Increased Regulatory T Cell Subsets (TReg and Tr1 Cells) in the Peripheral Blood of Patients with Multiple Myeloma Correlate with Disease Stage.
Authors: Feyler Sylvia , Marles Lee , Richards Stevie , Cook Gordon 
Published: April 2020

Abstract
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 TReg 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&lt;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&lt;0.001), MGUS (945±90 cells/μl, p&lt;0.001) and controls (849±95 cells/μl, p&lt;0.001). Using a sequential gating strategy, TReg 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 TReg cell population compared to the control group (15.0%±2.5 vs. 7.2%±1.1, p&lt;0.001). The increased TReg 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&lt;0.001), MGUS (10.8%±1.7, p=0.03) and controls (7.2%±1.1, p&lt;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.