Myeloid-derived Suppressor Cell-mediated Immunosuppressive Pathways In Myelodysplastic Syndromes

Objective:Myelodysplastic syndromes（MDS）is a heterogeneous group of clonal hematopoietic stem cell disorders.Antitumor immunity impairment is considered to play an important role in the development of MDS.However,the specific mechanisms are not clear.In this study,myeloid-derived suppressor cells（MDSC）and CD8⁺T cells which are from bone marrow of MDS patients were sorted and co-cultured in vitro.We detected the changes of the function of CD8⁺T cells blocking or don’t blocking the PD-1/PD-L1 pathway and C-myc pathway to explore the mechanism of MDSC-mediated immunosupression in MDS.Materials and methods:Sixteen MDS patients newly diagnosed in Tianjin Medical University General Hospital and six healthy volunteers were included in the study,and the clinical data of MDS patients were collected.Part one:CD8⁺T cells were isolated from bone marrow of MDS patients and healthy controls,then cultured separately in vitro.CD8⁺T cells were separated by MACS.Flow Cytometry was conducted to detect the expression of PD-1,perforin and Granzyme B in CD8⁺T cells.The apoptosis rate of CD8⁺T cells was detected by apoptosis kit.The function of CD8⁺T cells was compared between MDS patients and healthy controls.Part two:MDSC and CD8⁺T cells were isolated from bone marrow of MDS patients and healthy controls.Isolation of CD8⁺T cells and MDSC were carried out by MACS and FCM respectively.In vitro,CD8⁺T cells were cultured separately in one group as control.Co-culture of MDSC and CD8⁺T cells in a ratio of 3:1 in another group.Flow cytometry was conducted to detect the expression of PD-1,perforin and Granzyme B in CD8⁺T cells in both the individual culture group and the co-culture group,whose apoptosis rate was measured by apoptosis kit.Detecting the function of CD8⁺T cells in single culture group and co-culture group,to explore the immunosuppression of MDSC on the function of CD8⁺T cells.Part three:MDSC and CD8⁺T cells were isolated from bone marrow of MDS patients and healthy controls.Isolation of CD8⁺T cells and MDSC were carried out by MACS and FCM respectively.In vitro,the co-culture of MDSC and CD8⁺T cells by a ratio of 3 to 1 with PD-1 inhibitor and C-myc inhibitor.Flow cytometry was conducted to detect the expression of PD-1,perforin and Granzyme B in the following 4 groups:the MDSC+CD8⁺T group,the MDSC+CD8⁺T+PD-1 inhibitor group,the MDSC+CD8⁺T+C-myc inhibitor group and the MDSC+CD8⁺T+PD-1inhibitor+C-myc inhibitor group,whose apoptosis rate was measured by apoptosis kit.Detecting the function of CD8⁺T cells when blocking or don’t blocking the PD-1/PD-L1 pathway and C-myc pathway,and explore the effects of these two pathways on the immunosuppression of MDSC.Result:Part one:the function of CD8⁺T cells of MDS patients in vitro.（1）Purity of sorted CD8⁺T cells were generally≥90%.（2）The expression of PD-1 in CD8⁺T cells of MDS patients(CTL_MDS)was higher than that of healthy control(CTL_HC)（8.98±1.26%vs 4.41±0.97%,P<0.05）.And the expression of perforin and Granzyme B in CD8⁺T cells of MDS patients(CTL_MDS)were decreased compared to healthy control(CTL_HC)（11.12±4.14%vs25.14±2.56%,P<0.05;8.60±3.08%vs 15.55±2.60%,P<0.05;respectively）.（3）The apoptosis rate of CD8⁺T cells of MDS patients(CTL_MD)was higher than that of healthy control(CTL_HC)（19.19±2.39%vs 15.15±1.30%,P<0.05）.Part two:immunosuppression of MDSC of MDS on the function of CD8⁺T cells in vitro.（1）Purity of sorted MDSC and CD8⁺T cells were generally≥90%and the co-culture of MDSC and CD8⁺T cells by a ratio of 3 to 1 in vitro.（2）The expression of PD-1 in CD8⁺T cells in co-culture group(MDSC_MDS+CTL_MDS)was higher than that in single culture group(CTL_MDS)among MDS patients（17.68±2.22%vs 8.98±1.26%,P<0.05）,While the expression of perforin and Granzyme B in CD8⁺T cells were decreased（5.47±1.90%vs 11.12±4.14%,P<0.05;3.97±1.84%vs 8.60±3.08%,P<0.05;respectively）.For healthy control,the expression of PD-1 in CD8⁺T cell in the co-culture group(MDSC_HC+CTL_HC)was higher than that in the single culture group(CTL_HC)（6.48±1.38%vs 4.41±0.97%,P<0.05）,while the expression of perforin and granzyme B were decreased（17.09±2.06%vs 25.14±2.56%,P<0.05;10.56±2.37%vs 15.55±2.60%,P<0.05;respectively）;（3）The apoptosis rate of CD8⁺T cells in the co-culture group of MDS patients(MDSC_MDS+CTL_MDS)was higher than that the single culture group(CTL_MDS)（29.19±3.22%vs 19.19±2.39%,P<0.05）.The apoptosis rate of CD8⁺T cell in the co-culture group of healthy control(MDSC_HC+CTL_HC)was higher than that in the single culture group(CTL_HC)（19.88±1.54%vs 15.15±1.30%,P<0.05）.（4）The change rate of the expression of PD-1（96%vs 47%）,perforin and Granzyme B（51%vs 32%;53%vs 32%;respectively）and the change rate of apoptosis（52%vs 31%）in CD8⁺T cells between the co-culture group and the single culture group of MDS patients were greater than those of the healthy controls.Part three:the effect of PD-1/PD-L1 pathway and C-myc pathway on immunosuppression of MDSC.（1）Purity of sorted MDSC and CD8⁺T cells were generally≥90%and the co-culture of MDSC and CD8⁺T cells by a ratio of 3 to 1 in vitro.（2）In the co-culture group of MDS patients,the expression of PD-1 in CD8⁺T cells decreased in PD-1 inhibitor group(MDSC_MDS+CTL_MDS+PD-1 inhibitor)compared with that in non-inhibitor group(MDSC_MDS+CTL_MDS)（14.79±1.71%vs17.68±2.22%,P<0.05）,while the expression of perforin and Granzyme B increased（8.18±2.87%vs 5.47±1.90%,P<0.05;5.91±2.90%vs 3.97±1.84%,P<0.05;respectively）,and the apoptosis rate decreased（22.40±4.60%vs 29.19±3.22%,P<0.05）;（3）In the co-culture group of MDS patients,the expression of PD-1 in CD8⁺T cells decreased in C-myc inhibitor group(MDSC_MDS+CTL_MDS+C-myc inhibitor)compared with that in non-inhibitor group(MDSC_MDS+CTL_MDS)（15.34±2.01%vs17.68±2.22%,P<0.05）,while the expression of perforin and Granzyme B increased（8.12±2.45%vs 5.47±1.90%,P<0.05;5.56±1.76%vs 3.97±1.84%,P<0.05;respectively）,and the apoptosis rate decreased（23.88±6.38%vs 29.19±3.22%,P<0.05）.（4）In the co-culture group of MDS patients,the expression of PD-1 in CD8⁺T cells decreased while conducted with both the C-myc inhibitor and PD-1 inhibitor(MDSC_MDS+CTL_MDS+C-myc inhibitor+PD-1 inhibitor)compared with that in non-inhibitor group(MDSC_MDS+CTL_MDS)（13.53±1.87%vs 17.68±2.22%,P<0.05）,while the expression of perforin and Granzyme B increased（9.78±2.74%vs5.47±1.90%,P<0.05;7.52±3.05%vs 3.97±1.84%,P<0.05;respectively）,and the apoptosis rate decreased（21.08±4.36%vs 29.19±3.22%,P<0.05）.（5）For MDS patients,the change rate of expression of PD-1（23%vs 16%）,perforin and Granzyme B（78%vs 49%;89%vs 48%;respectively）and the change rate of apoptosis（27%vs 23%）in CD8⁺T cells between the co-culture with PD-1inhibitor and C-myc inhibitor group(MDSC_MDS+CTL_MDS+C-myc inhibitor+PD-1inhibitor)and co-culture without inhibitor group(MDSC_MDS+CTL_MDS)were greater than those between the co-culture with PD-1 inhibitor group(MDSC_MDS+CTL_MDS+PD-1 inhibitor)and co-culture without inhibitor group(MDSC_MDS+CTL_MDS).（6）For MDS patients,the change rate of expression of PD-1（23%vs 13%）,perforin and Granzyme B（78%vs 48%;89%vs 40%;respectively）and the change rate of apoptosis（27%vs 18%）in CD8⁺T cells between the co-culture with PD-1inhibitor and C-myc inhibitor group(MDSC_MDS+CTL_MDS+C-myc inhibitor+PD-1inhibitor)and co-culture without inhibitor group(MDSC_MDS+CTL_MDS)were greater than those between the co-culture with PD-1 inhibitor group(MDSC_MDS+CTL_MDS+C-myc inhibitor)and co-culture without inhibitor group(MDSC_MDS+CTL_MDS).（7）In the co-culture with PD-1 inhibitor group of healthy controls(MDSC_HC+CTL_HC+PD-1 inhibitor),the expression of PD-1（5.72±0.67%vs6.48±1.38%,P>0.05）,perforin and Granzyme B（19.53±3.19%vs 17.09±2.06%,P>0.05;12.12±3.00%vs 10.56±2.37%,P>0.05;respectively）in CD8⁺T cells did not change significantly compared with that without PD-1 inhibitor group(MDSC_HC+CTL_HC),the only difference was the decrease of apoptosis rate（17.20±1.02%vs 19.88±1.54%,P<0.05）in PD-1 inhibitor group compare with without PD-1 inhibitor group.（8）In the co-culture group of the healthy controls,the expression of PD-1（5.88±1.19%vs 6.48±1.38%,P>0.05）,perforin and Granzyme B（19.05±2.75%vs 17.09±2.06%,P>0.05;12.38±2.72%vs 10.56±2.37%,P>0.05;respectively）in CD8⁺T cells did not change significantly when C-myc inhibitor(MDSC_HC+CTL_HC+C-myc inhibitor)was used compared with that without C-myc inhibitor group(MDSC_HC+CTL_HC),but there had difference of the decrease of apoptosis rate（16.95±0.89%vs 19.88±1.54%,P<0.05）.（9）In the co-culture group of the healthy controls,the expression of PD-1（5.12±0.84%vs 6.48±1.38%,P>0.05）in CD8⁺T cells did not change significantly when C-myc inhibitor and PD-1 inhibitor(MDSC_HC+CTL_HC+C-myc inhibitor+PD-1 inhibitor)was used compared with that without inhibitor group(MDSC_HC+CTL_HC),while the perforin and Granzyme B expression increased（21.24±3.17%vs 17.09±2.06%,P<0.05;13.81±2.57%vs 10.56±2.37%,P<0.05,respectively）,the apoptosis rate decreased（15.42±0.64%vs 19.88±1.54%,P<0.05）.Conclusion1.The founction of CD8⁺T cells of MDS patients was weakened and the high expression of PD-1 in CD8⁺T cells of MDS patients suggesting the basis for the targeted therapy of PD-1 inhibitor in MDS patients.2.The MDSC exerts immunosuppression on CD8⁺T cells and the immunosuppression of MDSC of MDS patients was enhanced.3.After co-cultured MDSC and CD8⁺T cells in vitro and blocking the PD-1/PD-L1 pathway,the immunosuppression of MDSC on CD8⁺T cells was weakened,suggesting that MDSC can reduce the function of CD8⁺T cells and increase apoptosis through PD-1/PD-L1 pathway,which leads to abnormal anti-tumor immunity.4.After co-cultured MDSC and CD8⁺T cells in vitro and blocking the C-myc pathway,the immunosuppression of MDSC on CD8⁺T cells was impaired,This indicated that the expression of C-myc gene enhances the inhibitory effect of MDSC on CD8⁺T cell function,leading to the abnormal antitumor immunity.5.After co-cultured MDSC and CD8⁺T cells in vitro and blocking the PD-1/PD-L1 pathway and C-myc pathway,The immunosuppression of MDSC on CD8⁺T cells is weaker than that blocking PD-1/PD-L1 pathway alone or C-myc pathway alone.It suggested that the combined blocking of C-myc gene could enhance the effect of anti-PD-1 immunocheckpoint inhibitors.