Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma

AIM:To investigate the influence of autologous cytokine-induced killer (CIK) cells on the phenotypes of CIK effectorcells,peripheral T lymphocyte subsets and dendritic cellsubsets in patients with primary hepatocellular carcinoma (HCC).METHODS:Peripheral blood mononuclear cells (PBMC)were collected by a blood cell separator from 13 patientswith HCC,then expanded by priming them with interferon-gamma (IFN-γ) followed by monoclonal antibody (mAb)against CD3 and interleukin-2 (IL-2) the next day.Thephenotypic patterns of CIK cells were characterized by flowcytometry on d 0,4,7,10,13 and 15 of incubation,respectively.Then,5 mL of venous blood was obtained fromHCC patients before or 8-10 d after CIK cells were transfusedinto patients to assess the influence of CIK cells on thepercentages of effector cells,and proportions of DC1 or DC2in peripheral blood by flow cytometry.RESULTS:After two weeks of in vitro incubation,thepercentages of CD3~ CD8~ ,CD3~ CD56~ ,and CD25~ cellsincreased significantly from 33.5±10.1%,7.7±2.8%,and12.3±4.5% to 36.6±9.0% (P<0.05),18.9±6.9% (P<0.01),and 16.4±5.9% (P<0.05),respectively.However,thepercentages of CD3~ CD4~ and NK cells had no significantdifference.The percentages of CD3~ and CD3~ CD8~ cells werekept at high levels during the whole incubation period,butthose of CD25~ ,and CD3~ CD56~ cells began to decrease ond 7 and 13,respectively.The proportions of type Ⅰ dendriticcell (DC1) and type Ⅱ dendritic cell (DC2) subsets increasedfrom 0.59±0.23% and 0.26±0.12% before CIK cell therapyto 0.85±0.27% and 0.43±0.19% (all P<0.01) after CIK celltransfusion,respectively.The symptoms and characteristicsof HCC patients were relieved without major side effects.CONCLUSION:Our results indicated that autologous CIKcells can efficiently improve the immunological status inHCC patients,and may provide a potent approach for HCCpatients as the adoptive immunotherapy.     

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402／R

ABM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2 000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhlFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.     

Analysis for expression and significance of interleukin-17, RORγt in CD4+ T cells from patients with ankylosing spondylitis

Objective To study the expression of IL-17, RORγt in CD4+T cells from patients with ankylosing spondylitis (AS). Methods The specimens of venous blood PBMC were collected from 28 patients with AS and 15 healthy subjects. Intracellular flow cytometry detection of IL-17 was established after isolation of human CD4+ T cells from PBMC. The expression level of IL-17, RORγt mRNA in CD4+ T cells was determined from 28 AS patients and 15 healthy controls by real-time fluorescence quantitative RT-PCR using Anti -CD3/Anti -CD28 as stimulators or not. Analysis of variance and Pearson correlation were selected. Results The isolation of human CD4+ T cells from PBMC was effective and its purity reached 90%. The percentage of intracellular IL-17 in CD4+ T cells from AS pati-ents in the AS active group was higher than that of the AS stable group and healthy control group (P<0.01). The expression level of IL-17, RORγt mRNA in CD4+ T cells was significantly higher in patients with AS than in controls. After stimulated with anti-CD3/ anti-CD28 stimulation, the percentage of IL-17, RORγt mRNA was increased significantly (P<0.01). The percentage of IL-17, RORγt mRNA in the 12 h group was higher than that of the 24 h group, while both of them were higher than those without stimulation (P<0.05). Conclusion There is an abnormal expression of IL-17, RORγt in human CD4+ T cells from AS patients. Our results indicate that the abnormal expression of IL-17 might play a role in the development and progression of AS.     

Analysis for expression and significance of interleukin-17, RORγt in CD4+ T cells from patients with ankylosing spondylitis

Objective To study the expression of IL-17, RORγt in CD4+T cells from patients with ankylosing spondylitis (AS). Methods The specimens of venous blood PBMC were collected from 28 patients with AS and 15 healthy subjects. Intracellular flow cytometry detection of IL-17 was established after isolation of human CD4+ T cells from PBMC. The expression level of IL-17, RORγt mRNA in CD4+ T cells was determined from 28 AS patients and 15 healthy controls by real-time fluorescence quantitative RT-PCR using Anti -CD3/Anti -CD28 as stimulators or not. Analysis of variance and Pearson correlation were selected. Results The isolation of human CD4+ T cells from PBMC was effective and its purity reached 90%. The percentage of intracellular IL-17 in CD4+ T cells from AS pati-ents in the AS active group was higher than that of the AS stable group and healthy control group (P<0.01). The expression level of IL-17, RORγt mRNA in CD4+ T cells was significantly higher in patients with AS than in controls. After stimulated with anti-CD3/ anti-CD28 stimulation, the percentage of IL-17, RORγt mRNA was increased significantly (P<0.01). The percentage of IL-17, RORγt mRNA in the 12 h group was higher than that of the 24 h group, while both of them were higher than those without stimulation (P<0.05). Conclusion There is an abnormal expression of IL-17, RORγt in human CD4+ T cells from AS patients. Our results indicate that the abnormal expression of IL-17 might play a role in the development and progression of AS.     

Study on immune function of dendritic cells in patients with esophageal carcinoma

AIM:To investigate the immune function of dendritic cellsfrom both peripheral blood and operated tissues ofesophageal carcinoma patients in order to find therelationship between the immune function of dendritic cellsand the pathogenesis of esophageal carcinoma.METHODS:The expression of CD83,CD80,and CD86 onthe surface of dendritic cells cultured from the peripheralblood of patients was detected compared with that fromhealth donors using flow cytometry.The ability of dendriticcells to induce T lymphocyte proliferation was evaluated bya liquid scintillation counter.The expression of CD80,CD86,CD83,and S-100 proteins was assessed in esophagealcarcinoma tissues using immunohistochemical method.RESULTS:Compared with those from healthy donors,dendirtic cells cultured from the peripheral blood of patientsexpressed lower CD80 and CD86.Furthermore,the abilityof dendritic cells in patients to induce T lymphocyteproliferation was significantly lower than that of the controlgroup.Compared with the control group,the positiveexpression ratio and frequencies of CD80,CD86,and S-100 in esophageal carcinoma tissues were significantlydown regulated.The expression of CD83 was up-regulatedin the pericancerous tissues,but no expression was foundin the cancerous nodules.CONCLUSION:The impaired immune function and thedecreased number of dendritic cells cause pathogenesisand progression of esophageal carcinoma.     

Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma

AIM: To induce efficient expansion of natural killer (NK) cells from peripheral blood mononuclear cells (PBMCs) using a culture of anchorage-dependent Wilms tumor cell lines, and to provide a reliable supply for adoptive immunotherapy of hepatocellular carcinoma.METHODS: Culture expansion of NK cells was achieved using PBMCs cultured with Wilms tumor cells. Cytotoxicity was measured using a standard 54^Cr release assay and crystal violet staining technique. The proportions of CD3+,CD4+,CD8+,CD16+, and CD56+ cells were determined by flow cytometry.RESULTS: After PBMCs from healthy donors and hepatocellular carcinoma (HCC) were cultured with irradiated HFWT cells for 10-21 d, CD56+ CD16+ cells shared more than 50% of the cell population, and more than 80% of fresh HFWT cells were killed at an effector/target ratio of 2 over 24 h. NK-enriched lymphocyte population from HCC patients killed HCC-1 and 2 cells with sensitivities comparable to fresh TKB-17RGB cells. HCC cells proliferated 196-fold with the irradiated HFWT cells at 18 d. Stimulation by HFWT cells required intimate cell-cell interaction with PBMC. However, neither the soluble factors released from HFWT cells nor the fixed HFWT cells were effective for NK expansion. The lymphocytes expanded with IL-2 killed fresh HFWT target cells more effectively than the lymphocytes expanded with the 4-cytokine cocktail(IL-1 β, IL-2, IL-4 and IL-6). IL-2 was the sole cytokine required for NK expansion.CONCLUSION: Wilms tumor is sensitive to human NK cells and is highly efficient for selective expansion of NK cells from PBMCs.     

Usefulness of liver infiltrating CD86-positive mononuclear cells for diagnosis of autoimmune hepatitis

AIM: Although the pathogenic mechanism underlying autoimmune hepatitis (AIH) remains unclear, the immune system is thought to be critical for the progression of the disease. Cellular immune responses may be linked to the hepatocellular damage in AIH. Recently, much attention has been focused on the critical functions of costimulatory molecules expressed on mononuclear cells in the generation of effective T cell-mediated immune responses. Analysis of costimulatory molecule expressed on mononuclear cells from the patients with AIH may give us insight into the pathogenic mechanism of hepatocellular damage in AIH. METHODS: Peripheral blood mononuclear cells (PBMC) were taken from the patients with AIH (34 cases) and healthy controls (25 cases). Liver infiltrating mononuclear cells (LIMCs) were taken from the patients with AIH (18 cases), the patient with chronic hepatitis C (CH-C) (13 cases) and the patients with fatty liver (2 cases). Using flow cytometry, the cells were analyzed for the expression of costimulatory molecules, such as CD80, CD86, and CD152 (CTLA-4). The results were compared with clinical data such as the level of gammaglobulin, histological grade, presence or absence of corticosteroids administration and the response to corticosteroids. RESULTS: The levels of CD80 , CD86 and CD152 PBMC were significantly reduced in the patients with AIH as compared with healthy controls. By contrast, those cells were significantly higher in LIMC than in PBMC of the patients with AIH. Especially, the level of CD86 LIMC showed a marked increase irrespective of the degree of disease activity in the patients with AIH, although CD86 cells were rarely present in PBMC. The levels of CD86 cells were present in significantly higher frequency in patients with AIH than in the patients with CH-C. Furthermore, the patients with AIH with high levels of CD86 LIMC showed good responses to corticosteroids, whereas 2 cases of AIH with low levels of CD86 LIMC did not respond well. CONCLUSION: These results suggest that LIMC overexpressing costimulatory molecules such as CD80 and CD86 appears to play a role in the pathogenesis of AIH. Especially, CD86 molecule expressed on the LIMC may be useful for the diagnosis of AIH and for the prediction of the therapeutic effects of corticosteroids on AIH.     

Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma

AIM:To induce efficient expansion of natural killer(NK)cells from peripheral blood mononuclear cells(PBMCs)using a culture of anchorage-dependent Wilms tumor celllines,and to provide a reliable supply for adoptiveimmunotherapy of hepatocellular carcinoma.METHODS:Culture expansion of NK cells was achieved usingPBMCs cultured with Wilms tumor cells.Cytotoxicity wasmeasured using a standard ~(51)Cr release assay and crystal violetstaining technique.The proportions of CD3 ,CD4 ,CD8 ,CD16 ,and CD56 cells were determined by flow cytometry.RESULTS:After PBMCs from healthy donors andhepatocellular carcinoma(HCC)were cultured withirradiated HFWT cells for 10-21 d,CD56 CD16 cellsshared more than 50% of the cell population,and morethan 80% of fresh HFVVT cells were killed at an effector/target ratio of 2 over 24 h.NK-enriched lymphocytepopulation from HCC patients killed HCC-1 and 2 cells withsensitivities comparable to fresh TKB-17RGB cells.HCC cellsproliferated 196-fold with the irradiated HFWT cells at18 d.Stimulation by HFVVT cells required intimate cell-cellinteraction with PBMC.However,neither the soluble factorsreleased from HFWT cells nor the fixed HFWT cells wereeffective for NK expansion.The lymphocytes expanded withIL-2 killed fresh HFWT target cells more effectively thanthe lymphocytes expanded with the 4-cytokine cocktail(IL-1 β,IL-2,IL-4 and IL-6).IL-2 was the sole cytokinerequired for NK expansion.CONCLUSION:Wilms tumor is sensitive to human NK cellsand is highly efficient for selective expansion of NK cellsfrom PBMCs.     

Increasing the frequency of CIK cells adoptive immunotherapy may decrease risk of death in gastric cancer patients

AIM: To analyze the correlation between cytokineinduced killer (CIK) cells adoptive immunotherapy and cancer-related death in gastric cancer patients. METHODS: One hundred and fifty-six gastric cancer patients after operation at the Third Affiliated Hospital of Soochow University were enrolled in this study. Their clinical data including demographic characteristics, operation time, tumor size, pathological type and staging, tumor metastasis, outcome of chemotherapy or CIK cells adoptive immunotherapy, survival time or time of death were collected with a standard structured questionnaire. Kaplan-Meier method was used to estimate the median survival time, and the 2- and 5- year survival rates. Hazard risk (HR) and 95% confidence interval (95% CI) of CIK cells adoptive immunotherapy for gastric cancer were calculated using the two-stage time-dependent covariates Cox model. RESULTS: The survival time of gastric cancer patients was longer after CIK cells adoptive immunotherapy than after chemotherapy (χ 2 = 10.907, P = 0.001). The median survival time of gastric cancer patients was also longer after CIK cells adoptive immunotherapy than after chemotherapy (49 mo vs 27 mo, P < 0.05). The 2- and 5-year survival rates of gastric cancer patients were significantly higher after CIK cells adoptive immunotherapy than after chemotherapy (73.5% vs 52.6%, 40.4% vs 23.9%, P < 0.05). A significant difference was observed in the survival curve for patients who received CIK cells adoptive immunotherapy (0, 1-10, 11-25, and over 25 frequencies) (χ 2 = 14.534, P = 0.002). The frequencies of CIK cells adoptive immunotherapy were significantly related with the decreasing risk of death in gastric cancer patients after adjustment for sex and age of the patients, tumor stage and relapse (HR = 0.54, 95% CI: 0.36-0.80) when the first stage Cox model was used to define the subjects who remained alive beyond 36 mo as survivors. However, no correlation was observed between the frequencies of death in CIK cells adoptive immunotherapy a     

Dendritic cells from chronic hepatitis B patients can induce HBV antigen-specific T cell responses

AIM: To determine whether dendritic cells (DCs) fromchronic hepatitis B patients could induce HBV antigen-specific T cell responses or not.METHODS: DCs were generated from peripheral bloodmononudear ceils of patients with chronic hepatitis B (CHB)infection and healthy donors.We compared the phenotypesof these Des and their ability to secrete cytokines and toparticipate in mixed lymphocyte reactions.In addition,autologous lymphocytes were cultured with DCs loaded withHBV core region peptide HBcAg8-27,an epitope recognizedby cytotoxic T lymphocytes (CTL),and bearing human leucocyteantigen (HLA)-A2 for 10 d.Cytokine secretion and lytic activityagainst peptide-pulsed target cells were assessed.RESULTS: DCs with typical morphology were generatedsuccessfully by culturing peripheral blood mononuclear cells(PBMCs) from CHB patients with AIM-V containing GM-CSFand IL-4.Compared with DCs from normal donors,thelevel of CD80 expressed in DCs from CHB patients waslower,and Des from patients had lower capacity ofstimulate T cell proliferation.When PBMCs isolated frompatients with chronic or acute hepatitis B infection and fromnormal donors were cocultured with HBcAg18-27 peptide,the antigen-specific memory response of PBICs from acutehepatitis B patients was stronger than that of PBMCs fromchronic hepatitis B patients or normal donors.PBMCscocultured with DCs treated with HBcAg18-27 CTL epitopepeptide induced an antigen-specific T cell reaction,in whichthe level of secreted cytokines and lyric activity were higherthan those produced by memory T cells.CONCLUSION: DCs from patients with CHB can induceHBV antigen-specific T cell reactions,including secretionof cytokines essential for HBV clearance and for killing cellsinfected with HBV.     

Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma

AIM: To investigate the influence of autologous cytokine-induced killer (CIK) cells on the phenotypes of CIK effector cells, peripheral T lymphocyte subsets and dendritic cell subsets in patients with primary hepatocellular carcinoma (HCC). METHODS: Peripheral blood mononuclear cells (PBMC) were collected by a blood cell separator from 13 patients with HCC, then expanded by priming them with interferon-gamma (IFN-gamma) followed by monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2) the next day. The phenotypic patterns of CIK cells were characterized by flow cytometry on d 0, 4, 7, 10, 13 and 15 of incubation, respectively. Then, 5 mL of venous blood was obtained from HCC patients before or 8-10 d after CIK cells were transfused into patients to assess the influence of CIK cells on the percentages of effector cells, and proportions of DC1 or DC2 in peripheral blood by flow cytometry. RESULTS: After two weeks of in vitro incubation, the percentages of CD3(+)CD8(+), CD3(+)CD56(+), and CD25(+) cells increased significantly from 33.5+/-10.1%, 7.7+/-2.8%, and 12.3+/-4.5% to 36.6+/-9.0% (P<0.05), 18.9+/-6.9% (P<0.01), and 16.4+/-5.9% (P<0.05), respectively. However, the percentages of CD3(+)CD4(+) and NK cells had no significant difference. The percentages of CD3(+) and CD3(+)CD8(+) cells were kept at high levels during the whole incubation period, but those of CD25(+), and CD3(+)CD56(+) cells began to decrease on d 7 and 13, respectively. The proportions of type I dendritic cell (DC1) and type II dendritic cell (DC2) subsets increased from 0.59+/-0.23% and 0.26+/-0.12% before CIK cell therapy to 0.85+/-0.27% and 0.43+/-0.19% (all P<0.01) after CIK cell transfusion, respectively. The symptoms and characteristics of HCC patients were relieved without major side effects. CONCLUSION: Our results indicated that autologous CIK cells can efficiently improve the immunological status in HCC patients, and may provide a potent approach for HCC patients as the adoptive immunotherapy.     

Patient-derived dendritic cells transduced with an a-fetoprotein-encoding adenovirus and co-cultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against hepatocellular carcinoma cells.

BACKGROUND/AIMS: Breaking immunologic tolerance towards the hepatocellular carcinoma (HCC)-associated alpha-fetoprotein (AFP) antigen is possible. The use of this potential for the treatment of immunocompromised HCC patients is limited. In this study, we analyzed whether dendritic cells (DCs) from HCC patients transduced with a human AFP (hAFP)-expressing adenovirus and co-cultured with cytokine-induced killer (CIK) cells can induce a strong specific immune response against HCC-cells. METHODS: An hAFP-encoding adenovirus (Ad-hAFP) was generated. DCs from healthy donors or patients were transduced at a very high efficacy. Afterwards, DCs were co-cultured with autologous CIK-cells, and their ability to lyse HCC-cells was analyzed. RESULTS: AFP-transduced DCs stimulated CIK cells strongly to lyse about 70% of AFP-expressing HCC cells. Cytotoxicity was significantly higher when lymphocytes were co-cultured with Ad-hAFP-transduced DCs than with Ad-mock-transduced DCs, indicating an AFP-specific immune response. More interestingly, CIK cells from patients with AFP-positive HCC could be stimulated to lyse AFP-expressing HCC cells as effectively as CIK cells from healthy individuals and stronger than CIK cells from patients without AFP-expressing HCC. CONCLUSIONS: The data demonstrate that patient-derived DCs that were transduced with an AFP-expressing adenovirus and co-cultured with autologous CIK cells induce an AFP-specific, strong immune response against HCC cells. Therefore, this approach may have a potential for an adoptive and/or DC-based immunotherapy for HCC patients.     

树突状细胞与同源细胞因子诱导的杀伤细胞共培养细胞在肿瘤免疫治疗中的作用

目的观察树突状细胞(DC)与同源细胞因子诱导的杀伤 (CIK)细胞共培养后,共培养细胞(DC-CIK细胞)的表型、体外增殖活性和细胞毒活性的变化,并探讨CIK细胞在肿瘤治疗中的作用.方法 (1)提取3名健康献血者的外周血单个核细胞(PBMC),常规诱导出DC与CIK细胞后,将其共培养,动态观察CIK细胞和DC-CIK细胞增殖活性和表型变化;并用四甲基偶氮唑蓝(MTT)法测定其体外细胞毒活性;(2)80只BALB/c裸鼠随机分为2组,分别在前肢腋下接种A549肺腺癌细胞和BEL-7404肝癌细胞.10 d后分别选择荷A549和BEL-7404瘤大小相似的裸鼠各30只,全部一次性腹腔注射化疗药物后,每组再随机分成3组.①单独化疗组;②CIK治疗组;③DC-CIK治疗组,每组10只. 单独化疗组注射化疗药物后不再进行任何处理,另2组分别于化疗后的第2、4、6、8、10天腹腔注射CIK细胞和DC-CIK细胞进行免疫治疗.结果 (1)经DC作用后的CIK细胞CD +3CD +56双阳性细胞和CD +8细胞的数量明显升高;(2)DC-CIK细胞的增殖速度明显快于同源CIK细胞,DC-CIK细胞的细胞毒活性远高于CIK细胞;(3)在肿瘤的治疗中,CIK细胞可提高化疗的效果.化疗后25 d,CIK治疗组和DC-CIK治疗组肿瘤受抑制的程度均明显大于单独化疗组,DC-CIK组大于CIK治疗组(P<0.05).结论 DC与CIK细胞共培养细胞是一种强于CIK细胞的免疫活性细胞,在肿瘤治疗中具有更广阔的应用前景.     

慢性丙型肝炎患者CIK细胞的诱导培养及细胞亚群分析

目的 探讨慢性丙型肝炎患者细胞因子诱导的杀伤(CIK)细胞体外诱导培养过程中各细胞亚群的变化.方法 外周血单个核细胞(PBMC)在体外应用混合细胞因子(rhlFN-γ、IL-2和α-CD3)诱导培养,分别于0、7、10、13、15d时收集培养细胞,应用流式细胞技术检测各细胞亚群的频率.结果 动态分析发现培养过程中CD3+细胞频率迅速增加,至10d时到达平台期;在整个培养过程中,CD3+ CD8+T细胞和CD3+ CD56+细胞比例持续增加,表现出良好的扩增能力,且与患者基线ALT水平无关.结论 慢性丙型肝炎患者CIK细胞在体外培养的第10天,CD3+细胞呈现高扩增,且CD3+ CD8+T细胞和CD3+ CD56+细胞扩增状态良好,此时回输治疗可能效果较好.     

Cytotoxicity of interleukin 2-activated lymphocytes for leukemia and lymphoma cells

Studies were undertaken to determine whether leukemia and lymphoma cells would be lysed by autologous and allogeneic lymphokine-activated killer (LAK) cells. Peripheral blood mononuclear cells (PBMC) from patients and normal donors were cultured for five days, 2 weeks, and 4 weeks with medium containing 2,500 units of recombinant interleukin 2 (IL-2) per mL, and their cytotoxicity was assayed by a five-hour 51Cr-release test. Of primary tumors isolated from patients with acute nonlymphoblastic leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma, tumors of 37 out of 40 patients tested were shown to be susceptible to normal donors' LAK, and tumors of 18 of 20 patients tested were shown to be susceptible to autologous LAK. LAK cultured for longer periods showed a tendency to have lower cytotoxicity. LAK had also low, but significant, levels of cytotoxicity for nonmalignant target cells. Because PBMC expanded in IL-2-containing medium consisted mainly of OKT3-positive pan T cells, OKT8-positive suppressor/cytotoxic cells, and Leu-11-positive natural killer (NK) cells, and treatment with OKT3 and Leu-11 monoclonal antibodies (mAb) reduced LAK activity for autologous and allogeneic tumor cells, both T and NK cells appeared to be effector cells for LAK activity. Mechanisms of target-cell recognition in the LAK system seem to be different from those in alloreactive cytotoxic T lymphocytes (CTL) based on the results that, while cytotoxicity of alloreactive CTL was inhibited by the treatment of effector cells with mAb, OKT3, and OKT8, and by the treatment of target cells with a mAb that reacts with HLA class I antigen, LAK activity was not inhibited by the above treatment. When chromosomes of IL-2-expanded PBMC in nine patients and two normal individuals were analyzed, PBMC from one patient showed chromosomes of clonal abnormalities, and PBMC from five donors showed those of nonclonal abnormalities.     

Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells

BACKGROUND AND OBJECTIVES: There are numerous reports of in vitro and in vivo usage of dendritic cells (DC) pulsed with idiotype, the tumor-specific antigen of multiple myeloma (MM), for immunotherapy of MM. Data suggest that not only T-cells, but also the innate immune system reacts against MM. Here, we examined the cytotoxic activity of cytokine-induced killer (CIK) cells against myeloma cells. This heterogeneous effector population consists of T-, NK- and NKT-cells. DESIGN AND METHODS: CIK cells generated from buffy coats or blood from patients with MM were co-cultured with autologous idiotype-pulsed DC. The cytotoxic activity was investigated in lactate dehydrogenase release assays against cell lines or autologous CD138 positive cells from bone marrow. RESULTS: CIK cells were able to lyse MM cells at low effector to target ratios. This effect was significantly enhanced by co-culturing with specifically pulsed DC (83.8% lysis at an effector to target ratio of 16:1). Using an interferon-g secreting MACS separation assay, the cytotoxic activity of CIK cells was enhanced to maximal lysis at the lower effector to target ratio of 5:1. High cytotoxic activity was also shown in a completely autologous setting against enriched CD138+ cells from a patient with MM (54.4% lysis at an effector to target ratio of 6:1). Interestingly, there was no cytotoxic activity against the CD138- fraction of the bone-marrow. INTERPRETATION AND CONCLUSIONS: Using a heterogeneous population of effector cells, we were able to activate the innate and the adoptive immune-system against myeloma cells. CIK cells showed high lytic activity against MM cells, which could be enhanced by co-culturing with antigen-specific pulsed DC.     

Enhanced cytotoxicity of IL-24 gene-modified dendritic cells co-cultured with cytokine-induced killer cells to hepatocellular carcinoma cells

As a novel human cytokine found recently, IL-24 could selectively kill tumor cells by multiple ways. Dendritic cells (DCs) are the major antigen-presenting cells. Recent studies have revealed that IL-24 can promote the antigen-presenting function of DCs. In this study, we evaluated the antitumor effect and mechanism of co-cultured cytokine-induced killer (CIK) cells and autologous DCs modified with IL-24 gene on hepatocellular carcinoma (HCC) cells. DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells. Recombinant adenovirus AdVGFP/IL-24 (Ad-IL24) was constructed expressing IL-24. IL-24 gene was transduced into DCs via Ad-IL24, the cells obtained were named DC-IL-24. We demonstrated that the expression rates of CD80, CD83, CD1a, HLA-DR, CD40, CXCR4 on DC-IL-24 were significantly increased compared with those of the control group. DC-IL-24 produced markedly higher levels of IL-24, IL-12 and TNF-α as compared with those of DCs. On comparison with non-transfected DCs co-cultured with CIK cells, transfected DCs co-cultured with CIK cells had a significant higher lytic activity against SMMC7721 cells, a HCC cell line.     

免疫活性细胞回输法治疗慢性乙型肝炎不引起肝组织损害

目的 明确免疫活性细胞回输法治疗慢性乙型肝炎(CHB)时肝组织损害情况。方法 随机选择16例CHB患者用自体免疫活性细胞回输法治疗,追踪52周。治疗前后行两次肝穿刺获得肝组织,苏木精-伊红染色评价肝脏组织炎症情况,Masson染色评价肝组织纤维化情况,检测乙型肝炎病毒(HBV)复制指标以及肝功能,评价免疫治疗对肝组织的影响。结果 完整随访14例患者,4例患者治疗前后完成2次肝穿刺,其中1例患者结果显示治疗后肝细胞坏死和小叶内炎症程度明显减轻(G_3→G_1),纤维化程度改善(S_2→S_1)。其余3例肝脏组织学变化不明显,但无加重或恶化。免疫活性细胞体外培养前后,进行免疫荧光抗体染色,经流式细胞仪分析,细胞表型有明显的改变,CD_4′细胞比例逐渐下降,CD_8′细胞比例从20%上升至60%～80%。回输治疗后52周的随访结果,HBV DNA阴转率为42.86%(6/14),乙型肝炎e抗原阴转并伴有抗-HBe阳性的血清转换率为42.86%(6/14),无1例发生乙型肝炎表原抗原阴转。丙氨酸氨基转移酶的复常率为42.86%(6/14)。在回输治疗时以及治疗后的随访中未发生严重的不良反应。结论 免疫活性细胞回输法治疗CHB不引起肝组织损害,其免疫调节作用是通过非细胞毒机制途径达到对病毒抑制或清除的作用。     

Inducibility of lymphokine activated killer (LAK) cells in patients with acute myelogenous leukaemia in complete remission and its clinical relevance

Peripheral blood mononuclear cells (PBMC) from 42 patients with acute myelogenous leukaemia (AML) in complete remission (CR) and from normal donors were activated into LAK cells in the presence of 1000 U/ml of recombinant interleukin-2 (rIL-2). Cytotoxicity of LAK cells was assayed against K562, Daudi, and Raji cell lines, and autologous and/or allogeneic thawed leukaemic blasts. Fresh unactivated PBMC from normal donors and AML patients served as controls. Mean +/- standard deviation (SD) percentage lysis of the different targets by patient LAK cells were: K562 61 +/- 20%, Daudi 62 +/- 23%, Raji 48 +/- 24%, autologous blast cells 12 +/- 16% and allogeneic blast cells 13 +/- 10%. Lysis of the different targets by LAK cells from normal donors was similar to that achieved with LAK cells from AML patients. Overall there was a good correlation between the lysis of the different targets. There was no significant difference between the percentage lysis of autologous and allogeneic thawed blast cells, although LAK cells from seven out of the 18 patients tested were unable to lyse autologous leukaemic cells. Activity of patient LAK cells did not correlate with the initial characteristics of the patient nor with the time spent in CR before harvesting PBMC for activation. At the time of analysis, 32 patients were in continuing CR and 10 had relapsed. Multivariant analysis for prognostic factors showed that patients whose LAK cells had more lytic activity on K562 (P = 0.005) and fresh blast cell (P = 0.02) targets had significantly less risk of relapse than patients with little inducible LAK cell activity.(ABSTRACT TRUNCATED AT 250 WORDS)