טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHaj Tharwat
SubjectHuman Regulatory B Cells (CD19+CD25high) Regulate the
Proliferation of CD4+ T Cells and Enhance the
Expression of Foxp3 and CTLA-4 in
Regulatory T Cells
DepartmentDepartment of Medicine
Supervisors Professor Elias Toubi
Professor Doron Melamed
Full Thesis textFull thesis text - English Version


Abstract

Studies in both animal models and humans have shown subsets of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-β.

Our research aims were to establish the presence of human regulatory B (Breg) cells and to define their phenotypic characteristics, to study their regulatory role by assessing their ability to suppress the proliferation of CD4 T cells and to mediate the regulatory properties of regulatory T (Treg) cells in-vitro, and to define the mechanisms by which Breg cells modify the properties of Treg cells.

For this study, human B cells were purified from peripheral blood mononuclear cells, then stained and analyzed using flow cytometer for the expression of different molecules on gated CD19CD25high B (Breg) cells. Next, human Breg, CD4 T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4 T cells were stimulated with anti-CD3 and anti-CD28 and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for the expression of Foxp3 and CTLA-4.

We found that CD19CD25high B (Breg) cells expressed higher levels of CD27, CD86, CD1d, IL-10, TGF-β and semaphorin 3A compared with CD19CD25low B (non-Breg) cells. In addition, the histomorphologic structure of Breg cells differ from this of non-Breg cells. The co-culture of Breg cells with autologous stimulated CD4 T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4 T cells. Furthermore, non-stimulated Breg cells led to a mild, but yet statistically significant, increase of Foxp3 levels while ODN-CD40L stimulated Breg further increased Foxp3 expression in Treg cells compared with Treg cells cultured alone. Stimulated Breg cells also enhanced the expression of CTLA-4 on Treg cells. We also found that the regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-β but not IL-10 dependent.

In conclusion, human Breg cells, defined as CD19CD25highCD27highCD86highCD1dhighSema3AhighIL-10highTGF-βhigh B cells, decrease the proliferation of CD4 T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact. Finally, we believe that this unique subset of human B cells (Breg cells), play a major homeostatic role during the immune response and may be an option for treating autoimmune diseases.