Expression And Mechanism Of PD-1/PD-L1 And Treg In JAK2 V617F-positive Myeloproliferative Neoplasms

Objective:To Test the expression of programmed death-1(PD-1),programmed death ligand-1(PD-L1)and CD4+CD25+Foxp3+ regulatory T cells(Treg)in JAK2 V617F-positive myeloproliferative neoplasms(MPN),and to explore the effect and the mechanism of interferon(IFN-alpha 2b)and ruxolitinib on the expression of PD-1,PD-L1 and Treg.Methods:1 The MPN patients and some healthy volunteers in the No.1 hospital of Baoding were selected from Sep.of 2016 to Dec.of 2018.All MPN patients were JAK2 V617 F positive at first diagnosis by fluorescence quantitative PCR.In the newly diagnosed group,41 patients(23 males and 18 females,aged 27-82,median age 60 years)were enrolled,including 11 cases of Polycythemia vera(PV),12 cases of Primary Myelofibrosis(PMF),18 cases of Essential Thrombocythemia(ET).20 cases of IFN-a2 b treatment group(11 males and 9 females,aged 27-81,median age 62 years).There were 6 cases of Polycythemia vera(PV),6 cases of Primary Myelofibrosis(PMF),8 cases of Essential Thrombocythemia(ET).20 healthy volunteers were enrolled as control group(10 males and 10 females,aged 25-66,median age 59 years).Another 5 patients(3 males and 2 females,aged 68-81,average age 74)with advanced or transformed leukemia.All patients signed the informed consent and were approved by the Ethics Committee of No.1 hospital of Baoding.2 Experimental groups:The 41 case of MPN patients with JAK2V617 F positive were selected as the newly diagnosed group(untreated patients group);20 cases of treatment group(all patients received IFN-a2b(interferon alpha2b)subcutaneous or intramuscular injection,treatment for more than 6 months,3 times per week,3×106U/time;20 healthy volunteers were selected as the control group.3 The JAK2 V617F/JAK2 mutation rate(ratio of mutant JAK2 to wild JAK2)were detected in MPN patients by fluorescence quantitative PCR.4 The expression levels of PD-1 and PD-L1 in bone marrow and Treg in peripheral blood of different groups were detected by flow cytometry.5 Culture in vitro:The bone marrow and peripheral blood samples from 15 newly diagnosed patients were selected and before and after treated with 1×106U/L IFN-α2b for 48 h,and then test the expression levels of PD-1,PD-L1 and Treg.Bone marrow and peripheral blood samples from 12 newly diagnosed patients were selected and before and after treated with 250nmol/L Ruxolitinib for 48 hours,and then test the expression levels of PD-1,PD-L1 and Treg.6 All data were analyzed with SPSS19.0 software.All data conforming to normal distribution were compared by variance analysis.Q test was used for comparison between two groups.Pearson linear correlation analysis was used for correlation degree between two variables.IFN-a2 b and Ruxolitinib in vitro intervention data conformed to non-normal distribution.Wilcoxon rank sum test was used for all non-normal distribution data.P<0.05 was considered statistically significant.Results:1 JAK2V617 F mutation in patients with myeloroliferative neoplasms(MPN).PCR assessment showed that all patients with MPN were positive for JAK2 V617 F mutation by fluorescence quantitative PCR at the time of initial diagnosis.The mutation of JAK2 V617 F was 54.12%±24.86% in the newly diagnosed group and 40.12%±20.55% in the IFN-alpha 2b treatment group.The mutation of JAK2 V617 F in the newly diagnosed group was significantly higher than that in the IFN-a2 b treatment group,the difference had significance of statistics(P<0.05).2 Flow cytometry was used to detect the expression of PD-1,PD-L1 and Treg in MPN patients.PD-1 was highly expressed on activated effector T lymphocyte cells,B lymphocyte cells,and natural killer(NK)cells,PD-1 was also expressed on some tumor cells,PD-L1 was mainly expressed on antigen presenting cells(APC)and tumor cell.The positive rates of PD-1 and PD-L1 expression in bone marrow myeloid cells in the newly diagnosed group(40.87%±23.11%;44.79%±19.15%)were higher than those in the treatment group(17.86%±10.59%;29.95%±14.83%)and the control group(1.62%±0.78%;1.63%±0.84%)(P<0.01);The difference was statistically significant(P<0.01).The positive rate of Treg expression in the newly diagnosed group(12.24%±7.41%)was higher than that in the treatment group(5.26%±3.64%)and control group(1.24%±0.83%)(P < 0.01).The difference was statistically significant(P<0.01).The expression levels of PD-1 and PD-L1 in lymphocyte of the newly diagnosed group and IFN-a2 b treatment group were higher than those of the control group,but the expression level was still low,and there was no statistical significance(P > 0.05).3 The correlation of JAK2 V617 F,PD-1,PD-L1 and Treg expression in JAK2 V617 F positive MPN patients.Pearson correlation analysis showed that PD-L1 in myeloid cells was positively correlated with PD-1 in myeloid cells(r = 0.758,P < 0.01);PD-1 in lymphocytes was positively correlated with PD-1 in myeloid cells(r = 0.382,P < 0.05);PD-1 in myeloid cells was positively correlated with JAK2 V617 F mutation(r = 0.498,P < 0.01);PD-L1 in myeloid cells was positively correlated with JAK2 V617 F mutation(r = 0.686,P < 0.01);JAK2 V617 F mutation was positively correlated(r = 0.406,P < 0.01).There was no correlation between Treg expression and the above groups(P > 0.05).There was no correlation between lymphocyte PD-L1 and the above groups(P > 0.05).41 patients in the newly diagnosed group were divided into JAK2 V617 F mutation≥50% group and JAK2 V617 F mutation <50% group,with 21 and 20 cases respectively.Results: The expression of PD-1 in myeloid cells in JAK2≥50% group and JAK2<50% group were(42.86%±19.61%;26.25%±22.28%);the expression of PD-1 in lymphocyte were(4.20%±4.62%;1.99%±2.01%);the expression of PD-L1 in myeloid cells were(51.43%±17.12%;31.38%±15.79%);and the expression of Treg were(11.61%±8.70%;8.72%±5.65%).The expressions of PD-1,PD-L1,PD-1 and Treg in myeloid cells in JAK2≥50% group were significantly higher than those in JAK2<50% group,The difference was statistically significant(P < 0.05).There were 5 patients with progressive or transformed leukemia,including 3 males and 2 females,aged 68-81 years,with an average age of 74 years.The expression levels of JAK2V617 F mutation,PD-1 in myeloid cells,PD-L1 in myeloid cells,PD-1 in lymphocyte and Treg were respectively(84.72%±5.70%;76.50%±5.36%;12.74%±4.44%;27.25%±7.92%).JAK2V617 F mutation,PD-1 in myeloid cells,PD-L1 in myeloid cells,PD-1 in lymphocyte and Treg expression in this group were significantly higher than those in the newly diagnosed group,the treatment group and the control group.Because of there were fewer cases,no statistical analysis was made.4 Effects of IFN-a2 b and Ruxolitinib on the expression of PD-1,PD-L1 and Treg in primary MPN cells cultured in vitro.The expression of PD-1,PD-L1 in myeloid cells and Treg in peripheral blood decreased after 1×106U/L IFN-α2b intervention in vitro.The expressions of PD-1,PD-L1 in myeloid cells and Treg in peripheral blood in the pre-intervention group(48.48%±19.70%;42.54%±22.14%;10.0%±19.40%)were higher than those in the post-intervention group(45.55%±19.59%;35.79%±19.12%;5.27%±6.54%),The difference was statistically significant(P < 0.05).The expression of PD-1 in lymphocyte of the two groups were respectively pre-intervention group(4.21%±4.32%)and post-intervention group(1.67%±1.28%).There was no significant difference in expression between the two groups(P > 0.05).The expression of PD-1,PD-L1 and Treg in myeloid cells decreased after 250nmol/L Ruxolitinib intervention in vitro.The expressions of PD-1,PD-L1 and Treg in myeloid cells in the pre-intervention group(42.88%±24.92%;46.73%±21.76%;10.14%±10.92%)were higher than those in the post-intervention group(25.54%±19.70%;34.68%±23.51%;5.16%±7.52%),The difference was statistically significant(P < 0.05).The expression of PD-1 in lymphocyte of the two groups were respectively pre-intervention group(2.65%±2.82%)and post-intervention group(1.40%±1.47%).There was no significant difference in expression between the two groups(P > 0.05).Conclusion:1 PD-1,PD-L1 and Treg are involved in the pathogenesis of angiogenesis of myeloroliferative neoplasms.2 In patients with myeloroliferative neoplasms,the expression of PD-1 and PD-L1 in tumor cells(myeloid cells)was high,while the expression of Treg cells in peripheral blood was increased.3 JAK2 V617 F mutation was positively correlated with PD-1 and PD-L1 gene expression.High expression of PD-1 or PD-L1 may be associated with poor prognosis and leukemia transformation.4 After treatment with IFN-a2 b,the JAK2 V617 F mutation and the expression of PD-1,PD-L1 and Treg cells in MPN patients were decreased.5 Ruxolitinib can reduce the expression of PD-1,PD-L1 in primary bone marrow cells and Treg in peripheral blood of MPN patients in vitro.