The Mechanism And Clinical Study Of NKTm Non-specific Adoptive Cellular Immunotherapy Treatment On Malignant Tumor

Objective: To evaluate the method of in vitro induction and generationg NKTmcells from human peripheral blood mononuclear cells(PBMCs) and thendeterminate the cell phenotypes of the NKTm cells.Methods: In GMP laboratory system,54non small cell lung cancer patients’PBMCs were cultured in OKM-100and OKM-200medium and induced andamplified by stimulating factor OKM-24. After effector cells were generated, weanalysed the cycle and multiple of amplifying, counted and compared thenumber of cells before and after culturing, detected the survival rate of theeffector cells, and determinated the cell phenotypes by flow cytometry.Results: Effector cells were found proliferated from the second day to the end;the cycle of amplifying was13.46±1.20d; before the amplifying, the number oflymphocyte cells was6.60±2.23×10~6, after amplifying the number was increasedto2.99±0.65×109, the multiple of amplification was488.06±191.25; survival ratedetected by Trypan Blue staining was97.71±1.07％; the examination ofendotoxin, bacterium, fungus, mycoplasma and adventitious viruses were allnegative. After in vitro culture and amplification, the percentage of CD3~+,CD3~+CD8~+, CD45RO~+, CD25~+, CD29~+and CD3~+CD16~+/CD56~+cells increasedgreatly(P<0.01), while the percentage of CD19~+, CD3~+CD4~+, CD45RA+,CD4~+CD25~+, CD4~+CD29~+, CD3~-CD16~+/CD56~+(natural killer cells, NK) andCD3~+CD4~+/CD3~+CD8~+ratio decreaded obviously(P <0.01). Repeatability testsof cell phenotypes showed that the changes of percentage of each subgroup cellswere not obvious in different batch of NKTm cells cultivation.Conclusion: The in vitro induction and expansion efficiency of sensitizedlymphocyte cells from NSCLC patients peripheral blood under this culturesystem is pretty high, the operation process is simple, and the effectors cells after amplification are of good repeatability and biologic security. Objective: To study of the mechanisms of antitumor cytotoxicity of NKTmadoptive immune effector cells.Methods: NKTm cells were marked with granzyme B-FITC, perforin-PE,Fas-FITC and FasL-PE fluorescein antibody respectively; human lung cancercell lines A549, H2228and H661were marked with Fas-FITC and FasL-PEfluorescein antibody respectively; then analyzed by flow cytometry.Results: In NKTm cells, the percentage of granzyme B positive cells andperforin positive cells was3.07%and8.58%respectively; CD4~+Fas+cells andCD4~+FasL+cells accounted for99.9%and76.3%of the overall CD4~+CD8-cells,CD8~+Fas+cells and CD8~+FasL+cells accounted for99.9%and42.6%of theoverall CD8~+CD4-cells; CD3~-CD56~+Fas+cells and CD3~-CD56~+FasL+cellsaccounted for95.5%and14.6%of the overall CD3~-CD56~+cells,CD3~+CD56~+Fas+cells and CD3~+CD56~+FasL+cells accounted for99.6%and9.82%of the overall CD3~+CD56~+cells. In A549cells, the percentage of Fas+and FasL+cells accounted for86.3%and1.22%respectively; in H2228cells,the percentage of Fas+and FasL+cells accounted for74.4%and0.052%respectively; while in H661cells, the percentage of Fas+and FasL+cells onlyaccounted for0.667%and1.32%respectively.Conclusion: Low expression of granzyme B and perforin and high expressionof FasL in NKTm cell suggested that Fas/FasL-induced apoptosis might be the main way that NKTm killing tumor cells. Whether NKTm cells could or notpromoting tumor cells apoptosis by releasing some kinds of cytokines and howNKTm cells regulating human immune function after they reinfusing back to thebody still need further study. Objective: To retrospectively analysed the clinical efficacy of NKTm cellsadoptive cellular immunotherapy on the patients with malignant tumor throughcase-control study.Methods:54non-small cell lung cancer (NSCLC) patients,16small cell lungcancer (SCLC) patients,42gastric cancer patients,38colorectal cancer patientsand24pancreatic cancer patients who with definite pathological diagnosis andcomplete follow-up data and had been treated with NKTm adoptive cellularimmunotherapy were selected for this retrospective study as NKTmimmunotherapy group. Another174patients who with the same pathoogicaldiagnosis and admitted to the hospital at the same period as NKTmimmunotherapy group patients but had not been treated with NKTmimmunotherapy were selected as control group. Visiting all subjects, using theKaplan-Meier method to analyse the difference in overall survival (OS) of thetreatment group and the control group, using COX multivariate regressionmethod to analyse the factors that might affect patients’ OS, and stratifiedanalysing the OS in different subgroups of patients who accepted NKTm immunotherapy.Results: In addition to the number of NSCLC patients that receivedradiotherapy in the control group more than in the treatment group and wasstatistically significant (P=0.021), other clinical characteristics including age,sex, clinical stage, whether received surgery or not, the number of cycles ofchemotherapy and the application of targeted drug didn’t show significantdifference (P>0.05) between the NKTm immunotherapy group and controlgroup. In NSCLC, the OS of the treatment group was significantly longer thanthe control group (OS were31.1months and18.1months, P=0.008, HR=0.562,95%CI0.367to0.860), and the risk of death of the NKTm cell treatmentgroup compared with the control group decreased by43.8%; the OS of thetreatment group patients with gastric cancer patients was longer than the controlgroup (OS were27.0months and13.9months, P=0.028, HR=0.573,95%CI0.347to0.945), and the risk of death of the NKTm cell treatment groupcompared with the control group decreased by42.7%; the OS of treatment grouppatients with colorectal cancer was also longer than the control group patients(OS were44.0months and22.3months, P=0.008, HR=0.506,95%CI0.305to0.841), and the risk of death of the NKTm cell treatment group compared withthe controlgroup decreased by49.4%; the OS of the treatment group patientswith SCLC patients was slightly longer than the control group, but no significantdifference (OS were17.4months and10.0months, P=0.060, HR=0.487,95%CI0.228to1.037); the OS in treatment group and control group in patients withpancreatic cancer didn’t show significant difference (OS were8.5months and8.0months, P=0.309, hr=0.737,95%CI0.409to1.329).1to5year survivalrate in the treatment group was better than the control group numerically, but astatistical difference was only found in2-year survival rate of NSCLC patientsin the treatment group and control group (2-year survival rate,62.96%and35.44%respectively;95%CI,50.08%to75.84%and22.43%to48.45%,respectively, P <0.05). COX multivariate regression analysis showed that gender, clinical stage, whether accepted targeted therapy or not, number ofchemotherapy cycles, and whether accepted NKTm immunotherapy or not wereindependent prognostic factors for NSCLC patients; number of chemotherapycycles and whether accepted NKTm immunotherapy or not were independentprognostic factors for SCLC patients; clinical stage and whether acceptedNKTm immunotherapy or not were independent prognostic factors for gastriccancer patients; age, clinical stage, whether got surgery or not, whether gotNKTm immunotherapy or not were independent prognostic factors for patientswith colorectal cancer; only the number of cycles of chemotherapy was theindependent prognostic factor in patients with pancreatic cancer. Subgroupanalysis displayed that in NSCLC patients, the OS of male, age <60years,clinical stage III b+Ⅳ, without brain metastases, without radiotherapy,chemotherapy>6cycles, no application of TKI and TKI invalid subgroups whoaccepted the NKTm immunotherapy was significantly longer than the controlgroup (P <0.05); for patients with SCLC, the overall OS in treatment group andcontrol group was no significant difference, but subgroup analysis showed thatwomen, chemotherapy≤6cycles patients could benefit from the NKTmimmunotherapy (P <0.05); for patients with gastric cancaner, the subgroups ofclinical stage Ⅲc+IV and had received surgical treatment could benefit fromthe NKTm immunotherapy (P <0.05); for patients with colorectal cancer, thesubgroups of age <60years, female, clinical stage Ⅰ+Ⅱ+Ⅲa, had receivedsurgery and did not received radiotherapy could benefit from NKTmimmunotherapy (P <0.05); in all subgroups of patients with pancreatic cancer,the median OS of NKTm cell treatment group and the control group shew nosignificant difference (P>0.05). As far as security concerned, the incidence ofchills and fever was1.28%, the incidence of pruritus, erythema and papules was0.51%; no other significant adverse reactions were found.Conclusion:1.The result of retrospective case-control clinical study suggestedthat NKTm cell immunotherapy can prolong the OS of NSCLC, gastric cancer and colorectal cancer, but can not prolong the OS of patients with pancreaticcancer.2. COX multivariate regression analysis also showed that in addition topancreatic cancer patients, the the immune treatment of NKTm cells can prolongthe OS of NSCLC, SCLLC, gastric cancer and colorectal cancer patients.3.Subgroup analysis displayed, NKTm immunotherapy can prolong the OS ofmale, age <60years, clinical staging III b+Ⅳ, no brain metastases, noradiotherapy, chemotherapy>6cycles, no application of TKI and TKI invalidNSCLC subgroups patients; can prolong the OS of women and chemotherapy≤6cycles SCLC subgroups patients; can prolong the OS of clinical stage IIIc+Ⅳand had received surgery gastric cancer subgroups patients; can prolong the OSof age <60years, women, clinical staging Ⅰ+Ⅱ+Ⅲ, had received surgery, andno radiotherapy colorectal cancer subgroups patients; NKTm immunotherapycan not prolong the OS of all subgroups of patients with pancreatic cancer.4.Adverse reactions were mild and the security was good in the NKTm cellimmunotherapy.5. This study was only a retrospective case-control study, so it’sresults also need prospective cohort clinical study that with larger sample todemonstrate it.