Function And Mechanism Of Thymic B Cells In Immune Response And Regulation

Background:Thymus is one important central immune organ of the body, where supports T cell differentiation, growth and mature. After undergoing positive and negative selection, thymocytes gradually differentiate into mature T cells and migrate to the peripheral immune organs. Thymus provides a complex microenvironment for T cell development. Composed of thymic stromal cells, soluble molecules and extracellular matrix, the microenvironment orderly regulates T cell development, maturation and migration. In recent years a few researches have proved that there are about 0.1-0.5% B cell in the thymus of human and mouse. There are B cells of different developmental stages in the thymus, which have several physiological functions, such as involving in T cells differentiation. B cells are considered to reside in the junction of the cortex and medulla, the region for negative selection process of T cells differentiation and development. Thus,the researchers maintain that B cells in the thymus play a key role in the negative T cells selection. However, how B cells regulate the negative selection, differentiation and development of T cells remain unclear. In pathological conditions, B cells can abnormally occur in the thymus and lymphoid follicular. Several studies indicated that abnormal increase of B cells in thymus was closely associated with autoimmune diseases. However, the functions and mechanisms of B cells in autoimmune diseases still remain unknown. To resolve these questions, the phenotype, origin and function of B cells in the thymus need to be furtherly studied. Based on these, we mainly focus on the role and mechanism of thymic B cells subsets in immune regulation and response. Objective:To explore the specific mechanism and biological function of thymic B cell subsets in immune response and regulation. Material and Methods:1. First of all, to verify whether B cells exist in the thymus, thymocytes were analyzed for proportion and phenotype of B cells by flow cytometry.2. To identify regulatory B cells and effective B cells in thymus, we analyzed B cells in thymus of wide type mice and lupus like mice. To verify whether regulatory B cells exist in thymus, thymocytes were stimulated with LPS(10 mg/ml) for analyzing the expression of IL-10 by intracellular staining. Based on these, the phenotype of regulatory B cells was analyzed. On the other hand, antibody secreating plasma cells were also studied after lympnocytes isolated from lupus like mice thymus.3. To further detect IL-10 expression level in CD5+CD1dhi cells, CD5+CD1dhi cells were sorted and IL-10 m RNA relative expression quantity was analyzed by real time fluorescence quantitative PCR.4. To study function of B220+CD19+CD5+CD1dhi cells in vitro, after sorting CD19+CD5+CD1dhi cells and CD19+CD5-CD1 dlo cells by flow cytometry and CD4+T cells by microbeads, CD4+T cells labeled with CFSE were co-cultured with CD5+CD1dhi cells and CD5-CD1 dlo cells respectively for detecting inhibition of CD4+T cell proliferation. And also, the CD4+T cells were co cultured with CD5+CD1dhi cells and CD5-CD1 dlo cells respectively in vitro for 72 h, then regulatory T cells were analyzed by flow cytometry.5. To study the physiological function of CD5+CD1dhi cells in vivo, CD5+CD1dhi cells and CD5-CD1 dlo cells were transferred into CD19 cre mice and lupus mice via the caudal veil. Regulatory T cells, CD4-CD8+T cell and CD8-CD4+T cell were detected correspondingly in spleen, lymph node and thymus. The autoantibody titers in peripheral serum were tested by ELISA analysis; Kidneys were fixed by 4% formaldehyde for analysis of renal pathology by hematoxylin and eosin stain.6. To investigate affection of B cells from the lupus mice thymus on the differentiation of T cells, B cells were sorted and transfused into the wild-type mice via the tail vein. The mice were killed for analysis of ISP cells, CD4-CD8+ and CD8-CD4+T cells.7. To study the influence of m TOR deletion on B cells and ISP cells in the thymus, m TOR+/- mice were killed to detect proportion and numbers of B cell and ISP cell. Results:1. A certain number of B cells were detected in mouse thymus, including B220+ cells accounting for 13.35%, CD19+cells accounting for 4.7%. And the B cell phenotype was furtherly analyzed. Moreover, IL-10+B cells and Ig G+B cells in the thymus were found.2. When analyzing IL-10+B cell phenotype, we found that CD5+CD1dhiB cells in thymus expressed high level IL-10; We also found B220+CD5+GFP+B cells and CD19+CD5+GFP+B cell in IL-10-GFP transgenic mice, furtherly confirming the existence of regulatory B cells in the thymus.3. When co-cultured with CD4+T cells, CD19+CD5+CD1dhiB cells can significantly inhibit the proliferation of CD4+T cells compared with CD19+CD5-CD1 dlo cells; Moreover, CD19+CD5+CD1dhiB cells can promote CD4+T cells transforming to CD4+Foxp3+ regulatory T cell by interaction between surface CD5 and CD72 molecules.4. In CD19 cre mice, we found that CD4+Foxp3+ regulatory T cells decreased, while CD4+CD8-T cells and CD4-CD8+ T cells increased. Transfusion thymus CD19+CD5+CD1dhi B cells to CD19 cre mice by vail tail vein, number of CD4+Foxp3+Tregs increased, while CD4+CD8-T cells and CD4-CD8+ T cells decreased. Moreover, CD4+Foxp3+ Tregs also increased in the spleen and lymph nodes.5. When transfering thymus CD19+CD5+CD1dhi cells to lupus mice, CD4+Foxp3+ Tregs and Bregs increased. And the antibody titers reduced in peripheral serum, accompanied with renal pathology improved in certain degree.6. In lupus mice, CD19+CD5+CD1dhi cell in thymus decreased. However, the overall B cells abnormally increased and mainly changed into Ig G+ B cells.7. In lupus mice, CD4+CD8-T cells and CD4-CD8+T cells increased in thymus. When B cells in thymus decreased, CD4+CD8-T cells and CD4-CD8+T cells decreased.8. B cells in thymus of lupus mice can reduce the precursor ISP cells, increase CD4+CD8-T cells and CD4-CD8+T cells. When m TOR partially deleted in the thymus(m TOR+/- mice), B cells decreased and ISP cells increased, which suggests that m TOR signaling may participate in the regulating process. Conclusion:According to our results, we proved that a small amount of B cells exist in thymus of mice and found that B cells abnormally increased in the thymus. Therefore, B cell abnormality may closely correlates with the development of some autoimmune diseases. Further study found that the existence of a group of CD19+CD5+CD1dhiIL-10+B cell can regulate the differentiation of regulatory T cell through direct contact between CD5 and CD72. In lupus mice, CD19+CD5+CD1dhiIL-10+B cells reduced in thymus, and Ig G+ B cells increased. Further studies proved that B cells promote the differentiation of CD4+CD8- and CD4-CD8+T cells by reducing ISP cells. Transfusion of CD19+CD5+CD1dhi cells through tail vein to lupus mice can reduce the antibody titers; improve the symptoms of lupus-like mice. Our results show that B cells in thymus play a critical role in the maintenance of immune homeostasis.