Study of TIM-3 in Pathogenesis of Myelodysplastic Syndrome

Objective 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. TIM3+ and TIM3-stem cells were sorted from the bone marrow of 10 unteated patients with MDS, CD34+ stem cells were sorted out from the bone marrow of four normal controls. We subjected them to Wright's staining, colony-forming unit culture, and fluorescence in situ hybridization (FISH) respectively. The TIM3+ , TIM3- stem cells, and MDSC in the bone marrow of 8 unteated patients with MDS were sorted. 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. We sorted TIM3+ , 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. 10-12 weeks later, the differentiation of human-derived cells was detected by flow cytometry.

Results The TIM3+ and TIM3- stem cells were stained by Wright's stain respectively and observed under high magnification are same. The 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 CFU-E was (3 vs 6 vs 9), CFU-GM was (5 vs 17 vs 23). 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 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]. 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+ , 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). 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).

Conclusions

(1) The TIM3+ 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 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. 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) The number of human-derived cells detected was the largest. And 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.