Regulatory T Cells (Tregs) Are Highly Preserved in Multiple Myeloma Patients and Can Dominate Effector Functions.

Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells playing a key role in lymphocyte homeostasis and maintenance of tolerance. Tregs play a key role in hampering anti-tumor immune surveillance by regulating immune responses against tumor cells. Depletion of Tregs yields to improved anti-tumor immune responses in both preclinical and clinical settings. Contradictory data have been reported in the frequency and function of Tregs cells in multiple myeloma (MM). The aim of this study was to characterize MM Tregs from the phenotypic, molecular and functional standpoints. We studied both peripheral blood (PB) and bone marrow (BM) samples from patients with symptomatic MM and from healthy donors. CD4+CD25high Tregs were normally represented in both PB and BM of MM patients and expressed a memory and activated phenotype. No differences were observed between PB and BM Tregs of healthy donors and MM patients, based on the expression of CD45R0, HLADR, CD40L and CD69 cell surface antigens. Flow cytometry was also used to assess the intracellular expression of Foxp3, a transcription factor which is crucial for the inhibitory function of Tregs cells. More than 90% of CD4+CD25high T cells were Foxp3+ in the PB and BM of MM patients and healthy donors. CD4+CD25+ T cells were purified from PB of newly diagnosed MM by MACS sorting. These cells were anergic to the stimulation via TCR and as effective as normal donor-derived CD4+CD25+ cells in inhibiting the TCR-mediated proliferation of autologous CD4+CD25- counterparts. To investigate whether clonal restriction had occurred in MM Tregs, we studied TCR diversity of CD4+CD25+ and CD4+CD25- cells by determining the reciprocal usage of BV gene segments (TCRBV repertoire) with a novel multiplex polymerase chain reaction assay recently developed in our laboratory. Our results demonstrate an highly preserved polyclonal TCRBV repertoire, providing the first evidence in cancer patients that TCR diversity of Tregs is not skewed by the long lasting exposure to tumor cells. Based on these data, we propose that inhibitory signals delivered by the highly preserved Tregs subset in MM can easily overwhelm the effector mechanisms of antitumor immunosurveillance which are deeply compromised in MM. Thus, depletion or neutralization of Tregs should be considered in future trials aimed at controlling the disease in MM patients by immune intervention.