Study On Mechanism Of Action Of PD-L1 Monoclonal Antibody Combined With Chidamide On Diffuse Large B-cell Lymphoma

Objective:Diffuse large B-cell lymphoma(DLBCL)is one of the most prevalent and aggressive subtypes of non-Hodgkin’s lymphomas,and in particular,double-expression diffuse large B-cell lymphoma(DE DLBCL)has a poor prognosis.R-CHOP regimen is used as the firstline medication for diffuse large B-cell lymphoma.However,40% of patients,especially those with DE DLBCL,exhibit a poor response,gaining limited benefit from tumor biological heterogeneity,host microenvironment complexity,and drug resistance.Immunotherapy has demonstrated efficacy in various cancers,specifically blocking the programmed death 1(PD-1)or the ligand programmed death-ligand 1(PD-L1).Chidamide,a histone deacetylase inhibitor,has been shown to increase immunotherapy sensitivity.This study explored the effect of combining PD-L1 monoclonal antibody with chidamide on DLBCL cells and its potential molecular mechanism.Methods and Materials:Using a Ficoll gradient centrifuge to separate peripheral blood mononuclear cells from healthy individuals,CD8+T cells were subsequently enriched through magnetic bead sorting.After enrichment,CD8+T cells reached at least 90%purity and were stimulated for activation and expanded.All DLBCL cells(SU-DHL-4 and OCI-LY-10)used in experiments are in the logarithmic growth phase to ensure good cell viability.Activated CD8+T cells were co-cultured with SU-DHL-4 and OCI-LY-10 cells for 48 h at a ratio of10:1,and the co-culture system was treated with anti-PD-L1 monotherapy,or anti-PD-L1 combined with chidamide,as the experimental group.CCK-8 method was used to evaluate the effect of different concentrations of PD-L1 monoclonal antibody on the activity of DLBCL cells and to screen the optimal drug concentration.The apoptosis rate of tumor cells was quantified by double staining of 5(6)-Carboxyfluorescein diacetate succinimidyl ester/Propidium Iodide(CFSE/PI).Western blotting was conducted to analyze the expression of the protein of JAK2,STAT3,p-JAK2,p-STAT3,Bcl-2,c-Myc,PD-L1,PD-1,and pro-and anti-apoptotic genes.Real-time fluorescent polymerase chain reaction(q RTPCR)was used for analyzing PD-L1,PD-1,c-Myc,JAK2,STAT3,Bcl-2,Bax,IFN-γ,Granzyme B,TNF-α m RNA.Results:PD-L1 monoclonal antibody can inhibit the growth of SU-DHL-4 and OCI-LY-10,and the optimal concentration is 10 μg/m L.After co-culture of activated CD8+T cells and tumor cells for 48 h,the relative expression of PD-1 m RNA increased in CD8+ T cells,but PD-1protein was down-regulated,which was considered related to protein degradation,and PDL1 m Ab or with Chidamide could prevent this process.In addition,CD8+T cells showed decreased IFN-γ,Granzyme B,and TNF-α m RNA after co-culture.On the other hand,the co-culture of CD8+T cells with tumor cells(positive control group)upregulated the PD-L1 expression of tumor cells’ surface and drug groups.Compared to PD-L1 alone,coadministration with chidamide increased apoptosis rate in tumor cells(SU-DHL-4:21.8%±2.54% vs 29.2%±0.64%,OCI-LY-10: 18.5%±1.98% vs 24.3%±2.60%),and upregulated IFN-γ,Granzyme B,TNF-α m RNA expression in CD8+T cells,which are possibly associated with JAK2/STAT3 signalling pathway activation.Furthermore,results demonstrated decreased c-Myc,Bcl-2,Caspase3,Caspase9,and Bax proteins expression.Cleaved-PARP protein expression increased in the experimental groups,compared with the negative and positive control groups.Conclusion:PD-L1 monoclonal antibody in combination with Chidamide can increase the efficacy of its anti-PD-L1 monotherapy on diffuse large B-cell lymphoma,possibly associated with JAK2/STAT3 pathway activation,recovery of immune cell activity,and tumor cell apoptosis.Consequently,the PD-1/PD-L1 blockade combined with oral histone deacetylase inhibitor is a promising approach to improve patients’ therapeutic outcomes with DE DLBCL.