Association Of Telomere Lengths With Quantities And Functions Of CD4~+/CD8~+T Lymphocytes In Severe Aplastic Anemia

Objective:This study aims at measuring the relative telomere length(RTL)of CD4~+T lymphocytes and CD8~+T lymphocytes between severe aplastic anemia(SAA)patients and normal people,and comparing RTL,the quantity,expression level of functional molecules on the cell membrane surface and the ability of primary cell secretion of cytokines in SAA patients and control,then we explore the relationship between the RTL and the hyper-function of T lymphocyte,and illustrate that the shorter telomere have important function in the process of the onset of SAA.Methods:All the patients with SAA were diagnosed in the Hematology Department of General Hospital Tianjin Medical University from Jun 2015 to Jun 2016 according to Chinese AA Study Group Criteria.Healthy volunteer as health controls whose race,living area,gender and age were same as those of SAA patietns were also enrolled in this study.Part 1 Pheripheral blood mononuclear cells(PBMCs)were isolated from heparin anticoagulant venous blood of SAA and normal controls and health controls using density gradient centrifugation.CD4~+T,CD8~+T lymphocytes were purified using the respective anti-CD4 and anti-CD8 m Ab-conjugated microbeads according to the manufacturer’s instructions.We measure the relative telomere length(TRF)by Flow-FISH.Part 2 The quantity and cell membrane superficial molecule such as CD28,CD158 and CD70 expression level were analyzed by flow cytometry.IFN-γ,TNF-α of the post-stimulate CD8~+ T lymphocytes were analyzed by ELISA.We analyzed the correlation between RTL and the quantity function of CD4~+/CD8~+ T lymphocytes.Results 1.RTL of CD4~+T and CD8~+T lymphocytes RTLs of CD4~+T lymphocytes in untreated SAA patients were no statistical difference those of healthy controls((89.85±21.71)%vs(95.01±13.18)%,P=0.383),recovering SAA patients were also no statistical difference untreated SAA patients((84.18±10.01 1)% vs(89.85±21.7)%,P=0.315),recovering SAA patients were also no statistical difference those of healthy controls(P=0.107).RTLs of CD8~+T lymphocytes of untreated SAA patients were significant shorter than the healthy controls((76.57±16.88)% vs(95.71±9.11)%,P<0.001),RTLs of recovering SAA patients were longer than untreated SAA patients,but there are no statistical difference((86.49±11.36)%vs(76.57±16.88)%,P=0.06),recovering SAA patients were also no statistical difference those of healthy controls(P=0.49)2.The quantity of CD4~+T and CD8~+T lymphocytes Flow cytometric analysis the quanyity of CD4~+T lymphocytes from untreated,recovering SAA and normal control group were(359.17±267.58)/ul、(388.08±268.81)/ul and(327.12±74.36)/ul,untreated SAA was no significantly with recovering SAA(P=0.69)and normal control(P=0.58),recovering SAA was no significantly with normal control(P=0.37).Flow cytometric analysis the quanyity of CD8~+T lymphocytes from untreated,recovering SAA and normal control group were(871.70±473.03)/ul、(307.16±187.77)/ul and(269.51±96.53),untreated SAA was significantly higher than recovering SAA(P<0.001)and normal control(P<0.001),recovering SAA was no significantly with normal control(P=0.61).3.The function moleculors of CD4~+T and CD8~+T lymphocytes Flow cytometric analysis revealed the costimulatory signals CD28 expression on CD4~+Tlymphocytes in SAA was no significantly with the health controls((96.70+5.58)% vs(96.93+2.62)%,P=0.83);In SAA patients,the expression of CD70 was also no significantly with the health controls((2.26 +1.93)% vs(2.01+1.70)%,P=0.70);CD158 of SAA patients was no significantly with the health controls((3.61+2.81)% vs(3.98+2.18),P=0.68).Flow cytometric analysis revealed the costimulatory signals CD28 expression on CD8~+T lymphocytes in SAA was significantly lower than those in health controls((56.56+20.89)% vs(66.53+17.82)%,P=0.033).In SAA patients,expression of CD70 was ignificantly higher than those in health controls((8.50 +7.17)% vs(4.77+4.67)%,P=0.006);Expression of CD158 on CD8~+T lymphocytes was no significantly with the health controls((11.35+5.92)% vs(9.82+8.80)%,P=0.146).4.The secretion level of post-stimulate CD8~+ T lymphocytes The microplatereader shew that CD8~+T lymphocytes secreted IFN-γin SAA were apparently higher than the health controls((20.59+6.05)U/ml vs(15.82+4.21)U/ml(P=0.018);The TNF-αIncreased secretion in SAA(18.12+7.96)U/ml,which were significantly higher than those in normal controls(11.49+4.72)U/ml(P=0.002).5.The correlation between RTL and the quantity of CD4~+ T and CD8~+ T lymphocytes Using linear regression analysis,we made the correlation between telomere length and the quantity of CD4~+T lymphocytes.We found there no significant correlations with RTL for the quantity(r=-0.12,P=0.38).However,there have been significant negative correlations with RTL of CD8~+T lymphocyte for the quantity(r=-0.28,P=0.03).6.The correlation between RTL and the function of CD4~+ T and CD8~+ T lymphocytes Using linear regression analysis,we made the correlation between telomere length and those,such as the level of expression of CD28,CD158 or CD70 on CD4~+T lymphocytes,apoptosis rate of the primary CD4~+T lymphocytes.We found there no significant correlations with RTL for CD28,CD158,CD70(P>0.05).we made the correlation between telomere length and those,such as the level of expression of CD28,CD158 or CD70 on CD8~+T lymphocytes,and the level of secretion IFN-γ,TNF-α after stimulating CD8~+T lymphocytes.We found significant positive correlations with RTL for CD28(r=0.61,P=0.001).However,there have been significant negative correlations with RTL for CD158(r=-53,P=0.005),CD70(r=-0.51,P=0.016),IFN-γ(r=-0.63,P=0.005),TNF-α(r=-0.58,P=0.006)of CD8~+T lymphocytes.Conclusions 1.RTLs of CD4~+T cells have no difference with controls,but RTLs of CD8~+T lymphocytes in untreated SAA patients was significantly shorten compared with recovering SAA patients and health controls,and RTLs of CD8~+T lymphocytes in recovering SAA patients have a tendency to prolong,implied that it is primary change by the telomere shorten of CD8~+T cells in different subtypes of CD3+T lymphocytes.2.In SAA pantients,CD8~+T lymphocytes were sustained activated.The quantity increases,CD28 expression decreases,CD70 expression increases,type Ⅰ cytokines including IFN-γ,TNF-α secretion increase be high in CD8~+T lymphocytes.3.We analyze the relationship between RTLs and the quantity and the function of CD8~+T lymphocytes in SAA.We found that there have been significant positive correlations with RTL for CD28.However,there have been significant negative correlations with RTL for the quantity,CD158,CD70,IFN-γ,TNF-α.This phenomenon shows in SAA patients,CD8~+T lymphocytes are closely related to T cell hyper-function,which is related to the RTL.So the shorter RTLs of CD8~+ T lymphocytes in SAA may be associated with hyper-function of these cells,which contribute to the pathogenesis of SAA.