The Effect Of HDAC Inhibitors On PD-1(+) Cells In Peripheral Blood Of Patients With PTCL And Preliminary Exploration Of Its Mechanism

BACKGROUND:Peripheral T cell lymphoma(PTCL)is a group of non-Hodgkin's lymphomas(NHLs)originated from mature T cells with high heterogeneity.Although the incidence is low in western countries,but the incidence in China is significantly higher.Patients with PTCL have a poor prognosis and a high rate of relapsing,but the choice of effective salvage therapy was very limited.Peripheral blood lymphocytes are an important component of the human immune system.Among them,Tregs are involved in multiple tumors.Previous studies have shown that PD-1/PD-L1 may be an important mechanism for regulatory Tregs to exert immunosuppressive effects.PD-1 is an important immune checkpoint.It participates in tumor escape by inhibiting the function of tumor-infiltrating lymphocytes in the tumor microenvironment.Based on this theory,PD-1 monoclonal antibody has been used in lung cancer,melanoma and Hodgkin lymphoma,and it has achieved a amazing results.In our previous study,we found that PD-1 expression in peripheral blood CD4+ lymphocytes and Tregs of PTCL patients was significantly higher than the normal and correlated with the prognosis,indicating that circulating PD-1(+)cells play important role in PTLC.HDAC(histone deacytelase inhibitor,HDAC)inhibitors are novel small-molecule-targeted drugs that exert anti-tumor effects through the regulation of epigenetics.They can maintain DNA transcriptional activity by blocking the deacetylation of HDAC and cause changes in multiple signaling pathways which can induce the apoptosis and differentiation of tumor cell and inhibit angiogenesis[1-3].At the same time,it can also enhance the function of NK cells and CTLs.HDAC inhibitors as new drugs for the treatment of PTCL are gradually showing satisfied clinically effects.Romidepsin and Belinostat have obtained FDA approval for the treatment of relapsed and refractory PTCL.Chidamide as a domestic HDACi has also been approved for treatment of refractory/relapsed PTCL in our country.Our previous study found that the expression of PD-1 on T cells in peripheral blood was significantly higher in patients with PTCL than in normal controls,and the expression of PD-1 in peripheral blood T cells was decreased after treatment with HDACi and was positively correlated with the prognosis.It suggested that the immune function of PD-1(+)cells in circulation of PTCL patients was insufficient,and chidamide may regulate the function of PD-1(+)cells and restore its anti-tumor effects.Based on the above theories and our previous findings,we will perform gene expression profiling of PD-1(+)cells in this research to clarify its significance in PTCL,and further study the influence of chidamide on PD-1(+)cells and the mechanisms.OBJECTIVE:To figure out the role of circulating PD-1(+)cells in PTCL and the mechanism of chidamide influenced on PD-1(+)cellsMethods:1.The peripheral blood was collected from 77 newly diagnosed PTCL patients and50 healthy controls.Flow cytometry was used to analyze the expression of PD-1 on the surface of Tregs in peripheral blood of patients with PTCL.The relationship between the expression and the prognosis of patients was analyzed.2.18ml peripheral blood samples were collected from 22 newly diagnosed patients PTCL and 13 normal controls.Mononuclear cells were extracted from human lymphocytes.PD-1(+)lymphocytes were selected by PD-1 magnetic beads and than extract RNA for gene expression profiling.Gene expression profiles of peripheral blood PD-1(+)lymphocyte in patients and healthy subjects were analyzed by digital gene expression profiling technique,in order to explore the molecular basis of functional differences.FPKM(FragmentsPer Kilobases per Millionreads)value>1 is defined as the expression of the gene,edgeR software is used for significant differences in expression analysis,padj<0.05 as the difference significance standard;the differential genes were analyzed by GO,KEGG,Reactome enrichment analysis.3.Peripheral blood was collected from 57 PTCL patients treated with CHOP or combined with chidamide,flow cytometry was used to analyze the changes of expression of PD-1 on Tregs in circulation.18ml peripheral blood samples were collected from 5 newly diagnosed PTCL patients,and PD-1(+)cells were selected by magnetic beads.The cells were plated in 96-well plates at 2×10 5 cells/well.PD-I(+)lymphocytes were treated with 0.3?M and 0.5?M chidamide respectively according to the protocol of chidamide and references.PD-1(+)cells were labeled with CFSE-FITC and cultured for 48 for cell proliferation test,7-aad-PerCp were labeled for apoptosis test.ELISA was used to detect the changes of IFN-y levels in the cell supernatant;CFSE/7aad was used to detect the killing effect on target cells:PD-1(+)and PD-1(-)cells were seeded in 96-well plates at 2×105 cells/well seperately.The experimental group was treated with 0.3 ?M of chidamide for 48 hours.The control group was cultured in 10%FBS in 1640 complete medium.Tumor cells were labeled with CFSE and the ratio of PD-1,(+)or PD-1(-)cells:tumor cells was 50:1 and 25:1 respectively,afer co-cultured for 4 hours,these cells were labeled with 7-aad-PerCp,and flow cytometry was used to detected the ablity of PD-1(+)or PD-1(-)cells to kill tumor cells.The killing rate =(CFSE/7-aad double positive cell number)/CFSE positive cell number × 100%.0.3 and 0.5 ?M chidamide were treated on PD-1(+)cells for 48h,RT-PCR was used to detect the expression of Racl and MMP-9;ELISA was used to detect the section of MMP-9 on the cell supernatant.Result:1.The expression of PD-1 on Tregs in circulation of PTCL patients(1)The proportion of PD-1 on Tregs in peripheral blood of newly diagnosed patients who were classified into high-risk group was significantly higher than that in normal subjects(P= 0.0036),while the proportion of low-risk patients showed no difference compared with normal people(P= 0.5571);and the proportion of PD-1 on Tregs of high-risk patients was also significantly higher than that of low-risk patients;(2)The proportion of PD-1 expressed on Tregs of patients with progressive disease(PD)before treatment was significantly higher than that of patients with complete response(CR)or partial response(PR)(p<0.001).When the proportion was higher than 19.6%,the sensitivity was 80%and the specificity was 90.6%to predict progression of disease2.Gene expression profiling of circulating PD-1(+)cells of PTCL(1)The gene expression profiling in patients group was different,the expressions of ENTPD-1,BCL-6,MCL1,CD55 were high in 3 patients while the expressions of FZD5,CD274,PDCD1LG2,CD38,and CCL252 were high in other 2 patients and these genes may be related to drug resistance;(2)The expression of miRNA23A,HES6,CHI3L1,miRNA24-2,and SOCS1 in patients with progression disease was significantly higher than that in unadvanced patients,whereas the expression of TNF was significantly decreased;(3)Compared with healthy controls,PD-1(+)lymphocytes in peripheral blood of PTCL patients had distinct gene expression profiles.There were 2099 differentially expressed genes of which 1485 genes were highly expressed and 614 genes were lowly expressed.Enrichment analysis showed that the differential gene function was related to cell cycle,innate immune response,and interferon-y(IFN-y)signaling pathways.And we also found that the expression of Racl,MMP8,Racl,MMP-9 and CD177 was extremely high in PTCL patients while the expression of PVRL2 was low in PTCL patients.3.The influence of chidamide on PD-1(+)cells of PTCL patients(1)The proportion of PD-1 expressed on Tregs in peripheral blood of patients with CR/PR was significantly lower after chidamide treatment(P= 0.0066);while the phenomenon was not observed in patients treated with traditional chemotherapy(P=0.0533).(2)0.3?M chidamide treated on PD-1(+)cells for 48 hours exerted minimal cytotoxicity and significantly increased IFN-y secretion;0.5 ?M chidamide presented a killing effect on PD-1(+)cells(P=0.0013);PD-1(+)cells of PTCL patients had no proliferative capacity,and neither 0.3 ?M nor 0.5 ?M chidamide could promote its proliferation.(3)The secretion of IFN-y and the ability to kill K562 of PD-1(+)cells is weaker than PD-1(-)cells and 0.3 ?M chidamide could restore these functons of PD-1(+)cells(P=0.0006).(4)The expression of Racl and MMP-9 was decreased and PVRL2 was upregulated after the treatment of 0.3 ?M chidamide,and the secretion of MMP-9 was decreased.CONCLUSIONS:1.The proportion of PD-1 expressed on Tregs in peripheral blood of newly diagnosed PTCL patients may relate to prognosis;2.Gene expression was dramatically different between patients and the healthy,and the enrichment analysis showed that these genes were related to cell cycle,innate immune response,and interferon-y(IFN-y)signaling pathways.3.The proportion of PD-1 can be reduced after chidamide treatment,indicating that the funciotn of PD-1(+)cells could be regulated by chidamide.The cell experiment showed that the immune function of PD-1(+)cells is weaker than PD-1(-)cells,and chidamide can restore anti-tumor function of PD-1(+)cells by promoting the production of IFN-y and increasing their direct killing effect on tumor cells.Expression of Racl and MMP-9 was extremely high and PVRL2 was lowly expressed in PTCL patients and chidamide can reduce the expression of Racl and MMP-9 while upregulated PVRL2,and this maybe a mechanism of chidamide treating PTCL.