Project 1 : Molecular mechanism induced by CD9+ regulatory B cells to induce effector T cell apoptosis: a new therapeutic strategy for severe asthma.
We have characterized a new population of regulatory B cells (Breg), secreting IL-10 and expressing the CD9. CD9+ Breg are able to regulate inflammation in a murine model of severe asthma by reducing the number of effector T cells. The aim of our study was first, to decipher the molecular mechanism induced by CD9+ Breg cells in the control of inflammation on effectors T cells. Second, we wanted to determine whether uncontrolled inflammation was due to a decrease in Breg homeostasis and/or a deficit of suppressor properties of these cells.
CD19+CD9+ and CD19+CD9- B cells and CD3+CD4+CD25- effector T cells from the spleen of Balb-c mice were sorted and activated for 48h (a-CD40+LPS for B cells and IL-2 for T cells). T cells were co-cultured 48h with CD9+, CD9- or alone as control. Cell death, cell cycle arrest, apoptosis, and mitochondrial depolarization were determined by Yellow Dye, propidium iodide, Annexin V and JC-1 staining respectively. Caspases cleavage and protein expression of the Bcl-2 family members were estimated by western blotting. MAPK activation was measured by flow cytometry. The percentage of CD9+ Breg was estimated in the lung and the spleen of asthmatic (HDM model) and control mice.
CD9+, but not CD9- cells, induce effector T cells death, and inhibit proliferation with a cycle arrest in G0/G1. CD9+ Bregs activate T cell apoptosis through both intrinsic and extrinsic apoptosis pathways as illustrated by the mitochondrial depolarization and the cleavage of caspase 8-9 and PARP. We also showed that CD9+ Bregs induce expression of pro-apoptotic members of the Bcl-2 family and downregulated expression of anti-apoptotics. Moreover, CD9+, but not CD9- cells induce MAPK phosphorylation, showing that MAPK are the main actors of apoptosis activation. Finally, there was less CD9+ Breg cells in the spleen and the lung of asthmatic mice compared to control mice 4.7% and 3.1% compared to 7.6 and 6.8 respectively).
These data clearly show that CD9+ Bregs are able to exert a powerful anti-inflammatory function by inducing effector T cell apoptosis. Thus, an uncontrolled inflammation in asthma could results from a deficiency in CD9+ Bregs.
Project 2 : Characterization of a B lymphocyte signature predictive of chronic lung allograft dysfunction.
The occurrence of chronic lung allograft dysfunction (CLAD) is the main complication after lung transplantation (LT) and remains the most common cause of graft failure and death. Currently, the available immunosuppressive therapies cannot prevent its occurrence in 50% of patients at 5 years. Conversely, some LT recipients, remain free of CLAD for a long time, and are considered as stable (STA). Whereas in kidney transplantation, predictive B-cell signature of long term or poor graft outcome have been identified by us and others, nothing has been done yet in lung transplantation. We aim to identify and validate predictive B lymphocyte signatures in order to identify and lighten the risk of CLAD and allow for early intervention before irreversible damages.
PBMCs from the Lung Transplanted patients (COLT) COhort were immunophenotyped before LT, one month after LT, at the visit for which CLAD was diagnosed, 6 months before and 6 months after CLAD. PBMCs from patients with long-term functional stability (STA) and healthy volunteers (HV) were analyzed as controls. Cells were stained for the B-cell markers CD19, CD27, CD38, CD138, CD22, CD24, IgD, IgM, CD5 and CD9 to determine the percentage of transitional, naive, plasmablast, memory and regulatory B-cells.
No difference was observed for total CD19+ B cells, memory, naïve, and plasma cells between the three groups of patients at the different time points. No significant difference was observed for any B cell subtypes either, except for the CD24hiCD38hi transitional B cells that stayed low during time in BOS patients (1.1%±0.1) whereas increasing in patients with a stable graft function to reach a maximum peak at 24 month (6.3%±0.1; p≤0.001), with a level of CD24hiCD38hitransitional B cells superior to HV (3.3%±0.4; p≤0.01). These transitional express CD9 regulatory markers and secrete IL-10+. Finally, at 24 months post- LT, CD24hiCD38hi transitional B cells and CD9+ discriminate BOS and STA and are predictive of long-term BOS-free survival.
In summary, these data are the first to reveal that CD24hiCD38hi transitional B cells are associated with better lung allograft outcome. They may act as key actors in creating a favorable environment in which regulation of inflammation may be achieved and might be essential for the maintenance of long-term stable graft function after lung transplantation. Finally, the entirely new identification of CD9 expressing B-cells as predictive biomarkers of long-term BOS free survival will be an adjunct tool to risk-stratify patients. These data could help early prediction of BOS and development of personalized medicine, a major breakthrough, based on risk stratification and personalized pathways modulation.