Study Of TIM-3 In Pathogenesis Of Myelodysplastic Syndrome

Objective Previous studies have confirmed that TIM-3 is highly expressed on hematopoietic stem cells in bone marrow of patients with MDS,and TIM3+ stem cells are characterized by high proliferation,poor differentiation,and low apoptosis.In this study,the biological characteristics of TIM3+ stem cells were further explored from the aspects of cell morphology,cytogenetics,and animal models,providing a theoretical basis for the early identification of malignant MDS clones.Methods The subjects of the study were 44 newly diagnosed MDS patients who were admitted to the General Hospital of Tianjin Medical University from July 2016 to February 2018 and 17 normal controls.Part I Using flow cytometry,TIM3+stem cells(TIM3+CD34+CD38-Lin-cells)and TIM3-stem cells(TIM3-CD34+CD38-Lin-cells)were sorted from the bone marrow of 10 unteated patients with MDS,CD34+ stem cells(CD34+CD38-Lin-cells)were sorted out from the bone marrow of four normal controls.In order to understand the biological characteristics of TIM3+ stem cells,We subjected them to Wright's staining,colony-forming unit culture,and fluorescence in situ hybridization(FISH)respectively.Part II The expression of Gal-9 on MDSC(CD33+Lin-HLADR-cell population)in bone marrow of 15 patients with MDS and 8 normal controls was detected by flow cytometry.The TIM3+ stem cells,TIM3-stem cells,and MDSC in the bone marrow of 8 unteated patients with MDS were sorted by flow cytometry.TIM-3 inhibitors,Gal-9 inhibitors,and both of the two inhibitors were added into the three kinds of cells separately and they were cultured in groups.We examined the apoptosis of TIM3+ stem cells and TIM3-stem cells,and then analyzed the role of TIM-3/Gal-9 pathway in TIM3+ stem cells and MDSC in patients with MDS.Part III By flow cytometry,We sorted TIM3+ stem cells,TIM3-stem cells,and MDSC from the bone marrow of 11 untreated patients with MDS and sorted CD34+ stem cells from the bone marrow of 5 normal controls.The above cells were transplanted into 32 NCG mice.Peripheral blood and bone marrow were collected 10-12 weeks later.The differentiation of human-derived cells was detected by flow cytometry and the role of TIM3+ stem cells in the pathogenesis of MDS was explored.Results Part I The purity of the sorted TIM3+ stem cells and TIM3-stem cells was above 90%.The TIM3+ and TIM3-stem cells were stained by Wright's stain respectively.Observed under high magnification,the cells are round or oval,the cell volume is small,the nucleus is large,the nucleoli is obvious,the nuclear chromatin is fine,the cytoplasm is light blue without granules,and the high nucleus pulp ratio is in the form.They appear morphologically as primitive naive cells.The colony forming unit(CFU)assay showed that the average number of BFU-E formed by TIM3+ stem cells,TIM3-stem cells and normal bone marrow stem cells was(6 vs 11 vs 12),and the average number of CFU-E was(3 vs 6 vs 9),the average number of CFU-GM was(5 vs 17 vs 23).At the same time,it was observed that the single colonies formed by TIM3+ stem cells also contained fewer cells than TIM3-stem cells and normal bone marrow stem cells,and that some TIM3+ stem cell cells did not form colonies.In the FISH test,one of the patients with MDS showed karyotypic abnormalities(-7).Among the patient's TIM3+ stem cells,TIM3-stem cells,and conventional bone marrow cells,the proportion of karyotypic abnormal cells was 30%,20% and 16% respectively.We detected abnormal karyotypes(+8)and(20q-)in another MDS patient.The percentage of abnormal karyotypes(+8)in TIM3+ stem cells,conventional bone marrow cells,and TIM3-stem cells was 5%,3%,and 0% respectively.The percentage of abnormal karyotype(20q-)in TIM3+ stem cells,conventional bone marrow cells,and TIM3-stem cells in this patient was 26.7%,0%,and 0% respectively.No abnormal karyotype was detected in CD34+ stem cells in bone marrow of normal controls.Part II Our previous work has found that the number of MDSC in the bone marrow of MDS patients has increased,their function has been enhanced,and they have risen with the increase in the risk of disease.The proportion of MDSC in diseaseimproving patients has decreased,and the proportion of disease-progressors in MDSC has increased,and there has been no significant change in patients with stable disease.The expression of Gal-9 on MDSC was significantly higher than that of normal controls(0.62±0.64% vs 0.22±0.18%,P<0.05).The purity of MDSC in bone marrow of MDS patients after sorting by flow cytometry was above 90%.The proportion of apoptotic TIM3+ stem cells in the TIM3+ stem cells mixed MDSC group was significantly lower than that in the two inhibitor groups and that of apoptotic TIM3-stem cells in TIM3-mixed MDSC group [12.59(5.12,31.24)vs 42.54(29.41,58.39),65.86(24.44,99.43),P<0.05].Part III In the animal transplant model of our study,the expression rate of hCD45 in TIM3+ stem cells mixed MDSC group was significantly higher than that in TIM3+ stem cells group,TIM3-stem cells group and normal control CD34+ stem cells group(4.99±5.47% vs 0.65±0.22%,1.68±2.38%,0.55±0.40%,P<0.05).The expression rate of hCD19 in TIM3+ stem cells group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group(8.68±9.34% vs 27.46±18.06%,37.38±16.14%,P<0.01);The expression rate of hCD19 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group(4.76±4.78% vs 27.46±18.06%,37.38±16.14%,P<0.05).The expression rate of hCD33 in TIM3+ stem cells group was significantly lower than that in TIM3-stem cells group(11.34±14.70 % vs 22.45±8.73%,P<0.05),and lower than that in normal control CD34+ stem cells group(18.03 ± 10.05)%,even the difference was not statistically significant.The expression of hCD33 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group(5.07±5.29% vs 22.45±8.73%,P<0.05),which was lower than that of normal control CD34+ stem cells group,even the difference was not statistically significant.The expression rates of hCD13 in TIM3+ stem cell group,TIM3-stem cell group,TIM3+ stem cell mixed MDSC group and normal control CD34+ stem cell group were(18.50±22.54)%,(6.11±5.26)%,(1.13±0.62)%,(14.33±0.69)% respectively.There was no significant difference between these groups(F=1.101,P=0.367).The expression rates of hCD11 b in the TIM3+ stem cell group,TIM3-stem cell group,TIM3+ stem cell mixed MDSC cell group were(1.02±1.44)%,(10.19±6.30)% and(8.07±3.70)% respectively.Conclusions(1)The TIM3+ stem cells and TIM3-stem cells of MDS patients are difficult to distinguish morphologically,but the colony forming ability of the two are obviously different,and the karyotype abnormalities are also different.Therefore,for different types of stem cells,the TIM3+ stem cells may be the earlier karyotype anomaly "malignant" stem cells.(2)The expression rate of Gal-9 on MDSC cells in bone marrow of MDS patients was significantly increased.The apoptotic rate of TIM3+ stem cells in TIM3+ stem cells and MDSC mixed culture group was significantly lower than that of TIM3-stem cells in TIM3-stem cells and MDSC mixed culture group.In the TIM-3/Gal-9 pathway blocking group,the apoptotic rate of TIM3+ stem cells increased.Blocking the TIM-3/Gal-9 pathway can inhibit the anti-apoptotic effect of MDSC on TIM3+ stem cells,providing a new target for the clearance of malignant clones in patients with MDS.(3)After the TIM3+ stem cells and MDSC mixed group were transplanted into NCG mice,the number of human-derived cells detected was the largest.In mouse models,MDSC can promote the expansion of TIM3+ stem cells.Human-derived cells in the TIM3+ stem cell group and the TIM3+ stem cell and MDSC mixed group cannot undergo normal myeloid and lymphoid differentiation as well as the TIM3-stem cell group or the normal control CD34+ stem cell group.It is speculated that TIM3+ stem cells may have clonal growth,while MDSC has a promoting effect.