Supervisors info:
Ουρανία Τσιτσιλώνη, Αναπληρώτρια Καθηγήτρια
Τομέας Φυσιολογίας Ζώων και Ανθρώπου, Τμήμα Βιολογίας
Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Multiple Myeloma (MM) is an incurable malignancy characterized by the uncontrolled proliferation of aberrant plasma cells that infiltrate the bone marrow and overproduce abnormal immunoglobulin fragments. Modern therapeutic regimens that include proteasome inhibitors and immunomodulators allow for the achievement of a complete response (CR) in the majority of MM patients. Nevertheless, many patients often relapse after treatment. For this reason, the bone marrow microenvironment has also been implicated, due to its’ potential supportive role in contributing to the clonal expansion of myeloma plasma cells. Recent studies on the pathophysiology of MM indicate a possible involvement of two immunosuppressive populations, TH17 lymphocytes and Myeloid Derived Suppressor Cells (MDSCs), which appear to be increased in the bone marrow of MM patients. Surprisingly, studies performed on regulatory T cells (Tregs), which exhibit potent immunosuppressive activity, have not yet demonstrated the correlation of these cells with disease onset and progression. The purpose of this project was to develop a method based on Multiparameter Flow Cytometry (MFC) to study the levels and functionality of Tregs in the bone marrow and peripheral blood of MM patients, including those with detectable levels of Minimal Residual Disease (MRD+) or not (MRD-) in their bone marrow. For this purpose, we performed immunophenotypic analysis of Tregs in peripheral blood and bone marrow samples from 27 MM patients. The samples were collected at the Department of Clinical Therapeutics, School of Medicine, University of Athens. In order to identify Tregs and study their functional profile, samples of both biologic materials were labeled with 12 fluorescent antibodies comprising two different 8-color panels: CD3-FITC, CD4-APC / Cy7, CD25-APC, FoxP3-PE, CD8- Cy5-5, Ki67-AmCyan, CD45RA-PE / Cy7, CD39-Pacific Blue, CD45RO-PerCP / Cy5-5, CTLA-4-Pacific Blue, CD127-AmCyan, HLADR-PE / Cy7. Along with Treg immunophenotyping, we identified the paraprotein subtype in newly diagnosed patients and assessed the MRD status with MFC in those MM patients that achieved CR after treatment, thus grouping the latter based on the presence or not of MRD in their bone marrow. Based on the above, we determined the (%) levels of Tregs and studied their functional profile separately in three groups of patients: a) newly diagnosed MM patients, b) MRD+ patients, and c) MRD- patients. Our results revealed that in all three groups of patients (newly diagnosed, MRD+, MRD-) Tregs are numerically higher in the peripheral blood than in the bone marrow. Also, MRD- patients displayed the highest CD3+CD4+CD25hiFoxP3+ Tregs and CD3+CD4+CD25hiFoxP3+CD127lo Tregs, expressed as separate percentages on gated CD4+ T cells and total nucleated cells. The group of newly diagnosed patients was characterized by higher Treg ratio on gated CD4+ T cells in the peripheral blood rather than in the bone marrow, whereas MRD+ and MRD- patients displayed the opposite trend. Concerning the study of the functional profile of CD3+CD4+CD25hiFoxP3+ Tregs there were no differences between bone marrow and peripheral blood in each group; however MRD+ patients showed higher rates of CD39+ Tregs. The above results suggest that Tregs in MRD+ patients, despite not showing an increase in absolute numbers, may have a stronger immunosuppressive phenotype, therefore facilitating the escape of myeloma cells from immunosurveillance. It is also possible that the drugs used in MM therapeutics directly or indirectly alter the migration capacity and anchoring pattern of Tregs from the peripheral blood to the bone marrow or vice versa. Nevertheless, more samples need to be analyzed in order to decipher the exact role of Tregs in the pathophysiology of MM.
