Role Of Myeloid-Derived Suppressor Cells-Mediated Immunosuppression In Myelodysplastic Syndromes

Objective:MDS comprises a group of malignant clonal hematological disorder.Antitumor immunity impairment is supposed to be associated with the pathogenesis and progression of MDS,but the specific mechanism is far from clear.So in this study we worked on investigation of the quantities and function of bone marrow myeloid-derived suppressor cells（MDSC）in myelodysplastic syndromes（MDS）.Detection of functional molecules on both MDSC and CD8+T cells after co-culture of MDSC and CD8+T cells in vitro with/without STAT3 inhibitor to explore the mechanism of MDSC-mediated immunosuppression on CD8+T cells in MDS.Methods:1.The percentage of LIN^-HLA-DR^-CD33⁺MDSC in 36 MDS patients and 19 healthy controls were measured by flow cytometry（FCM）.The correlations between MDSC percentage and clinical hematological parameters of MDS patients were analyzed.2.Expression of STAT3,pSTAT3,PD-L1 and CCR2 in MDSC in both MDS patients and healthy controls were detected by FCM.The levels of CCL2 in bone marrow（BM）supernatant and IL-6 in both BM supernatant and serum were determined by ELISA.3.Expression of PD-1,CD3ζ,Perforin and Granzyme B in CD8⁺T cells were detected by FCM.Concentration of IFN-γin BM supernatant was determined by ELISA.4.Isolation of CD8⁺T cells and MDSC were carried out by MACS and FCM respectively.Expression of STAT3,ARG1 in MDSC after co-culture were detected by FCM.Relative ARG1 mRNA expression of MDSC was determined by RT-PCR.Expression of CD3ζ,Perforin and Granzyme B in CD8⁺T cells after co-culture were detected by DCM.Concentration of IFN-γin culture supernatant was determined by ELISA.Apoptosis rate of CD8⁺T cells after co-culture was measured by FCM.Results:Part 1.（1）The percentage of MDSC in MDS group was significantly higher than that in healthy controls（HC）group（22.52±13.08%vs 8.15±5.75%,P<0.0001）.According to IPSS score:The percentage of MDSC in high-risk（IPSS score>1,H-MDS）group was significantly higher than that in low-risk（IPSS score≦1,L-MDS）group（26.16±14.29%vs 16.82±8.57%,P=0.013）,and the MDSC percentage of H-MDS group and L-MDS group were both significantly higher than that of HC group（26.16±14.29%vs 8.15±5.75%,P<0.0001 and 16.82±8.57%vs 8.15±5.75%,P=0.024,respectively）.According to WPSS score:There is no significant difference between H-MDS and L-MDS group in the percentage of MDSC（23.42±13.22%vs 23.25±12.05%,P=0.970）,but MDSC percentage in H-MDS group and L-MDS group were both significantly higher than that in HC group（23.42±13.22%vs 8.15±5.75%,P=0.009 and 23.25±12.05%vs 8.15±5.75%,P=0.001,respectively）.（2）Correlation analysis between MDSC percentage and clinical parameters of MDS patients:The percentage of MDSC in the≥5%BM blasts group was significantly higher than that in the<5%group（26.53±13.48%vs 17.15±8.42%,P=0.033）,and the percentage of MDSC was positively correlated with BM blast percentage（r=0.3897,P=0.033）.There was no significant difference in the percentage of MDSC between the normal karotype group and the abnormal karotype group（23.54±16.83%vs 22.27±10.39%,P=0.603）,and there was no significant difference between the three prognosis-related karotype groups（22.62±14.74%vs27.11±14.30%,P=0.543;22.62±14.74%vs 21.54±10.12%,P=0.826;27.11±14.30%vs 21.54±10.12%,P=0.475）.The percentage of MDSC was negatively correlated with hemoglobin level and platelet counts（r=-0.345,P=0.039 and r=-0.331,P=0.049,respectively）.Part 2.（1）The activation rate of STAT3 in H-MDS,L-MDS and HC group were49.96±10.88%,41.89±13.20%and 32.02±15.27%,respectively;H-MDS and L-MDS group had significantly higher pSTAT3 level than HC group（P<0.01 and P=0.039,respectively）,and H-MDS group had a relatively higher level than L-MDS group（P=0.048）.（2）Expression of CCR2 on MDSC in H-MDS,L-MDS and HC group were 12.16±13.80%,20.47±10.12%and 6.94±8.54%,respectively.Level of CCR2 in L-MDS group was higher than that in HC group（P=0.0092）;But that in H-MDS had no significant difference from HC or L-MDS group（P=0.3232 and P=0.1537,respectively）;Expression of PD-L1 on MDSC in H-MDS,L-MDS and HC group were 32.79±13.84%,20.71±14.79%and 10.84±7.23%,respectively.H-MDS and L-MDS group had significantly higher PD-L1 level than HC group（P<0.001 and P=0.038,respectively）,and H-MDS group had a relatively higher level than L-MDS group（P=0.011）.（3）Concentration of BM supernatant CCL2 in H-MDS,L-MDS and HC group were 119.1±65.60pg/mL,132.7±77.82pg/mL and 61.39±23.84pg/mL,respectively.Level of CCL2 in both H-MDS and L-MDS group were significantly higher than in HC（P=0.035 and P=0.015,respectively）,but there was no significant difference between H-MDS and L-MDS group（P=0.577）.（4）Concentration of BM supernatantIL-6inH-MDS（169.4±101.8pg/mL）andL-MDSgroup（144.0±70.27pg/mL）were both higher than in HC group（57.02±48.18pg/mL）（P=0.005 and P=0.025,respectively）,but there was no significant difference between H-MDS and L-MDS group（P=0.459）;Concentration of serum IL-6 in H-MDS（153.75±71.32pg/mL）and L-MDS group（141.53±40.76pg/mL）were both higher than in HC group（80.89±39.85pg/mL）（P=0.008 and P=0.032,respectively）,but threr was no significant difference between H-MDS and L-MDS group（P=0.625）.Both the concentration of BM supernatant and serum IL-6 were positively correlated with STAT3 activation rate of MDSC（r=0.5424,P=0.0244 and r=0.5826,P=0.0226,respectively）.Part 3.（1）The expression of PD-1 on CD8⁺T cells in H-MDS group（31.97±18.60%）was significantly higher than that in L-MDS group（21.43±14.52%,P=0.043）,and were both significantly higher than that in HC group（10.28±7.04%）（P<0.0001 and P=0.030,respectively）.（2）The expression of CD3ζon CD8⁺T cells in H-MDS and L-MDS group were significantly lower than that in HC group（1.59±0.77%vs 10.55±11.10%,P=0.001 and 4.95±3.3%vs 10.55±11.10%,P=0.044,respectively）.However,there was no significant difference between H-MDS group and L-MDS group（1.59±0.77%vs 4.95±3.3%,P=0.219）.（3）Expression of Perforin and Granzyme B in CD8⁺T cells from MDS patients were both decrease in MDS patients compared with HC group（14.79±23.38%vs 34.74±26.79%,P=0.0449and 17.01±25.33%vs 41.71±35.40%,P=0.0465;respectively）.（4）Concentration of BM supernatant IFN-γwas decreased in MDS patients compared withHC group（325.1±145.8 vs 506.9±271.4pg/mL,P=0.0230）.Part 4.（1）Purity of sorted MDSC and CD8⁺T cells were generally≥90%;（2）Expression of STAT3 and ARG1 in MDSC were both decreased after STAT3blockade in vitro（3.112±2.018%vs 0.485±0.488%,P=0.0186 and 31.55±21.61%vs 21.15±16.69%,P=0.0031;respectively）.（3）Relative ARG1 mRNA expression in MDSC after STAT3 blockade in vitro was also decreased（1.878±2.064 vs 0.832±0.969,P=0.0297）.（4）Expression of CD3ζ,Perforin and Granzyme B in CD8⁺T cells in(MDSC_MDS+CTL_MDS)group were all lower than that in CTL_MDSDS group（3.543±2.056 vs 6.622±2.128%,P=0.020;8.390±5.181 vs 15.39±6.595%,P=0.0001;12.98±6.404 vs 22.31±8.412%,P=0.0003;respectively）;While the expression of CD3ζ,Perforin and Granzyme B in CD8⁺T cells got partially increased when blocking STAT3 pathway in(MDSC_MDS+CTL_MDS+STAT3 inhibitor)group than(MDSC_MDS+CTL_MDS)group（6.198±4.555 vs 3.543±2.056%,P=0.006;12.58±6.642vs 8.390±5.181%,P=0.0036;17.96±9.605 vs 12.98±6.404%,P=0.0030;respectively）.（5）.Concentration of IFN-γin culture supernatant was lower in(MDSC_MDS+CTL_MDS)group than that in CTL_MDSDS group（223.9±78.17 vs 331.8±45.46pg/mL,P=0.0044）;While the level of IFN-γgot partially increased when blocking STAT3 pathway in(MDSC_MDS+CTL_MDS+STAT3 inhibitor)group than(MDSC_MDS+CTL_MDS)group（273.9±310.8 vs 223.9±78.17pg/mL,P=0.0364）;Similarly,Concentration of IFN-γin culture supernatant was lower in(MDSC_MDS+CTL_HC)group than that in CTL_HCC group（326.2±181.7 vs 565.2±195.9 pg/mL,P=0.001）;While the level of IFN-γgot partially increased when blocking STAT3 pathway in(MDSC_MDS+CTL_MDS+STAT3inhibitor)group than(MDSC_MDS+CTL_MDS)group（426.7±203.4 vs 326.2±181.7pg/mL,P=0.0050）.（6）Apoptosis rate of CD8⁺T cells in(MDSC_MDS+CTL_MDS)group was higher than that in CTL_MDSDS group（22.80±8.300vs 16.04±4.415%,P=0.0011）;While the apoptosis rate got partially decreased when blocking STAT3 pathway in(MDSC_MDS+CTL_MDS+STAT3 inhibitor)group than(MDSC_MDS+CTL_MDS)group（18.39±7.721 vs 22.80±8.300%,P=0.0006）;Similarly,apoptosis rate of CD8⁺T cells was higher in(MDSC_MDS+CTL_HC)group than that in CTL_HCC group（16.91±6.623 vs 6.890±2.943%,P=0.0002）;While apoptosis rate got partially decreased when blocking STAT3 pathway in(MDSC_MDS+CTL_HC+STAT3 inhibitor)group than(MDSC_MDS+CTL_HC)group（11.76±4.231 vs 16.91±6.623%,P=0.001）.Conclusions:1.The percentage of MDSC was increased in MDS patients and correlated with the severity of disease;2.Level of IL-6 got increased in MDS patients and induced the activation of STAT3 pathway in MDSC;Higher level of CCL2/CCR2 was associated with recruitment and activation of MDSCs;Up-regulation of PD-L1 expression on MDSC in MDS patients might be associated with MDSC-mediated immunosuppression.3.Increased PD-1 expression and decrease CD3ζon CD8+T cells inhibited activation of CD8⁺T cells;Lower level of functional molecules Perforin,Granzyme B and IFN-γalso reflected the hypofunction of CD8⁺T cells in MDS patients;When co-cultured with MDSC from MDS patients,expression of CD3ζ,perforin,granzyme B and IFN-γof CD8⁺T cells got even lower,and apoptosis of CD8⁺T cells got increased;Meanwhile,function of CD8⁺T cells got partially restored when blocking STAT3 pathway in vitro.4.In MDS,activating STAT3 pathway could up-regulate the expression of ARG1in MDSC.MDSC could down-regulate the expression of CD3ζand decrease the production of perforin,granzyme B and IFN-γof CD8⁺T cells by STAT3/ARG1pathway to inhibit activation of CD8⁺T cells and finally impair antitumor immunity.PD-L1/PD-1couldbeanothercriticalpathwayforMDSCtoexert immunosuppression on CD8⁺T cells-dependent antitumor immunity in MDS..