4+ T cells while surprisingly about 8% of T cells also produced IL-17 when co-cultured with IL10+B cells. IL-17 ELISA results were parallel with the IL-17 intracellular cytokine staining analyses. Ror gamma T and IL-17 relative gene expression levels in co-culture groups showed similar results with flow cytometry and ELISA results. Main research focus of this study was to investigate the interaction between HACT-B cell subgroups and CD4+ T cells and understand the effects of these B cell subgroups on T cell differentiation ex vivo. The results revealed that both Helicobacter-activated IL-10+B and IL-10-B cells induce IL-10 production from CD4+T cells and it might suggest that T cells differentiate into Tr1-like cells in both co-culture conditions. Although the CD25 and CD62L expression on T cell surfaces show significantly higher activation/differentiation in Helicobacter-activated IL-10-B and T cell co-cultures, regulatory type indicator CD4-CD49b-LAG3 surface marker co-expressions were higher on T cells in the Helicobacter-activated IL-10+B and T cell co-cultures. Furthermore, intracellular IL-17A levels and relative gene expression experiments of IL-17A and RorgammaT showed significantly higher results in IL-10- B and T cell co-culture groups, as expected. On the other hand, interestingly IL-17 production was also observed in T cells co-cultured with IL-10- B cells. The reason behind the IL-17 and RorgammaT expressions in IL-10+ B–T cell co-culture groups was suggested to be the IL-6 and TGF- β produced by IL-10 negative B cell population in the IL-10 positive co-culture group. This study has contributed to the literature through providing a first step to show the ex vivo interactions of Helicobacter-activated B cells subgroups, IL10+ HACT B cells and IL-10- HACT B cells, with CD4+ T cells.