| Objective We aimed to investigate the quantities of CD56 bright Natural Killer(NK)cells in patients with Severe Aplastic Anemia(SAA),as well as their functional receptors,cytokines produced by them and the cell signal pathways.We also tried to demonstrate the immunoregulatory roles of NK cells in SAA patients on CD4+T cells,CD8+T cells and myeloid Dendritic Cells(mDC).Methods We successively collected patients with untreated SAA,SAA response to immunosuppressive therapy(SAA-CR+PR)who were hospitalized in Tianjin Medical University General Hospital from January 2015 to December 2016,and healthy controls(HC)as research participants.Part I Quantities of circulating NK cell subsets(CD56bright NK and CD56 d im NK)of patients with SAA,SAA-CR+PR and HC were identified by flow cytometry(FCM).Correlation of CD56 b right NK quantity with clinical parameters were analyzed.The expressions of cell surface receptors including NKp46,NKp44,NKG2 D,NKG2A,CD158 a,CD158b and DNAM-1,intracelluar protein including perforin and granzyme B,intracellular cytokines including IFN-?,IL-17 a,TNF-?,IL-4,IL-10,IL-13 and TGF-? of NK cell subsets were analyzed by FCM.Plasmatic IL-2 and IL-12 levels of SAA,SAA-CR+PR and HC were measured by ELISA.CD56 b right NK cells from HC were sorted and the expressions of IFN-? and IL-10 m RNA were analyzed by q RT-PCR.Part II Peripheral NK cells of SAA,SAA-CR+PR and HC were sorted by magnetic activated cell sorting system(MACS)and cultured with high concentration of recombinant human interleukin-2(rh IL-2)and interleukin-12(rh IL-12).The supernatants were collected for the detection of IFN-?,IL-10 and TGF-?.The m RNA expression of IFN-?,IL-10,TGF-?,STAT4,p38 and STAT6 of NK cells were analyzed by q RT-PCR.The expressions of STAT4,p-STAT4,p38,p-p38 were analyzed by Western Blot.Part III The quantities of Th1,Th2,Th17 and Regulatory T cells(Treg)weremeasured by FCM.Peripheral NK cells from SAA and HC,bone marrow(BM)CD4+ T cells and CD8+ T cells from SAA were sorted by MACS,and m DC from SAA were cultured and induced.NK cells from SAA and HC were co-cultured with CD4+ T cells,CD8+ T cells and m DC.Quantities of Th1,Th2,Th17 and Treg,expressions of perforin and Granzyme B of CD8+ T cells and expressions of CD80 and CD86 on DCs were analyzed by FCM.The m RNA expressions of T-bet,GATA3,ROR?t and FOXP3 were measured by q RT-PCR.Proliferation rates of CD8+ T cells and m DC were detected by CCK-8.Results Part I The percentage of CD56 bright NK / NK in SAA patients was(4.33±2.53)%,significantly lower than that in HC(7.46±2.53)%,(p=0.001).The percentage of CD56 dim NK / NK in SAA patients was(95.67±2.54)%,similar with that in HC.The absolute number of CD56 brightNK cells in SAA was(3.26±2.85)×106/L,lower than that in HC(10.94±5.34)×106/L(p<0.001),and elevated in SAA-CR+PR(10.13±6.81)×106/L.The absolute number of CD56 bright NK cells in SAA was positively correlated with PLT(R=0.735,p<0.01),absolute number of Reticular cells(Ret)(R=0.755,p<0.01)and percentage of Ret(R=0.670,p<0.01).CD56 bright NK cells in SAA expressed higher levels of NKp46,NKp44 and NKG2 D and lower level of NKG2 A compared with those in HC.The expressions of CD158 a,CD158b,perforin and Granzyme B were null-to-low in CD56 bright NK cells,and no differences were found among three groups.The percentage of DNAM-1+ CD56 b right NK / CD56 bright NK was(96.68±1.73)%,higher than that in HC(94.49±2.15)%,(p=0.020).After PMA stimulation,CD56 bright NK cells in SAA produced lower level of IFN-? and higher level of IL-10,IL-4 and TGF-? compared with those in HC.There were no differences of the productions of IL-17 a,TNF-? and IL-13 among three groups.The plasmatic levels of IL-2 and IL-12 in SAA were(156.3±23.5)pg/m L and(28.9±6.1)pg/m L,higher than in SAA-CR+PR and HC.Under the stimulation of IL-2 and IL-12,the relative expression of IFN-? m RNA in CD56 bright NK cells from HC on Day 3 was elevated(1.44±0.13)(p=0.009),and then decreased(0.79±0.12)on Day 7(p=0.002).The relative expression of IL-10 m RNA in CD56 bright NK cells from HC on Day 3 was almost unchanged(1.24±0.54),and then increased(2.67±1.29)onDay 7(p=0.026).Part II NK cells from SAA secreted lower level of IFN-?,expressed lower levels of IFN-? and STAT4 m RNA,and lower level of phosphorylated STAT4 compared with NK cells from SAA-CR+PR and HC.NK cells from SAA secreted higher levels of IL-10 and TGF-?,expressed higher levels of IL-10,TGF-? and p38 m RNA,and higher level of phosphorylated p38 compared with NK cells from SAA-CR+PR and HC.Part III IL-2 stimulated NK cells from SAA expressed higher percentage of CD107a+(28.68±12.44)than from HC(17.05±9.04)(p=0.017)when cocultured with autologous activated CD4+T cells.The quantity of Th1 and the ratio of Th1/Th2 were elevated in SAA.After cocultured with NK cells from SAA,the quantity of Th1 and the relative expression of T-bet m RNA were downregulated,the quantity of Th2,the relative expression of GATA3 m RNA were upregulated,and the ratio of Th1/Th2 was downregulated.In SAA,the number of Th17 cells and the ratio of Th17/Treg was upregulated.After cocultured with NK cells from SAA,the quantity of Th17 and the relative expression of ROR?t m RNA were downregulated,the quantity of Treg,the relative expression of FOXP3 m RNA were upregulated,and the ratio of Th17/Treg was downregulated.The cell inhibition rates of CD8+ T cells were not different between coculturing groups of CD8+ T cells with NK from SAA(+SAA NK)and with NK from HC(+HC NK).The expressions of perforin and Granzyme B in CD8+ T cells was higher in(+HC NK)group than in non-coculture group(Ctrl).The cell inhibition rate of m DC in(+SAA NK)group was higher than in(+HC NK)group(p=0.047).The expression of CD80 on m DC in(+HC NK)group was upregulated compared with in(+SAA NK)group.Conclusions 1.Insufficient amount of CD56 bright NK cells was shown in SAA patients.The lower quantity of CD56 bright NK cells the patients had,the more severe the disease was.After intensive immune suppression therapy(IST),the quantities of CD56 bright NK cells recovered.2.The activating receptors and the adhesion molecule DNAM-1 on CD56 bright NK cells were elevated while the inhibiting receptor was decreased,indicatingCD56 NK cells in SAA were functionally activated.Compared with HC,CD56 bright NK cells in SAA produced less Type ? cytokines and more Type ? cytokines.3.Plasmatic levels of IL-2 and IL-12 were elevated in SAA.Under the stimulation of rh IL-2 and rh IL-12,CD56 b right NK cells from HC produced IFN-? and IL-10 in a time-dependent fashion.4.The cell signal pathway JAK-STAT4—IFN-? in NK cells from SAA was down-regulated while pathway p38 MAPK—IL10/TGF-? was up-regulated under the stimulation of rh IL-2 and rh IL-12.5.NK cells from SAA mediated in the differentiation of CD4+ T cells by correcting the balance of Th1/Th2 and Th17/Treg.6.NK cells from SAA might be able to down-regulate the cytotoxicity activity of CD8+ T cells.7.NK cells from SAA showed an inhibition effect on m DC cells,and down-regulated the expressions of co-stimulatory molecules on m DC. |
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