Role Of TIM3/Gal-9 Signaling Pathway In Cellular Immune Tolerance Of The Patients With Myelodysplastic Syndromes

Objective Myelodysplastic Syndromes（MDS）are a group of malignant clonal diseases of hematopoietic stem cells.Malignant clonal cells proliferation and cellular immune tolerance are the main pathogenesis.Hyperactivity of immunoregulation in regulatory T cells（Treg）and Myeloid-derived suppressor cells（MDSCs）,increased expression of cytokines such as IL-12,TGF-β,and high expression of immune checkpoint proteins（ICP,such as PD-1,TIM3,CTLA-4,etc.）could lead to cellular immune tolerance in MDS.PD-1 and CTLA-4 inhibitors have been approved for clinical treatment,but increased expression of TIM3 has been detected in patients resistant to PD-1 inhibitors,and studies have reported that combined with TIM3 inhibitor achieved a 50%remission rate in high-risk MDS^[1],making the relationship between TIM3 and cellular immune tolerance attracted much attention,while the relationship between CD8⁺T cells and TIM3 in MDS patients is still unclear.Our research tried to explore the relationship between CD8⁺T cells exhaustion and MDSCs in MDS patients.The expression of immune checkpoint T cell immunoglobulin mucin 3（TIM3）and its ligand galectin 9（Gal-9）in MDS was detected.The change of CD8⁺T cells after co-cultured with MDSCs in vitro with or without TIM3/Gal-9 pathway inhibitors was examined,in order to clarify the role of TIM3/Gal-9 pathway in CD8+T cells exhaustion further.The restoration of the exhaustion of CD8⁺T cells might provide a new target for immune therapy of MDS.Methods The subjects were 110 MDS patients newly diagnosed in the Hematology Department of Tianjin Medical University General Hospital from October 2016 to January 2020 and 73 normal controls（NC）.Part I The percentage of CD3⁺CD8⁺T cells in MDS patients and normal controls was detected by flow cytometry（FCM）.The expression of TIM3,Perforin,Granzyme B and CD95 in CD8⁺T cells were detected by FCM.Part II MDSCs were sorted by FCM.The expression of Gal-9 in MDSCs of MDS and NC were determined by FCM and real-time fluorescence quantitative PCR（RT-PCR）.Relative IL-10,IL-12,TGF-β,and TNF-αm RNA expression in MDSCs were examined by RT-PCR.The levels of IL-10,IL-12,TGF-β,TNF-αand Gal-9 in MDSCs culture supernatants and the levels of Gal-9 in bone marrow（BM）supernatants,serum were detected by ELISA.The ratio of IL-10/IL-12,TGF-β/TNF-αwere used to evaluate the polarization direction of MDSCs,and the clinical data of MDS patients were collected for correlation analysis with the functional factors of MDSCs.Part III Co-culture CD8⁺T cells with MDSCs or exogenous Gal-9 with or without TIM3/Gal-9 pathway inhibitors in vitro.CD8⁺T cells in BM and peripheral blood（PB）of MDS and NC were separated by magnetic beads,MDSCs in BM and PB of MDS were sorted by FCM.The expression of Perforin and Granzyme B in CD8⁺T cells and the apoptosis of CD8⁺T cells were detected by FCM after co-cultured with MDSCs.Expression of Notch1,EOMES,p-m TOR and p-AKT in CD8⁺T cells after co-culture were determined by Western blotting（WB）.PartⅣExpression of TIM3 and Gal-9 in CD8⁺T cells in MDS patients before and after treatment with demethylation drugs（mainly DAC）were detected by FCM.ResultsPart I Study on the number and function of CD8⁺T cells in MDS（1）The proportion of CD3⁺CD8⁺T cells in peripheral blood mononuclear cells（PBMC）in MDS patients was significantly lower than that in the NC group（17.97±8.47%vs.26.35±8.30%,P=0.0355）.According to the IPSS score,the percentage of CD3⁺CD8⁺T cells in high-risk MDS（IPSS>1）patients was slightly less than that in low-risk group MDS（IPSS≤1）,but there was no difference（16.05±9.75%vs.20.07±9.12%,P=0.5217）.（2）Expression of INF-γsecreted by CD8⁺T cells in MDS patients was significantly less than that in the NC group（15.05±9.20%vs.25.5±11.4%,P=0.0039）,there is no significant difference between high-risk and low-risk MDS（15.0±8.07%vs.15.11±10.52%,P>0.05）.（3）The expression of TIM3 on CD8⁺T cells in MDS patients was significantly higher than that in the NC group（TIM3⁺CD3⁺CD8⁺T cells/CD3⁺CD8⁺T cells=22.06（15.77-30.41）%vs.11.55（6.78-15.13）%,P=0.0013）,there was no difference between the low-risk group and the high-risk group（21.82（17.14-27.12）%vs.26.29（12.04-34.25）%,P>0.05）.TIM3⁺CD8⁺T cells in MDS compared with TIM3^-CD8⁺T cells in MDS and NC were overexpressed CD95（P=0.0014）,lower expression of Perforin（P=0.0106）and Granzyme B（P=0.0129）.Part II Study on the functional status of MDSCs in MDS（1）The expression of Gal-9 on MDSCs in MDS patients（MDS-MDSCs）was significantly higher than that in the NC group（NC-MDSCs）（17.21%（8.74%-35.77%）vs.2.61%（1.19%-3.39%）,P<0.0001）,but there was no significant difference between the low-risk and high-risk MDS group（16.86%（7.15%-39.19%）vs.20.72%（10.74%-36.35%）,P=0.7148）.The relative expression of Gal-9 m RNA on MDSCs in MDS was significantly higher than that in NC（3.49（2.09-12.57）vs.0.99（0.50-2.50）,P<0.05）.The levels of Gal-9 in BM supernatant,serum,and MDSCs culture supernatant of MDS were higher than those in NC group,and there was no statistical difference between high-risk MDS and low-risk MDS group（BM supernatant（high-risk MDS vs.low-risk MDS vs.NC）:323.6±42.6 pg/ml vs.381±55.98 pg/ml vs.154.9±23.72 pg/ml,P=0.0042;serum（high-risk MDS vs.low-risk MDS vs.NC）:194.5±16.13 pg/ml vs.237.1±7.18 pg/ml vs.69.81±11.93 pg/ml,P<0.0001;MDSCs culture supernatant（MDS vs.NC）:147.7±5.24 pg/ml vs.14.2±0.55 pg/ml,P<0.0001）.（2）The ratio of relative expression of IL-10/IL-12 and TGF-β/TNF-αm RNA in MDSCs in the high-risk MDS was higher than that in the NC,there was no difference between the low-risk MDS and NC group.And the IL-10/IL-12 concentration ratio in high-risk MDS was higher than NC,but there was no difference in the TGF-β/TNF-αconcentration ratio.（IL-10/IL-12 m RNA:20.2（8.54-75.12）vs.5.13（2.23-25.63）vs.1.80（0.33-8.93）,P=0.0145;TGF-β/TNF-αm RNA:2.69（1.56-6.35）vs.0.64（0.37-0.99）vs.0.73（0.30-2.79）,P=0.0055;IL-10/IL-12 concentration:4.06±1.50 vs.0.23±0.02 vs.0.22±0.01,P=0.0060;TGF-β/TNF-αconcentration:4.21（0.66-21.52）vs.7.26（1.46-27.37）vs.3.30（1.54-3.74）,P=0.2569）.（3）The TGF-β/TNF-αm RNA ratio in MDS was negatively correlation with the percentage of CD3⁺CD8⁺T lymphocytes in PB,and was positively correlation with the percentage of CD3⁺CD4⁺T cells and CD4⁺/CD8⁺T cells（R²=0.3831,P=0.0423;R²=0.4747,P=0.0132;R²=0.4529,P=0.0233）.The percentage of regulatory T cells（Treg%）in CD4⁺T cells in high-risk MDS（3.17±0.73%）was higher than NC（0.98±0.30%,P=0.0345）,but there was no difference between the low-risk MDS group（1.44±0.62%）and high-risk MDS group and NC group（P=0.1456 and P=0.8284,respectively）.The Treg%in PBMC from NC（NC-PBMC）was increased after co-cultured with MDS-MDSCs compared with NC-PBMC cultured alone（16.03±5.15vs 9.79±3.36,P=0.0305）.（4）Both IL-10/IL-12 m RNA ratio and TGF-β/TNF-αm RNA ratio were positively correlated with the proportion of bone marrow blasts（IL-10/IL-12:R²=0.2853,P=0.0491;TGF-β/TNF-α:R²=0.4867,P=0.0038）.The ratio of TGF-β/TNF-αm RNA in MDS patients with absolute neutrophil absolute value（ANC）≤0.8×10^9/l was significantly higher than MDS patients with ANC greater than 0.8×10^9/l（2.41（0.96-12.83）vs.0.91（0.37-1.38）,P=0.0360）,there was no differences in IL-10/IL-12m RNA（14.08（4.88-56.87）vs.9.22（3.35-17.59）,P=0.3884）.Part III Study on the role of TIM3/Gal-9 pathway in MDS（1）The purities of MDSCs and CD8⁺T cells sorted in vitro were both above 90%.Expression of Perforin in CD8⁺T cells from NC（NC-CD8）co-cultured with MDSCs from MDS patients（MDS-MDSCs）was lower than that in the NC-CD8 cultured alone group（16.02±5.86%vs.22.02±5.96%,P=0.0271）,early apoptosis was increased（38.66±7.91%vs.16.18±3.84%,P=0.0075）,no difference in Granzyme B and proliferation（Granzyme B:20.1%（14.08-37.12%）vs.31.68%（24.17-42.32%）,P=0.0605;proliferation:953075（533581-4433061）vs.757364（330311-1596830）,P>0.05）.（2）In the CD8⁺T cells and MDSCs from MDS co-cultivation system,the expression of Perforin and Granzyme B in CD8⁺T cells were lower in MDS-CD8+MDS-MDSCs group than the MDS-CD8 group（P=0.0001 and P<0.0001,respectively）and increased early apoptosis（P=0.0450）;the function of CD8⁺T cells was partially restored in the CD8+MDSCs+TIM3 inhibitor（F38-2E2）group,CD8+MDSCs+Gal-9 inhibitor（9M1-3）group and CD8+MDSCs+TIM3/Gal9inhibitors（F38-2E2/9M1-3）group,higher expression of Perforin and Granzyme-B than CD8+MDSCs co-culture group without inhibitors,and decreased early apoptosis rate（Comparisons between groups:Perforin%:P<0.0001,P=0.0001,P=0.0002;Granzyme-B%:P=0.0021,P=0.0050,P=0.0075;early apoptosis rate:P=0.0130,P=0.0029,P=0.0071）.（3）Expression of Perforin and Granzyme B were lower in the MDS-CD8+excess exogenous Gal-9 group than that in the MDS-CD8 group,the CD8+Gal-9+F38-2E2group,and the CD8+Gal-9+9M1-3 group（15.33±2.83%vs.21.79±3.35%vs.20.63±3.37%vs.19.01±3.49%,P<0.0001;15.95±5.47%vs.21.32±6.56%vs.20.18±6.44%vs.18.38±6.07%,P=0.0019）.（4）Expression of Notch1,EOMES,p-m TOR,p-AKT were lower in the MDS-CD8+Gal-9 group than that in MDS-CD8 group（1.7±0.24%vs.5.01±0.98%,P<0.01;26.37±6.65%vs.34.81±7.96%,P<0.05;3.15±0.88%vs.6.44±2.30%,P<0.01;15.78±6.21%vs.25.57±9.73%,P<0.01）.PartⅣStudy on the expression of TIM3 and Gal-9 on CD8⁺T cells in MDS patients before and after treatment with DAC（1）The expression of Gal-9 on CD8⁺T cells in MDS patients was significantly higher than that in NC group（15.55±2.01%vs.1.64±0.21%,P<0.0001）.（2）Expression of TIM3 on CD8⁺T cells in MDS who treated with a standard dose of DAC was higher than that before DAC treatment（30.51±3.61%vs.23.57±3.24%,P=0.0441）,while the expression of Gal-9 on CD8⁺T cells was no statistical difference between newly diagnosed and after DAC treatment（11.70±6.30%vs.7.26±2.61%,P=0.3709）.（3）The expression of TIM3 on CD8⁺T cells in the DAC-sensitive group decreased after the second course of treatment,and increased after the second course of treatment in the DAC-resistant group.Conclusions:1.Highly expression of TIM3 in CD8⁺T cells of MDS patients might be induce CD8⁺T cells exhaustion.2.Increased expression of Gal-9 in MDSCs of MDS,and MDSCs in higher-risk MDS tend to M2-type polarization direction which might related to immune cells and disease risk.It suggested that MDSCs might inhibit immune cells through TIM3/Gal-9 pathway,and inhibit or convert the direction of MDSCs polarization might helpful for anti-tumor treatment in MDS.3.The expression of Perforin and Granzyme B in CD8⁺T cells after co-culture with MDSCs were decreased and early apoptosis of CD8⁺T cells was increased.The function of CD8⁺T cells could be partially restored by TIM3/Gal-9 pathway inhibitors.It suggested that MDSCs might inhibit the function of CD8⁺T cells through the TIM3/Gal-9 pathway.TIM3/Gal-9 pathway inhibitors might be helpful for immunotherapy of MDS.4.CD8⁺T cells in MDS patients also overexpress Gal-9,which indicates that CD8⁺T cells in MDS might form a TIM3/Gal-9 autocrine loop to accelerate their own exhaustion.The expression of TIM3 on CD8⁺T cells was increased after treatment in DAC resistance group,it suggested that DAC treatment might affect immune cells in MDS patients,DAC resistance in MDS patients might related to the high expression of TIM3 on CD8⁺T cells.DAC combined with immune checkpoint inhibitors might help the treatment of resistance MDS patients.