Study on Quantity and Function of Regulatory T Cell Subsets in Multiple Myeloma and MGUS

Introduction

Almost all multiple myeloma (MM) cases were progressed from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). So far, the pathogenesis of myeloma is not yet clear. The immune cells in the tumor microenvironment, such as regulatory T (Treg) cells with a unique immunosuppressive function, play an important role in myelomagenesis. Although there have been reports on Treg cells in MM patients, the results were still in debate. In this study, we performed a comprehensive analysis of peripheral blood (PB) and bone marrow (BM) Treg subsets and aging Treg-like cells in untreated MM patients and individuals with MGUS, which might help further elucidate mechanisms of immune dysfunction during myelomagenesis.

Methods

Our study included 20 MGUS patients and 26 newly diagnosed MM patients. Flow cytometry was applied to determine the proportion of Treg cell subsets and aging Treg-like cells in PB and BM. Flow sorting technology was used to separate Treg cell subsets and effector T cells in the bone marrow of newly diagnosed MM patients. The inhibitory function was indirectly calculated by detecting proliferation rate of CFSE-labelled effective T cells which were cocultured with different Treg cell subsets. Concentration of IL-10 from the culture supernatants of proliferation assay was measured using ELISA.

Results

In PB, the proportion of activated Tregs (aTregs, CD4⁺CD45RA^-FoxP3^hi) in CD4⁺ T cells was significantly higher in MGUS and untreated MM patients than healthy controls (P=0.01, P<0.001); there was no difference in the proportion of resting Tregs (rTregs, CD4⁺CD45RA⁺FoxP3^lo) between MGUS and untreated MM patients compared with healthy adults (P=0.72, P=0.07). There was also no significant difference in the frequencies of non-Tregs (CD4⁺CD45RA^-FoxP3^lo) from MGUS and MM patients with normal controls (P=0.22, P=0.67). The proportion of CD4⁺CD28^-FoxP3⁺ Treg-like cells in CD4⁺ T cells was gradually increased in MGUS, untreated MM patients than healthy controls (P<0.01, P<0.01); Treg-like cells in newly diagnosed MM patients were significantly higher than those in MGUS patients (P=0.01).

In BM, the proportion of aTregs was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01); the proportion of rTregs in MGUS, untreated MM patients was significantly lower than that of controls (P=0.02, P<0.01). However, there was no significant difference in the frequencies of non-Tregs in BM from MGUS and MM patients with normal controls (P=0.14, P=0.88). The proportion of Treg-like cells in CD4⁺ T cells was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01, P<0.01). Treg-like cells in untreated MM patients were significantly higher than those in MGUS patients (P<0.01).

The inhibition rate of aTreg in bone marrow of newly diagnosed MM patients was significantly higher than that of rTreg (P<0.01), while the inhibition rate of non-Treg was significantly lower than that of rTreg cells (P<0.01). The inhibition rates of aTreg (P=0.21), rTreg (P=0.08) and non-Treg (P=0.09) in healthy controls were no difference from those in MM patients. The level of IL-10 secreted by non-Treg in untreated MM patients was notably higher than that of aTreg and rTreg; the ability of cytokine secretion of Treg subsets in MM patients was similar with that of healthy controls.

Conclusions

1. There were significant changes in the frequencies of Treg cell subsets and Treg-like cells in peripheral blood and bone marrow of MGUS and MM patients, suggesting that immunomodulatory abnormality has existed in patients at premalignant stage.

2. The immunosuppressive and cytokine secretory functions of Treg subsets in bone marrow of untreated MM patients were intact compared with that in healthy adults.

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