Therapeutic Effect Of Depleting MDSC And Promoting DC Maturation With Gemcitabine On Mouse B-cell Lymphoma

The relapsed or refractory lymphomas are still lack efficient clinical treatment and novel strategies are required to develop.Vaccination withtumor antigen-pulsed dendritic cells(DC)has been shown to elicit anti-tumor immunity and to induce tumor regression in certain cancer patients.However,a number of clinical trials have failed to show significant superiority of DC immunotherapy to traditional therapy on lymphomas.One reason is that tumors can form immunosuppressive environment rendering them insensitive to immunotherapy.Another possible reason is that tumor antigen-pulsed,in vitro matured DC may decrese their function and quantity.Finally,it is proposed thatimmunotherapy is more efficient to eliminate small residual tumors rather than relapsed,refractory huge lymphomas.One strategy to enhance the efficacy of DC immunotherapy is to eliminate the immunol suppressor cells in host.Myeloid derived suppressor cells(MDSC)are often observed accumlating in many tumor-bearing mice and cancer patients.These cells had the ability to suppress immune responses and to promote tumor growth.Gemcitabine has been shown to selectively deplet MDSC,while preserving T cells and B cellsin the mice bearing mesotheliomas.It has been proposed that elimination of MDSCwith gemcitabine may enhance effects of cancer immunotherapy.Another strategy to enhance the efficacy of DC immunotherapy is to change the application method of DC vaccine.Traditional method is usually using tumor antigen peptides or tumor lysates to puls DC in vitroand vaccination by subcutaneous injection.This method is not consistent with natural antitumor process ofDC in vivo and exits obvious defects.While intratumoral injection of semi-mature naiveDC can maintain the activities and functions of DC better.The intratumoral injected DC may direct uptake antigens and also DAMP release from chemo-treated tumor cells,and further migrationand maturation to activating immune response.Purpose:To establish a new culture method for amplify a large number of high purity MDSC in vitro by mimicking the microenvironment of MDSC expansion in vivo,then to study the properties of cultured MDSC including the developmental biology in vitro and in vivo,the immune function,and whether cultured MDSC promote the growth of lymphoma in mice;whether the number of MDSC increase in lymphoma bearing mice;whether gemcitabine eliminate MDSC in lymphoma bearing mice and promote maturation of intratumoral injected semi-maturenaiveDC;whether gemcitabine chemotherapy combined with intratumoral injection of semi-mature DC play a synergistic effect on lymphoma,and boost the IFN-y secretion and CTL activity;whether T cells and NK cellsparticipate in the antitumor immunity.Methods:1.For expansion MDSC in vitro,mouse bone marrow cells cultured with serum-free medium and mGM-CSF(30ng/ml)on themitomycin C-inactivated splenic endothelial stromal cell line 107B as feeder cells.The ratio of CD1 lb+Gr-1+ cells in the harvested cells was detected by flow cytometry.MDSC were further purificatedby magnetic cell sorting the Gr-1+cells and their immunophenotype were analysized by flow cytometry.Colony-forming unit were detected using CFC assays.CFU-S were detectedby intravenous infusion of MDSC to the total body irradiated(8Gy)mice.ROS were detectedby fluorescence probe DCFDA.Argininase were detectedby test urea method.Suppression effect of MDSC on T cells proliferation stimulated by Con-A were detection by CCK8.Suppression effect of MDSC on OT-1 mouse derived OVA peptide specific CD8+T cells(CFSE labed)proliferation were detectedby flow cytometry.Percentage change of Treg cells in peripheral blood of Foxp3-IRES-GFP mice after MDSC infusion were detectedby flow cytometry.Observe the symptoms and survival rate of acute GVHD mice after MDSC transfusion.Observe the growth speedof lymphoma in mice after subcutaneous inoculating MDSC plus A20 lymphoma cells together.2.BALB/c mice were inoculated subcutaneously with 2×105 A20 lymphoma cells.Large tumor blocks with a minimal volume of 700-1,000 mm3 were usually detected 30 days after inoculation.Mouse spleen was grinded and red blood cells were eliminated by Tris-NH4C1,incubated with anti-Gr-1-PC5,anti-CD11b-PE antibody and analyzed MDSC ratio by flow cytometry.Spleen cells were incubated with non-specific blocking buffer and magnetic beads conjugated Gr-1 antibody.Gr-1+ cells were purified by magnetic sorting.Purified MDSC were then analyzed by flow cytometry for their immunophenotype.Purified MDSC were incubated with gemcitabine(lOug/ml)for 24h cells or 48h,apoptosis were detectedby AnnexinV-FITC/PI.Thirty days after inoculation the lymphoma bearing mice were intra-peritoneal injected of 120mg/kg gemcitabine.MDSC ratio in the spleen and apoptosis of A20 lymphoma cells were detected by flow cytometry after 48h.Bone marrowderived murine semi-mature dendritic cells were generated by culturing bone marrow cells from the femur of BALB/c mice in RPMI-1640/10%FCS supplemented with mGM-CSF.DC were incubated with gemcitabine pre-treated A20 cells for 48h and then labeled with anti-CD llc-APC and anti-CD86-PE antibody and analyzed by flow cytometry.Thirty days after inoculation the lymphoma bearing mice were intra-peritoneal injected of 120mg/kg gemcitabine and 48h after intratumoral injected with 5×106 CFSE labed DC.The mice peripheral blood,spleen and tumor tissue cell suspension was preparedafter 48h,labeled with anti-CD 1 lc-APC anti-CD86-PE antibody and analyzed by flow cytometry.3.Thirty days after inoculation the lymphoma bearing mice were randomly divided into 5 groups:A.intra-peritoneal injection of saline plus intratumoral injection of saline(NS);B.intra-peritoneal injection of saline plus intratumoral injection of 5×105 DC(DC);C.intra-peritoneal injection of gemcitabine plus intratumoral injection of saline(GEM);D.intra-peritoneal injection of gemcitabine plus intratumoral injection of 5×105 DC(GEM+DC);e.intra-peritoneal injection of gemcitabine plus intratumoral injection of 5×105 DC+5×106 MDSC(GEM+DC+MDSC).Observation the growth of tumor and the survival time of mice.Sevendays after DC intratumoral injection there mice of each group were taken and spleen cells suspension were prepared.IFN-?level in the cultured spleen cells supernatant were detected by ELISA.The CTL activity of the cultured spleen cellswere detected by LDH release assay.Single cell suspension of spleen or tumor tissue were incubated with anti-CD4-PE,anti-CD8-FITC and anti-DX5-PE antibody andthen analyzed by flow cytometry.Tumor tissues of each group were fixed and sliced and adding anti-DX5-PE antibody and anti-NKG2D-FITC antibody for incubation.NK cells in the tumor were detected by laser scanning confocal microscope.NK cell depletion was achieved using intra-peritoneal injection of 300 mg of anti-asialo-GM1 antibody or isotype control Ab on day 24,26,28,32 and day 35(DC intratumoral injection is day 32).CD4+ and CD8+ T cells were individually depleted by intra-peritoneal injection of 100 mg of anti-CD4(GK1.5),or anti-CD8(TIB105)mAbson day 24,26,28,32 and 35.Results:1.Application of mouse spleen endothelial stromal cells as feeder cells to mimic the microenvironment of MDSC development in vivocan acquire 4×108 cells from one mouse bone marrow cells after 9 days culture in vitro.The ratio of CD11b+Gr-1+ MDSC reached 90%.Their immunophenotype is similar to the MDSC found in tumor bearing mice.Cultured MDSC mainly consisted of myeloid progenitor cells because CFC assay showed that cultured for seventh days,a small amount of CFU-G and CFU-GM generated but without the formation of other colonies and 1×103 MDSC transfusion to irradiated mice can generate an average of 12-15 CFU-S.MDSC generated in culture can inhibit the proliferation of T cells stimulated by Con-A and also the proliferation of antigen-specific CD8+ T cells from OT-1 mice stimulated by OVA peptides.MDSC infusion can increase the proportion of Treg cells in the peripheral blood of Foxp3-IRES-GFP mice,and protect the mice from aGVHD as the murine mortalitydecreased from 100%to 40%.MDSC co-inoculation with A20 cells promoted lymphoma growth obviously in the mice,2.There were significantly increased percentages(nearly 30%)of Gr-1+CD11b+ MDSC in the spleen of the lymphoma-bearing mice on thirty days after A20 cells inoculation,and was approximately ten times more than that in the spleen of the control mice.In contrast to the control treatment,more than 83.2%of MDSC apoptosis after 48 hours ofgemcitabine treatment.Mice injected with gemcitabine markedly reduced the percentage of MDSC in spleen down to 6.9%.DC co-cultured with gemcitabine pre-treated,apoptotic A20 cells markedly increased the percentages of CD86+mature DC.Analysis of CFSE labeled DC 48 hours after intratumoral injection revealed that they were primarily mature DC with high CD11c and CD86 expression.3.The spleen cells from the lymphoma bearing mice that were sequentially treated with gemcitabine and DC showed a tenfold increase of IFN-yproduction and significantly increased the cytotoxic activity to A20 cells.Mice treated with both gemcitabine and DC demonstrated decreased size of tumor blocks and they continued to shrink.The mice survived significantly longer than the control treatment group,and 90%of these mice acquired longsurvival.The percentage of CD4+,CD8+Tcells was comparable in the tumor samples within all groups.In contrast,the percentage of DX5+NK cells increased significantly in the tumor sample of mice treated with gemcitabine and DC.Co-injection of anti-asialo-GM1 antibody,gemcitabine comined DC treatment failed to inhibit tumor growth,while CD8+T cells depletion resulted in delayed tumor growth compared with the tumor growth in the NK cells depletion experiment.Conclusions:1.Establishment a new culture method for expansion a large number of high purity MDSC in vitro by mimicking the microenvironment of MDSC amplified in mice.MDSC generated in culture can strongly inhibit the immunol response.MDSC co-inoculation with A20 cells promoting lymphoma growth obviously in the mice.2.Like other mouse tumors,A20 lymphoma can also induce MDSC accumulation in mice.Gemcitabine can induce apoptosis of MDSC and eliminate the majority of MDSC in lymphoma bearing mice,and promote the maturation of intratumoral injected semi-mature naive DC through inducing lymphoma cells apoptosis.3.Gemcitabine chemotherapy combined with intratumoral injection of semi-mature DC therapy has synergistic effect on eliminate giant mouse B-cell lymphoma and prevent its recurrence.NK cells appeared to be the major effector cells mediating tumor elimination.