| BackgroundMultiple myeloma(MM)is a common hematoligic malignancy which often occurs in the older adults.The main clinical manifestations include renal insufficiency,osteolytic lesions,anemia and hypercalcemia.Almost all 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.Changes in tumor microenvironment and genetic alterations synergically promote disease occurrence and progression.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 due to lack of analysis based on Treg cell subsets,and utilization of peripheral blood as study subject which cannot truly interpret the real size and function of Tregs pool in the tumor microenvironment.In this study,we performed a comprehensive analysis of peripheral blood(PB)and bone marrow(BM)Treg subsets and Treg-like cells in MM patients at diagnosis(MM-diag),in individuals with monoclonal gammopathy of undetermined significance(MGUS),in relapsed MM patients(MM-rel)and in treatment with good response(MM-plateau),ObjectiveTo investigate the frequencies and function of Treg cell subsets and aging Treg-like cells in the peripheral blood and bone marrow of multiple myeloma and MGUS patients;To analyze the possible changes of distribution and function of Treg subsets during the progression from MGUS to MM which might help further elucidate the mechanism of immune dysfunction during myelomagenesis.MethodsOur study included 20 MGUS patients and 60 MM patients diagnosed between November 2017 and May 2018 at Peking Union Medical College Hospital.Flow cytometry was applied to determine the proportion of Treg cell subsets and aging Treg-like cells in the peripheral blood and bone marrow.Clinical data were collected to analyze the corelation with disease status and risk stratification.Flow sorting technology was used to separate Treg cell subsets and effector T cells in the bone marrow of newly diagnosed multiple myeloma patients.The inhibitory function is 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 were measured using ELISA.Results1.The frequencies of Treg subsets in MGUS and MM patients(1)In peripheral blood,the proportion of activated Treg(aTreg)cells in CD4+ T cells was significantly higher in MGUS,untreated MM,and relapsed/refractory MM patients than healthy controls(P<0.01);there was no difference in the proportion of resting Treg(rTreg)cells between MGUS,untreated MM patients compared with healthy adults(P=0.7167,P=0.0740)while the proportion of rTreg in relapsed/refractory MM patients was significantly lower than that of controls(P=0.0108).There was no significant difference in the frequencies of non-Treg cells in PB from MGUS and MM patients with normal controls(P>0.05).(2)In bone marrow,the proportion of aTreg cells was significantly higher in MGUS,untreated MM,relapsed/refractory MM patients,as well as patient at plateau period compared with healthy controls(P<0.001);the proportion of rTreg in MGUS,untreated MM patients was significantly lower than that of controls(P=0.0185,P=0.0082),while relapsed/refractory MM patients had similar rTreg frequencies with healthy adults(P=0.0518).There was no significant difference in the frequencies of non-Treg cells in BM from MGUS and MM patients with normal controls(P>0.05).2.The function of Treg subsets in MGUS and MM patients(1)The inhibition rate of aTreg cells in the bone marrow of newly diagnosed MM patients was significantly higher than that of rTreg(P=0.0002)and the inhibition rate of non-Treg cells was significantly lower than that of rTreg cells(P<0.0001).The inhibition rates of aTregs(P=0.210),rTregs(P=0.0783)and non-Tregs(P=0.0894)in healthy controls were no difference from those in MM patients.(2)The level of IL-10 secreted by non-Tregs in untreated MM patients was significantly higher than that of aTregs and rTregs;the ability of cytokine secretion of Treg subsets in MM patients was similar with that of healthy controls.3.The frequencies of Treg-like cells in MGUS and MM patients(1)In peripheral blood,the proportion of CD4+ CD28-FoxP3+Treg-like cells in CD4+ T cells was gradually increased in MGUS,untreated MM,and relapsed/refractory MM patients than healthy controls(P<0.001);Treg-like cells in newly diagnosed MM patients were significantly higher than those in MGUS patients(P=0.0142).(2)In bone marrow,the proportion of aTreg cells in CD4+ T cells was significantly higher in MGUS,untreated MM,relapsed/refractory MM patients,as well as patient at plateau period compared with healthy controls(P<0.001).Treg-like cells in untreated MM patients were significantly higher than those in MGUS patients(P=0.0027).(3)In the low risk(P=0.0016),low-intermediate risk(P<0.001)and high-intermediate risk(P<0.001)group of MGUS patients,Treg-like cells in bone marrow increased compared to healthy adults.Conclusions1.There were significant changes in the quantity of Treg cell subsets and Treg-like cells in peripheral blood and bone marrow of MGUS and MM patients.These changes were related to disease status and treatment response.2.The immunologic function of Treg subsets in bone marrow of untreated MM patients was similar with that in healthy adults:activated Treg cells can effectively inhibit the proliferation of effector T cells,resting Treg cells can be partially inhibited;non-Treg cells cannot inhibit the proliferation of Teff cells,while they can exert immune-modulatory function by secreting cytokines.3.The proportion of aTreg cells and Treg-like cells in peripheral blood and bone marrow of MGUS patients was significantly higher than that of healthy control,suggesting that immunomodulatory abnormality has existed in patients at premalignant stage. |
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