The Effect And Mechanism Of Cytokines In Diffuse Large B Cell Lymphoma Microenvironment On CD19-CART Cells

BackgroundDiffuse large B cell lymphoma(DLBCL)is the most common subtype of NonHodgkin lymphoma(NHL),and accounts for 30-40% of all NHL.Rituximab in combination with CHOP is the first-line therapy,and 58%-75% of patients could achieve complete remission after 6 to 8 cycles of R-CHOP chemotherapy.However,50% DLBCL patients are refractory to or relapse after R-CHOP therapy.Salvage therapies for these patients include high dose chemotherapy and autologous hematopoietic stem cell transplantation.But the response rate and complete remission rate after salvage therapy are still unsatisfied.Chimeric antigen receptor-modified T cell(CART)is a newly emerging immunotherapy for refractory/relapsed B-cell malignancy.However,results of CART cell immunotherapy for B-cell NHL is inferior to B-cell acute lymphoblastic leukemia.The main factors causing this difference may lie in the complex lymphoma microenvironment,in which the inhibitory ingredients could negatively affect CART cell function.Cytokines are main components of lymphoma microenvironment,and the effects of different cytokines on CART cell remain to be clarified.Therefore,identification and clearance of inhibitory effects of cytokines on CART cells are the key point of improving CARTcells therapeutics effects for B-cell NHL.ObjectiveThe objectives of this study are to identify the effects of cytokines in lymphoma microenvironment on CART cells function,proliferation and apoptosis,and then to remold lymphoma microenvironment and to enhance CART cells capability to overcome suppressive lymphoma microenvironment by combining with other cytokines.Methods(1)Clinical data analysis: 41 cases of B-ALL and B-NHL patients who received CART cell therapy in Shanghai Changhai hospital are included in this study from Janurary 2014 to December 2018.Patients basic clinical information,diagnoses,therapeutic information and survival information were included.We used t test,Chi-square test and Fisher’s exact method to compare the basic clinical information of patients.The Kaplan-Meier method and the Log-rank test were applied to analyze and compare patients OS and PFS.(2)Experimental methods: CART cells were manufactured by using CD19-4-1BB lentiviral vector to transinfect T cells,and CART cells transinfection rate was detected by flow cytometry.CART cells were cultivated in medium containing defined cytokine cocktail for 48 h,and then mixed with target cells for the test of tumor killing and cytokines release analysis.CART cells were cultivated in medium containing different cytokines for 72 hours before proliferation and apoptosis analysis.CART cells tumor killing is assessed by CD107 a expression.Cytokines release is detected by Cytokines Beads Assay.CART cells proliferation is examined by CFSE dying.CART cells apoptosis is measured by Annexin V/PI.Results(1)B-ALL paitients show higher complete remission rate and longer PFS than BNHL with CART cells immunotherapy.Patients who received ASCT combining with CART have higher complete remission rate than those treated with CART alone.Lymphoma microenvironment may be the main explanation to these discrepancy.(2)CART cells manufactured in our experiments achieved high transfection rate to 70%-80%.CART cells could specifically recognize and kill CD19 positive target cells,at the same time,release cytokines IL-2,TNF-α and IFN-γ.(3)In the lymphoma microenvironment,IL-2 could enhance CART cell tumor lysis activity,cytokines release and CART cell proliferation,and reduce CART cells apoptosis.IL-15 could promote CART cell killing of tumor cells and inhibit CART apoptosis.IL-4 could decrease IFN-γ release and VEGF could accelerate CART cells apoptosis.Transforming growth factor(TGF-β)could enhance CART cells tumor lysis activity but strikingly inhibit CART cells cytokine release and proliferation,and increase CART cells apoptosis.(4)IL-15 could reverse TGF-β effects on CART cells.IL-15 combinating with TGF-β could remarkably enhance CART cells tumor lysis activity,cytokine release and proliferation,and reduce CART cells apoptosis.ConclusionLymphoma microenvironment is the main factor that compromise CART immunotherapy on B-NHL.TGF-β is a major inhibitory cytokine in lymphoma microenvironment,which could inhibit CART cells cytokine release and proliferation,and increse CART cells apoptosis.IL-15 could reverse the inhibiting effect of TGF-β on CART cells.Therefore,remolding microenvironment and enhancing CART cells capability to inhibitory microenvironment may be a valuable strategy to enhance CART cells therapeutic effects on B-NHL.