The Immune Response And Efficacy(Combined With TACE)of Multiple Antigen Stimulating Cellular Therapy On Hepatocellular Carcinoma

Background&AimCell therapy on cancer belong to the category of biotherapy for cancer,it can regulate the body's immune status by applying the principle and method of immunologic.According to different mechanisms,immunotherapy can be divided into two categories:active immunotherapy and adoptive cell therapies,it has been considered as a new treatment option following surgery,chemotherapy and radiotherapy.Primary liver cancer(PLC)is that malignant tumor of the liver cells or intrahepatic bile ducts,includes pathologic type of Hepatocellular carcinoma(HCC)?Intrahepatic cholangiocarcinoma(ICC)and mix type.And HCC accounts for about 90%of primary liver cancer.HCC is one of the most common of malignant tumors in our country.Most HCC patients had reached BCLC C or D stage when diagnosed,Current currtive therapies are utilized to treat patients with early stage cancer,and HCC is rarely cured and usually relapses quickly.Therefore,the immunotherapy of HCC has been the research focus in the area of medicine for the past few years.In our study,we treated patients with HCC with multiple antigen stimulating cellular therapy.During MASCT,mature DCs pulsed with a peptide pool of multiple tumor antigens and autologous T cells stimulated with theses DCs followed by ex vivo proliferation were sequentially injected to patients with HCC to elicit both active and passive immune responses in vivo.We launched the study to evaluate the clinical efficacy and immune response stimulatde by MASCT treated patients with HCC.Method:1.The immune responses in the patients' PBMCs induced by MASCT treatmentPeripheral blood mononuclear cells(PBMCs)were separated,and the adherent cells were differentiated into immature DCs.ImDCs were loaded with 14 kinds of antigen peptides and cultured with maturation factors to prepare mDCs,The un-adherent cells of PBMCs were co-cultured with mDCs,induced into CTL.Immunofluorescence experiments analyzed whether long peptides could be effectively internalized by the immature DCs and location of peptides in DCs.DCs and CTL had been quality controled.Besides,the CTL generated from HLA-A2+patients against the HCC cell line,HepG2(HLA-A2+)and Huh-7 cells(HLA-A2-),as effector-target cells killing model to analyze the HLA-restricted killing capacity.Prospectively analyzed 13 patients with HCC after curative treatment.All the patients are expected to accept 3 courses of MASCT treatment.During treatment,the patients were followed up,once tumor progression of a patient were diagnosed,the patient would be immediately refund group to received conventional treatment.The immune responses induced by MASCT in the HCC patients' PBMCs during treatment course were examined.Experiments were designed as(1)T cell phenotype detection of PBMCs;(2)Proliferation assay and IFN-y production of antigen-specific T cells;(3)Specific immune response of each kind of antigen peptide.2.Efficacy of transcatheter arterial chemoembolization combined with multiple antigen stimulating cellular therapy on hepatocellular carcinomaThe clinical data of 32 patients with HCC received TACE from August 2010 to March 2015 in the Hepatology Unit of Nanfang Hospital were retrospectively analyzed,the patients were divided into combination group(TACE+ MASCT treatment)(32 patients)and TACE group(34 patients),TACE group received TACE treatment alone,and combination group received MASCT treatment in the interval of TACE treatment.The progression-free survival(PFS)and overall survival(OS)were evaluated in the two groups as main outcome measures.To explore whether combined with MASCT treatment can improve the clinical efficacy of TACE,prolong the PFS and OS of HCC patients after TACE.Result:1.MASCT successfully induced mDC and CTL,the multiple treatments improved immunosuppressive state and stimulate specific immune responses in patients with HCCThe average amount of DCs received by patients was 2.23 × 107.Full immune functional properties are shown by the high expression of maturation signature molecules on the surface of DCs.Moreover,these DCs secreted high level of pro-inflammatory cytokine,IL12,but low levels of immunosuppressive cytokine,IL10.Particularly,immunofluorescence experiments demonstrated that long peptides could be effectively internalized by the immature DCs and were primarily localized in the cytosol instead of the lysosomes.After co-culturing with DCs pulsed with the peptide pool,the activated T cells proliferated to the average amount of 6.63 × 109 cells.The cells were almost exclusively CD3+,with a major percentage being CD8+cells and a minor proportion being CD4+ and CD3-CD56+cells,but an insignificant amount of regulatory T cells(Treg)was detected.Moreover,IFN-and TNF-a were detected in the supernatants of cultured CTLs,while the immune inhibitory cytokines IL10 and IL4,were hardly detected.Besides,the activated T cells generated from HLA-A2+ patients exhibited superior cytotoxic activity against the HLA-A2+ HCC cell line,HepG2,than against the HLA-A2-HCC cell line Huh-7 cells,suggesting an HLA-restricted killing capacity.We have observed that,after repeating of MASCT treatments,the frequency of CD56+NKG2D+ cell,CD3+CD56+ cell(NKT)and effector T cells in the patients'PBMCs was significantly increased,the frequency of regulatory T cells(Tregs)in the patients' PBMCs was significantly decreased.Moreover,the specific immune responses against tumor antigens in different HCC patients by both the the IFN-y stimulation assay,T cell proliferation assay and ELISPOT assay were gradually enhanced during multiple treatment of MASCT,different patients exhibited a diverse immune response to every peptide,and the antigen-specific immune responses observed in tumor-free patients'PBMCs were significantly stronger than that in the patients with recurrence.2.Combined with MASCT could robustly improve the clinical efficacy of TACE treatment on HCC,prolong the PFS and OS of HCC patients after TACE.The median PFS were 9.5 months in combination group compared with 5.5 months in TACE group(P<0.01).The median OS were 19.5 months in combination group compared with 10.5 months in TACE group(P<0.05).MASCT,potal vein invasion,serum AFP level before treatment and ECOG performance status were independent prognostic factors for PFS and MASCT,potal vein invasion and serum TBIL level before treatment for OS.Conclusion:1.MASCT successfully induced high quality mDC and CTL,the multiple-treatments improved immunosuppressive state and stimulate specific immune responses in patients with HCC.2.Combined with MASCT could robustly improve the clinical efficacy of TACE treatment on HCC,prolong the PFS and OS of HCC patients after TACE.