The role of complosome in CD4 T-cells
| Název česky | Role komplosomu u CD4 T-lymfocytů |
|---|---|
| Autoři | |
| Rok publikování | 2021 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | The serum complement system serves as an ancient humoral branch of innate immunity and represents the first line of defense against various pathogens. However, recent studies confirmed that also CD4 T-cells synthesize complement proteins intracellularly. This intracellular complement system refers to as complosome and regulates CD4 T-cell survival and Th1 differentiation. Cell purification: CD4 T-cells were separated from PBMC using a negative magnetic selection technique. The purity of cell suspension was typically over 93 % and was verified by a flow cytometry. Cell stimulation: CD4 T-cells were incubated in 96U well plates (150.000 cells/well). For the complosome activation, we stimulated cells with a combination of anti-CD3 (10ug/ml) + anti-CD46 (5ug/ml) + IL-2 (50U/ml) and with or without a Vitamin D (1*10-7 M). Complosome dynamics measurement: For determination of period with maximal complosome changes, we incubated cells with or without stimuli for 0, 12, 36, 60 and 108 hours. Results showed that maximal complosome changes were detected after 60 hours of stimulation. Complosome component detection: Following complosome components were measured: CD46, C3aR, C5aR, C3b and cytokines INF-? and IL-10. Cells were stained using a standard intracellular staining protocol with blocking of nonspecific monoclonal antibody binding using a monomeric human IgG (1ug/ml). Flow cytometry: The analysis of complosome components was measured in CD4+ positive cells after doublets and dead cells exclusion. The negativity for all measured CD markers was assessed using FMO controls. Conclusion: We were able to measure a complosome activation in CD4 T-cells, which drive their differentiation into Th1 proinflammatory phenotype (production of INF-?). High dose of IL-2 initiate the transition from Th1 into regulatory Tr1 phenotype (production of IL-10). We also confirmed that addition of vitamin D into wells supported the transition into Tr1 phenotype. Because complosome dysregulation and positive effect of vitamin D treatment has been described in autoimmune disorders, we focused on potential complosome pathology and vitamin D effect in allergic asthma patients |