Reguatory T-Cell Subsets Demonstrate Quantitative Differences but Not Functional Abnormalities in Patients with Multiple Myeloma (MM) Compared with Healthy Controls.

The immunologically hostile microenvironment of MM contributes to the limited success of immunotherapy strategies. In addition to direct tumour-induced immunosuppression, tumour cells may generate suppressor cells such as T_reg cells which can profoundly suppress immune responses and induce tolerance. This study aimed to determine if T_reg subsets are increased in the peripheral blood (PB) and bone marrow (BM) of patients with MGUS/MM and how this correlates with increasing disease burden. PB samples from 166 patients with MGUS/MM (Newly diagnosed (ND), n=34; Plateau/low disease burden (LD), n=63; Relapsed/refractory (R/R), n=27 and MGUS, n=42) with a median age of 69 years (range 39–89 yrs) were analysed by FACS and compared to PB from 32 age/sex matched controls. Using a sequential gating strategy, naturally occurring T_reg cells (nT_regs) were identified as CD4⁺/CD25⁺/FoxP3⁺ T-cells and expressed as a percentage of the CD4⁺ T-cells. Double negative T cells (DN T_regs) were identified as CD3⁺/CD4⁻/CD8⁻/alphabeta-TCR⁺/gammadelta⁻TCR⁻ and expressed as a percentage of the CD3⁺ cells. Sera were analysed by ELISA for IL10 and TGF-beta. nT_Reg cells were significantly increased in patients with MGUS/MM compared with controls (Controls 1.6% ± 0.2, MGUS 2.3% ± 0.3, ND 2.4% ± 0.2, LD 4.2% ± 0.7 & R/R 3.8% ± 0.5; p=0.003). There was a positive correlation of nT_Regs number with paraprotein level (R=0.3, p=0.005). Patients on Thalidomide at the time of sample collection had significantly higher numbers of nT_Regs of 6% in comparison to patients who never received Thalidomide (3%) and patients who previously received the drug (3.5%, p=0.005). Analysis of BM (n=12) demonstrated a significantly reduced number of nT_Regs in comparison to PB (1.6% vs 3.0%, p=0.025), which is higher than the number of nT_Regs in the BM of controls (0.7%). Functionally, nT_Regs from patients demonstrated suppressive activity of both autologous and allogeneic T-cells similar to nT_Regs cells from control PB (p=NS). In contrast, DN T_Regs were significantly reduced in patients with myeloma (ND 1.0% ± 0.2, LD 1.1% ± 0.1, R/R 1.3% ± 0.2) compared with MGUS and controls (2.1% ± 0.9 & 3.3% ± 0.7, respectively; p=0.02). Serum IL10 levels were significantly lower in ND (17 ± 38 pg/ml) and LD (29 ± 36 pg/ml) than in Controls (55 ± 89 pg/ml), MGUS (72 ± 151 pg/ml) and R/R (90 ±182 pg/ml, p=0.02). TGF-b levels differed significantly between groups (Controls 3765 ± 1593 pg/ml, MGUS 3506 ± 1504 pg/ml, ND 4774 ± 9879 pg/ml, LD 1915 ± 1324 pg/ml, R/R 2802 ± 2011 pg/ml, p=0.025). These results provide further evidence of immune dysregulation in MM. The association with advanced disease stage suggests a causal association.