Decreased TIM3 Expression Of NK Cells In The Immune Pathogenesis Of Severe Aplastic Anemia

ObjectiveSevere aplastic anemia（SAA）is an autoimmune disease characterized by severe pancytopenia and bone marrow failure.In our previous studies,we found NK cells were decreased in SAA patients.T cell immunoglobulin mucin-3（TIM3）,an important regulator of immunity,is widely detected on NK cells.In this study,we intend to detect the expression of TIM3 on peripheral blood NK cells in SAA patients to reveal the further immune pathogenesis of SAA.Furthermore,we tried to further elucidate the changes of functions of TIM3⁺NK and TIM3^—NK cells in SAA by measuring the functional molecules and cytotoxic activity of TIM3⁺NK and TIM3^—NK cells.Finally,SAA mice model was established.Then we observed the therapeutic effects of TIM3 blocker,TIM3⁺NK infusion and TIM3^—NK infusion on SAA mice model.At the same time,we determined whether combined CsA could further improve the therapeutic effect of SAA mice model.This study supplement the theory of SAA patients and provide a new treatment target to improve the efficacy of SAA treatment.MethodsA total of 36 SAA patients including 18 newly diagnosed cases and 18 remission SAA（R-SAA）cases from January 2017 to December 2018 in Hematology Department of Tianjin Medical University General Hospital were enrolled.A group of15 healthy controls（HC）participated in the research as controls.PartⅠ1.The TIM3 expression on the surface of total NK cells and CD56^bright,CD56^dim NK subsets were detected by FCM.2.TIM3 mRNA relative expression in peripheral blood NK cells in SAA patients and HC were measured by RT-PCR.3.The correlations of the expression of TIM3 on NK cells and clinical indicators in SAA patients were evaluated.PartⅡ1.The expressions of surface receptors and cytoplasmic protein of TIM3⁺NK and TIM3^—NK cells from peripheral blood were detected by FCM.2.Isolation and purification of TIM3⁺NK and TIM3^—NK cells,and the effect of TIM3⁺NK and TIM3^—NK cells for CD80 and CD86 expression on mDCs after co-culture were measured by FCM.3.The effect of TIM3⁺NK and TIM3^—NK cells for CTLA-4 and CD39 expression on Tregs after co-culture were measured by FCM.4.The effect of TIM3⁺NK and TIM3^—NK cells for apoptosis rate of K562 cells after co-culture were measured by FCM.5.Western-blot was used to detect the changes of TIM3⁺NK and TIM3^—NK signaling pathway proteins（AKT,P-AKT）and compare the functional activity of the two groups.6.NK cells in peripheral blood of SAA patients and normal controls were sorted by MACS.The expression levels of T-bet and Eomes mRNA in NK cells were detected by RT-PCR,and whether the changes of T-bet and Eomes mRNA were related to the expression of TIM3 in NK cells were compared.PartⅢ1.AA mice model was established.The TIM3 expression on the surface of NK cells and functional molecules on TIM3⁺NK and TIM3^—NK cells of SAA model,irradiated mice and normal mice were detected by FCM.Western-blot was used to detect the changes of TIM3⁺NK and TIM3^—NK signaling pathway proteins（AKT,P-AKT and PI3K）,and to compare the functional activity of the two groups.2.The SAA model mice were divided into normal control group（NC group）,pure irradiation group（TBI group）and aplastic anemia group（AA group）,CsA treatment group,TIM3⁺NK cells treatment group（TIM3⁺NK）,TIM3^—NK cells treatment group（TIM3^—NK）,CsA combined with TIM3^—NK cells treatment group（CsA+TIM3^—NK）,TIM3 blocker treatment group（TIM3 blocker）,CsA combined with TIM3blocker treatment group（CsA+TIM3 blocker）.After treatment,the general condition,blood routine and bone marrow count of mice in each group were compared.ResultsPartⅠ1.The TIM3 expression on the surface of total NK cells and NK subsets were measured by FCM.The TIM3 expression on peripheral blood NK cells in SAA untreated patients was significantly lower than that in SAA remission patients（P<0.05）and normal controls（P<0.01）.There was no difference between R-SAA and normal controls.The TIM3 expression on CD56^dim NK cells in SAA untreated patients was lower than that in normal controls（P<0.01）.We also detected TIM3expression on CD56^bright NK cells,but the result showed that there was no statistical difference between SAA untreated patients and normal controls（P>0.05）.The TIM3expression on both CD56^dim and CD56^bright NK cells in R-SAA patients showed an upward trend compared with SAA untreated patients,but there was no statistical difference.2.The purity of NK cells was>90%.The relative TIM3 mRNA expression was significantly increased in NK cells in R-SAA patients compared with SAA untreated patients and normal controls.The relative TIM3 mRNA expression in NK cells in SAA untreated patients was lower than normal controls,but the difference had no statistical significance.3.In all SAA patients,the expression of TIM3 on total NK cells was positively correlated with the WBC counts,the neutrophils proportion and the reticulocytes proportion.There was a negatively correlation between the TIM3expression on NK cells and the proportion of lymphocyte.The expression of TIM3 on NK cells was negatively correlated with the ratio of mDC/pDC cells,CD8+/CD3+T cells,and the expression of granzyme B in CD8+T cells and was positively correlated with the expression of CD86 on myeloid mDCs.We did not find that the TIM3expression was correlated with the expression of CD80 on mDCs,the expression of perforin in CD8+T cells and the number of Tregs.PartⅡ1.TIM3^—NK cells expressed higher NKG2D and Granzyme B than TIM3⁺NK cells in untreated SAA patients.As for the expressions of inhibitory receptors,we found NKG2A,CD158a and CD158b on TIM3^—NK cells surface,were lower than TIM3⁺NK cells.We did not find any differences of the expressions of NKp46,NKp44 and perforin between TIM3^—NK and TIM3⁺NK cells in untreated SAA patients.TIM3^—NK cells expressed higher NKG2D and Granzyme B than TIM3⁺NK cells in R-SAA patients.As for the expressions of inhibitory receptors,we found CD158a and CD158b on TIM3^—NK cells surface,were lower than TIM3⁺NK cells.We did not find any differences of the expressions of NKG2A,NKp46,NKp44 and perforin between TIM3^—NK and TIM3⁺NK cells in R-SAA.TIM3^—NK cells expressed higher NKG2D and Granzyme B than TIM3⁺NK cells healthy controls.As for the expressions of inhibitory receptors,we found NKG2A on TIM3^—NK cells surface,were lower than TIM3⁺NK cells.We did not find any differences of the expressions of CD158a,CD158b,NKp46,NKp44 and perforin between TIM3^—NK and TIM3⁺NK cells in healthy controls.We compared the functional molecules of TIM3^—NK and TIM3⁺NK cells in SAA,R-SAA and healthy control groups.The expression of NKP44 on TIM3⁺NK cells in SAA groups was higher than that of healthy control groups,while the expression of NKP44,NKP46 and perforin in TIM3^—NK cells was higher than that of healthy control groups.There was no significant difference in the expression of other functional molecules on TIM3^—NK and TIM3⁺NK cells among the three groups.2.The expression of CD80 and CD86 on mDC_alone,mDC+TIM3⁺NK and mDC+TIM3^—NK groups in newly diagnosed SAA patients were measured by FCM.The expression of CD80 and CD86 were significantly decreased after being incubated with TIM3^—NK and TIM3⁺NK cells in SAA,especially mDC+TIM3^—NK group,significantly lower than mDC+TIM3⁺NK group（P<0.01）.3.The expression of CTLA-4 and CD39 on Treg_alone,Treg+TIM3⁺NK and Treg+TIM3^—NK groups in newly diagnosed SAA patients were measured by FCM.The expression of CTLA-4 and CD39 were increased after being incubated with TIM3^—NK and TIM3⁺NK cells in SAA,especially Treg+TIM3^—NK group,but the result showed that there was no statistical difference among three groups（P>0.05）.4.The apoptosis rate（AR）of K562 cells were significantly increased after being incubated with TIM3^—NK and TIM3⁺NK cells in SAA,especially K562+TIM3^—NK group,significantly higher than K562+TIM3⁺NK groups（P<0.01）.5.There was no significant difference in the level of AKT of receptor post-signal pathway protein between TIM3^—NK and TIM3⁺NK cells in patients with SAA,but the level of P-AKT in TIM3^—NK cells is higher than TIM3⁺NK cells.6.The relative T-bet and Eomes mRNA expression was increased in NK cells in SAA untreated patients compared with SAA remission patients and normal controls,but the difference had no statistical significance（P>0.05）.PartⅢ1.AA mice model was established.The TIM3 expression on peripheral blood NK cells in SAA mice was significantly lower than that in TBI mice（P<0.05）and normal controls（P<0.05）.There was no difference between TBI mice and normal controls.We did not find any differences of the expressions of NKG2A between TIM3^—NK and TIM3⁺NK cells in SAA mice.TIM3^—NK cells expressed higher NKG2D than TIM3⁺NK cells（P<0.05）.There was no significant difference in the level of AKT of receptor post-signal pathway protein between TIM3^—NK and TIM3⁺NK cells in SAA mice,but the level of P-AKT and PI3K in TIM3^—NK cells is higher than TIM3+NK cells.These results suggest that the decreased level of TIM3 in NK cells of SAA mice,and the activity of TIM3^—NK cells is higher than that of TIM3⁺NK cells.2.On the 17th day of model establishment,the weight,hemogram and bone marrow cells count of AA mice were significantly lower than that of NC group（p<0.05）.The weight,hemogram and bone marrow cells count of CsA treatment group,TIM3⁺NK cell infusion treatment group,TIM3^—NK cell infusion treatment group,CsA combined with TIM3^—NK cell infusion treatment group,CsA combined with TIM3 blocker treatment group has some improvement,TIM3 blocker alone treatment of AA mice slightly increased,the effect is not significant（P>0.05）,and combined with CsA has no significant synergistic effect.The therapeutic effect of TIM3^—NK cell infusion group was better than that of TIM3⁺NK cell infusion group.The therapeutic effect of CsA combined with TIM3^—NK cell infusion group was more significant than that of CsA alone group.TIM3^—NK cell infusion therapy may have some synergistic effect with CSA.Conclusions1.In this study,we found that untreated patients with SAA had lower TIM3expression on NK cells and CD56^dim NK subsets compared with normal controls,and were correlated with the severity of pancytopenia of SAA.TIM3 expression recovered to normal after IST.The relative TIM3 mRNA expression in NK cells in untreated patients with SAA was lower than the normal control,but the difference had no statistical significance.2.We further confirmed that the expression of activation molecules on TIM3^—NK cells was increased and the killing function was enhanced compared with TIM3⁺NK cells.In addition,TIM3^—NK cells have enhanced inhibition of mDCs and K562 cells and play an immunomodulatory role in SAA.Therefore,TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA.Low expression of TIM3 contributes to the enhancement of NK cell function,which in turn inhibits the immune activation state of SAA and improves the disease level.3.The expression of TIM3 on NK cells of AA mice decreased,and the activity of TIM3^—NK cells was stronger than that of TIM3⁺NK cells,which was consistent with the decrease of TIM3 on NK cells of SAA patients and the strong activity of TIM3^—NK cells.After TIM3^—NK cell reinfusion,the general condition,blood cell count and bone marrow cell count of SAA mice were improved,and the combined treatment of CsA was more effective.It may further clarify the immune pathogenesis of SAA and provide a new treatment target to improve the efficacy of SAA treatment.