Increased Regulatory T Cell Subsets (T_Reg and Tr1 Cells) in the Peripheral Blood of Patients with Multiple Myeloma Correlate with Disease Stage.

The immunologically hostile microenviroment of multiple Myeloma may contribute to the limited success of immunotherapy strategies. In addition to direct tumour-induced immunosuppression, tumour cells may generate suppressor cells to further suppress the immune effectors. Regulatory T-cells profoundly suppress immune responses and induce tolerance and 2 main subsets have been identified: Naturally Occurring T_Reg cells and Inducible regulatory Tr-1 cells. The association between tumour cells and regulatory T cells has not been studied in haematological malignancies, especially those of B lymphocyte lineages. Therefore, this the aim of this study is to determine if regulatory T-cell subsets are increased in the peripheral blood of patients with MM and how this varies with increasing disease burden. Peripheral blood from patients with MM (De novo, n=3; Low Disease burden, n=19; Relapsed/Refractory Disease, n=11) and MGUS (n=6) with a median age of 69 years old (range 39–89 yrs) were analysed by flow cytometry and compared to age-sex matched controls (n=20, median age 60 yrs, range 33–80 yrs). Whilst there was no significant difference in the absolute lymphocyte counts between MM patients and controls (1.58x10⁹/l ±0.14 vs. 1.9x10⁹/l ±0.1, p=0.05) a significant CD4⁺ T-cell lymphopenia was noted in patients with MM compared to controls (393±62 cells/μl vs. 849±95 cells/μl, p<0.001). The CD4⁺ T-cell lymphopenia was most marked in patients with relapsed/refractory disease (462±114 cells/μl) and low tumour burden (380±85 cells/μl) compared with newly diagnosed patients (875±64 cells/μl, p<0.001), MGUS (945±90 cells/μl, p<0.001) and controls (849±95 cells/μl, p<0.001). Using a sequential gating strategy, T_Reg cells were identified as CD4±/CD25⁺/GITR⁺ T-cells and expressed as a percentage of the CD4⁺T-cell population. Overall, patients with MM demonstrated a significant increase in the T_Reg cell population compared to the control group (15.0%±2.5 vs. 7.2%±1.1, p<0.001). The increased T_Reg cell population was most marked in patients with relapsed/refractory disease (13.6%±1.5) and low tumour burden (16%±1.9) compared with newly diagnosed patients (6.7%±1.0, p<0.001), MGUS (10.8%±1.7, p=0.03) and controls (7.2%±1.1, p<0.001). Tr1 cells were analysed using an in-house assay and identified on a sequential gating strategy as CD4⁺/IL-10⁺/IL-4⁻ T-cells and expressed as a percentage of the CD4⁺T-cell population. Overall, patients with MM demonstrated an increase in the Tr1 cell population compared to the control group (14.5%±5.5 vs. 9.8%±1.0) though the trend did not reach statistical significance (p=0.23). Similarly, an alteration in the Th1/Th2 balance was seen with an increase in the Th2 cell population compared to the control group (6.3%±3 vs. 2.8%±0.1) though the trend did not reach statistical significance (p=0.15). These results provide further evidence of immune dysregulation in patients with MM and suggest that tumour-associated immunosuppression may be mediated through the actions of regulatory T-cell subsets. In particular, the association with advanced disease stage suggests a casual association between the malignant cells and induction of immune regulatory cells. Further work in establishing a casual association between MM tumour cells and regulatory T-cells is on-going and is essential if immunotherapeutic strategies are ever to reach their full potential.