A Phase IB Trial of Autologous Cytokine-Induced Killer Cells in Combination with Sintilimab, Monoclonal Antibody Against Programmed Cell Death-1, plus Chemotherapy in Patients with Advanced Non-Small-Cell Lung Cancer.

INTRODUCTION: Can the Cytokine-induced killer (CIK) cells in combination with immune checkpoint inhibitor further improve the efficacy of chemotherapy in non-small cell lung cancer (NSCLC) patients? What are the adverse reactions of this combination therapy? But these problems are not clear. Therefore, we conducted a phase 1b trial to evaluate the safety and efficacy of autologous CIK cells therapy combined with Sintilimab, antibody against programmed cell death-1, plus chemotherapy in untreated, advanced NSCLC patients. PATIENTS AND METHODS: Patients with stage IIIB/IIIC/IV NSCLC received Sintilimab, platinum-based doublet chemotherapy, and CIK cells every 3 weeks for 4 cycles, then maintenance treatment with Sintilimab in squamous and with Sintilimab plus pemetrexed in non-squamous NSCLC until disease progression or unacceptable toxicity or 2 years. The primary endpoints were safety and objective response rate (ORR). RESULTS: Thirty-four patients received the treatment. 94.1% of patients experienced treatment-related adverse events (TRAEs). Grade 3 or greater TRAEs occurred in 64.7% of patients. One (2.9%) patient died of grade 5 immune-related pneumonia. The ORR and DCR were 82.4% (95% CI, 65.5%-93.2%) and 100.0% (95% CI, 89.7%-100.0%), respectively. Objective responses were evaluated in 14 of 15 non-squamous patients (93.3%; 95% CI, 68.1%-99.8%) and in 14 of 19 squamous patients (73.7%; 95% CI, 48.8%-90.9%). Median PFS was 19.3 months (95% CI, 8.3 months to not available). CONCLUSION: Autologous CIK cells immunotherapy in combination with Sintilimab plus chemotherapy was well tolerable and showed encouraging efficacy in patients with previously untreated, advanced NSCLC (ClinicalTrials.gov number, NCT03987867).

Efficiency of Cytokine-Induced Killer Cells in Combination with Chemotherapy for Triple-Negative Breast Cancer.

PURPOSE: The treatment of triple-negative breast cancer (TNBC) remains challenging, due to the absence of estrogen, progesterone, and human epidermal growth factor receptors. This study was designed to evaluate the efficiency and safety of cytokine-induced killer (CIK) cell immunotherapy, following regular chemotherapy, for patients with TNBC. METHODS: A total of 340 patients with postmastectomy TNBC, from January 1, 2010 to June 30, 2014, were included in this retrospective study. Seventy-seven patients received CIK cell immunotherapy, following regular chemotherapy (arm 1), and 263 patients received regular chemotherapy alone (arm 2). The primary aim was overall survival (OS) and disease-free survival (DFS), and the treatment responses and adverse events were also evaluated. RESULTS: The 5-year DFS and OS rates in arm 1 were 77.9% and 94.3%, compared with 69.8% and 85.6% in arm 2, respectively (p=0.159 and p=0.035, respectively). This clearly shows that there was no statistical difference in the 5-year DFS between the two groups. Multivariate analyses of arm 1 indicated that a Karnofsky performance score (KPS) ≥90 and stage I/IIA disease were significantly associated with a prolonged DFS period (hazard ratio [HR], 0.25; 95% confidence interval [CI], 0.09-0.74; p=0.012; and HR 0.21; 95% CI, 0.06-0.82; p=0.024, respectively), but a KPS ≥90 and stage I/IIA disease were not independent prognostic factors for OS. Toxicity was mild in patients who received the CIK therapy. CONCLUSION: The data suggested that CIK cell immunotherapy improved the efficiency of regular chemotherapy in patients with TNBC, and the side effects of CIK cell immunotherapy were mild.

Cytokine-Induced Killer Cells Modulates Resistance to Cisplatin in the A549/DDP Cell Line.

Background Cytokine-induced killer (CIK) cells can potentially enhance the tumor-killing activity of chemotherapy. Objective This study aimed to evaluate the effects of CIK cells on cisplatin (DDP) resistance in the human lung adenocarcinoma cell line A549/DDP. Methods The detect resistance index, drug resistance related-genes and cytokine secretion of A549/DDP co-cultured with CIK cells were assayed in vitro. Results After A549/DDP co-culture with CIK cells, the DDP resistance of A549/DDP significantly decreased in a time-dependent manner. The DDP resistance of A549/DDP co-cultured with CIK cells for 20 h decreased 4.93-fold compared with that of A549/DDP cells cultured alone (P<0.05). The mRNA and protein expression levels of the glutathione-S-transferase (GST) -π gene in A549/DDP significantly decreased after co-culture with CIK cells (P<0.05). The secretion of interferon (IFN)- γ significantly increased along with the co-culture time of A549/DDP with CIK cells. The expression of GST-π was restored by adding the neutralizing IFN-γ. Conclusion CIK cells can reverse the drug resistance of A549/DDP in a time-dependent manner by reducing GST-π expression to increase the accumulation of DDP. The effect of CIK cells on re-sensitizing lung cancer cells to the chemotherapy drug was partially dependent on the secretion of IFN-γ.

Effect of miR-155 knockdown on the reversal of doxorubicin resistance in human lung cancer A549/dox cells.

Doxorubicin has been widely used in the treatment of cancer. However, acquired doxorubicin resistance severely hinders the application of the drug. In the present study, doxorubicin resistance was investigated in lung carcinoma. microRNA-155 (miR-155) was found to be upregulated in the doxorubicin-resistant A549/dox cell line. Suppression of miR-155 in this cell line considerably reversed doxorubicin resistance, and doxorubicin-induced apoptosis and cell cycle arrest were recovered. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis revealed that miR-155 suppression downregulated the expression of multidrug resistance protein 1, multidrug resistance-associated protein 1, breast cancer resistance protein, glutathione S-transferase-π, Survivin and B-cell lymphoma 2, and upregulated the expression of caspase-3 and caspase-8. In addition, it was found that miR-155 suppression inhibited the activation of AKT and extracellular signal-regulated kinase. The transcriptional activity of nuclear factor-κB and activator protein-1 was also downregulated. In summary, the present results indicate that miR-155 may participate in doxorubicin resistance in lung carcinoma. The current study provides a novel target for lung carcinoma treatment.

Maintenance therapy with autologous cytokine-induced killer cells in patients with advanced epithelial ovarian cancer after first-line treatment.

Cytokine-induced killer (CIK) cells have shown cytolytic ability against ovarian cancer cells in vitro and in vivo. This study was aimed to evaluate the clinical efficacy of maintenance therapy of CIK cells in patients with advanced epithelial ovarian cancer after first-line treatment. A paired study was performed in patients with stages IIB-IV epithelial ovarian cancer after cytoreductive surgery followed by 6-8 courses of carboplatin/paclitaxel chemotherapy. A total of 92 patients who achieved complete remission after first-line treatment were enrolled in this study. Forty-six patients in the treatment group received CIK cells transfusion monthly, whereas the other 46 patients in the control group received observation with follow-up. Progression-free survival (PFS), overall survival (OS), and toxicity were evaluated. Our results showed that median PFS was 37.7 months in the treatment group and 22.2 months in the control group (P=0.004). However, although median OS in the treatment group (61.5 mo) was longer than that in the control group (55.9 mo), there was no significant difference (P=0.289). The subgroup analysis revealed that the survival advantage of PFS from immunotherapy was independent of the extent of debulking surgery and pathologic stage. After 2 courses of CIK cells transfusion, the proportion of CD4CD25CD127 regular T cells in the peripheral blood significantly decreased (P=0.006). No grades III and IV adverse reaction were found during CIK cells infusion. Maintenance therapy with CIK cells improved the PFS in patients with advanced ovarian cancer after first-line treatment with slight side effects. However, the benefits with respect to OS are still pending.

A randomized phase II study of autologous cytokine-induced killer cells in treatment of hepatocellular carcinoma.

PURPOSE: This prospective study aims to explore the benefit of cytokine-induced killer cell (CIK) treatment in hepatocellular carcinoma patients, which has not yet been thoroughly studied before. METHODS: From January 2004 to May 2009, 132 patients who were initially diagnosed with hepatocellular carcinoma of Barcelona Clinic Liver Cancer (BCLC) stage A, B or C, Child-Pugh scores of A or B and without prior treatment were enrolled in the study. Patients were randomly assigned to either arm 1 (n = 66) to receive CIK treatment plus standard treatment, or arm 2 (n = 66) to receive standard treatment only. The primary end point was overall survival (OS) and the secondary endpoint was progression-free survival as evaluated by Kaplan-Meier analyses and treatment hazard ratios with the Cox proportional hazards model. RESULTS: The 1-year (OS: 74.2% vs. 50.0%, 95% CI: 63.6-84.8% vs. 37.8-62.2, p = 0.002), 2-year (OS: 53.0% vs. 30.3%, 95% CI: 40.8-65.2% vs. 19.1-41.5%, p = 0.002), 3-year (OS: 42.4% vs. 24.2%, 95% CI: 30.4-54.4% vs. 13.8-34.6%, p = 0.005) and median overall and progression-free survivals of arm 1 patients were significantly higher than those of arm 2. Therefore, in patients who are not suitable for surgery, significant benefit is obtained from CIK treatment. The main adverse effects of CIK included fever, allergy and headache pain. CONCLUSIONS: Hepatocellular carcinoma patients who were not suitable for surgery demonstrate prolonged overall and progression-free survival from CIK treatment.

Increased prevalence of regulatory T cells in the lung cancer microenvironment: a role of thymic stromal lymphopoietin.

Expansion of CD4+CD25+ regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. Thymic stromal lymphopoietin (TSLP) contributes to the generation of natural Tregs in thymus. Therefore, the purpose of this report was to investigate the role of TSLP in the increasing prevalence of Tregs in lung cancer microenvironment. The expression ratio of TSLP protein in tumor tissues was significantly increased compared with that in benign lesion and non-cancer lung tissue. The prevalence of Tregs in tumor microenvironment was correlated with the expression of TSLP in lung cancer. Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) collected from lung cancer patients and left unstimulated (imDCs) or exposed to hTSLP (TSLP-DCs) or LPS (LPS-DCs). TSLP-DCs expressed intermediate levels of CD83 and high levels of CD86, CD11C, and HLA-DR, which showed a characteristic of less mature DCs. TSLP-DCs secreted low levels of IL-6, IL-12, IL-10, TNF-α and IFN-γ, and high levels of TGF-ß and MDC. The percentage of Tregs in CD4+CD25- T cells cocultured with TSLP-DCs group was statistically higher than that of LPS-DCs and imDCs. Transwell assays showed that TSLP-DCs exhibited increased ability to attract the migration of CD4+CD25- Tregs, when compared with imDCs. These results indicated that TSLP proteins were expressed in lung tumor tissue and correlated with the prevalence of Tregs. TSLP-DCs could induce CD4+CD25- T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells.

Alloreactive natural killer cells promote haploidentical hematopoietic stem cell transplantation by expansion of recipient-derived CD4(+) CD25(+) regulatory T cells.

Alloreactive NK cells (Allo-NKs) have been shown to exert advantageous effects on the outcomes of haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) for cancer treatment. However, the mechanisms of action of Allo-NKs remain unclear. We established a novel Haplo-HSCT conditioning regimen composed of Allo-NKs and a low dose of immunosuppressive drugs (Allo-NKs + Chemo) to investigate alternative mechanisms besides direct cytotoxicity. The inhibitory effects of different cell subsets on the donor-recipient mixed lymphocyte reactions (MLRs) were evaluated after Haplo-HSCT. The quantities and functions of CD4(+) CD25(+) regulatory T cells (Tregs) and dendritic cells (DCs) in the spleen and the thymus were examined. Our results showed that the Allo-NKs + Chemo regimen induced systemic tolerance, and that CD4(+) CD25(+) Tregs played a significant role in inducing and maintaining systemic tolerance after Haplo-HSCT. Alloreactive NK cells promoted the expansion of recipient-derived CD4(+) CD25(+) CD127(-) Tregs in the thymus and the spleen which could be amplified in vitro by the immature donor-derived DC subset isolated from the thymus of Allo-NKs + Chemo-treated mice. Our findings suggested that Allo-NKs are capable of inducing systemic tolerance after Haplo-HSCT by assembling donor-derived immature DCs to expand recipient-derived Treg cells in the thymus.

Fetal-maternal microchimerism enhances the survival effect of interleukin-2-activated haploidentical peripheral blood stem cell treatment in patients with advanced solid cancer.

Killer immunoglobulin-like receptor (KIR)-ligand incompatibility in the graft-versus-host direction is associated with improved outcome in patients receiving hematopoietic stem cell transplants. Fetal-maternal microchimerism has been suggested to mediate acquired fetal-maternal tolerance. The goal of this study was to determine the clinical efficacy of KIR-ligand incompatibility and fetal-maternal microchimerism for interleukin-2-activated haploidentical peripheral blood stem cells (haplo-PBSCs) treatment for patients with advanced solid cancer. Forty-two (42) patients with advanced stage of solid cancer and refractory to standard chemotherapy were treated with haplo-PBSCs donated by their parents or children. Human leukocyte antigen typing, fetal-maternal microchimerism status, and engraftment were detected. Clinical outcomes including overall survival (OS), progression-free survival (PFS), and Karnofsky Performance Status (KPS) level were evaluated. Patients receiving haplo-PBSCs treatment with KIR-ligand incompatibility in the graft-versus-host direction had higher probability of OS (26.8 ± 3.1 months) and PFS (13.4 ± 1.3 months) when compared with those with KIR ligand compatibility (OS: 17.4 ± 3.0 months, p < 0.05 and PSF: 8.0 ± 0.9 months, p < 0.05). Further, OS (31.2 ± 4.3 months), PFS (14.7 ± 2.2 months), and KPS increase (27 points) in the microchimerism-positive group was improved compared with that in the microchimerism-negative group (OS: 16.9 ± 3.8 months, p < 0.01, PFS: 5.6 ± 1.4 months, p < 0.01, and KPS increase: 15 points, p < 0.01). Therefore, KIR-ligand incompatibility and fetal-maternal microchimerism are associated with better outcome for haplo-PBSCs treatment.

[A preliminary study on the effects of feto-maternal microchimerism in activated human leukocyte antigen haploidentical mobilized peripheral blood stem cells on treatment of solid tumors].

OBJECTIVE: To study the effect of feto-maternal microchimerism in the treatment of activated human leukocyte antigen (HLA) haploidentical mobilized peripheral blood cells against solid tumors. METHODS: Genomic DNA samples of 25 pairs of HLA haploidentical donors and recipients were extracted. The donor-derived HLA-DRB loci were detected with nested PCR-sequence specific primer (SSP) typing. The mixed lymphocyte proliferation action between the patients and respective donors, the engraftment of donor's cells and the serum levels of Th1/Th2 type of cytokines were measured with MTT, FISH and ELISA method respectively. The survival time of patients with or without feto-maternal microchimerism were compared as well. RESULTS: Using nested PCR-SSP typing, the positive rates of feto-maternal microchimerism in the 25 pairs of HLA haploidentical donors and recipients were 40% in the maternal/children pairs and 0 in the paternal/children pairs. The chimerism positive patients showed less proliferation activity when cocultured with respective donors as compared with unrelated ones (P = 0.03). Only one chimerism positive patient experienced the engraft of donor's cell 3 months after treatment as the donor derived XX chromosome was identified with FISH. When the data of chimerism positive patients were deleted, the serum levels of IFNgamma 1 month after treatment dropped dramatically from 171.4 (26.3 approximately 258.4) ng/L to 29.4 (1.2 approximately 39.9) ng/L. The survival time in chimerism positive patients of the maternal/children pairs was significantly longer than that in chimerism negative patients, which was (31.2 +/- 4.3) months and (11.1 +/- 3.3) months, respectively (P = 0.036). CONCLUSION: Feto-maternal microchimerism might induce anergy in the HLA haploidentical donors, favor the engraftment of donor's progenitors and maintenance of positive microenvironment and prolong the survival time.

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system.

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.

Experimental study on the treatment of intracerebral glioma xenograft with human cytokine-induced killer cells.

Objective. To investigate the phenotype changes and proliferation activities of cytokine-induced killer cells (CIKs) and lymphokine-activated killer cells (LAKs) from healthy donor, and the cytotoxicities of CIKs and LAKs to human in vitro glioma cell lines U251 and U87. Therapy of CIK intratumoral injection was evaluated in nude mouse models. Methods. CIK cells were induced from peripheral blood mononuclear cells (PBMC) of healthy donors with multiple cytokines. Phenotype analysis of CIKs and LAKs was performed with flow cytometer (FCM). The specific cytotoxicities of CIKs and LAKs against cell line U251 and U87 were determined by LDH method. After intracerebral injection of CIKs, the distribution of CIKs and the inflammatory reaction of their surrounding brain tissue were observed through continuous pathological sections. In vivo anti-tumor activity of CIKs was evaluated in athymic nude mice with intracerebral xenotransplanted U251 glioma by MRI. Results. Amount of CIKs was increased (49.83+/-2.04) times and double positive cells, CD3(+)/CD56(+) cells, were increased from (3.36+/-1.85%) to (44.07+/-14.14%) with elevated absolute amount over 1000 times after 2 week culture. In vitro experiments demonstrated that compared with LAK, CIKs possessed more obvious cytotoxic activity to U251 and U87. In vivo experiments showed that there was no severe inflammatory reaction in brain tissue. CIKs can markedly inhibit intracranial xenotransplanted glioma growth by intracranial injection (P<0.01). Conclusion. CIKs are a kind of highly effective immune cells which have a strong suppressive effect on growth for in vitro and in vivo glioma. Local injection of CIKs does not produce severe damage to normal brain tissue and is likely to be used in clinical adoptive immunotherapy of intracerebral glioma.

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine.

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.

[Application of low dose of fludarabine and cyclophosphamide combined with donor NK Cells as a non-myeloablative conditioning regimen for the haploidentical hematopoietic stem cell transplantation in mice].

This study was aimed to investigate the feasibility of low dose of fludarabine, cyclophosphamide combined with donor derived alloreactive NK cells as a new nonmyeloablative conditioning regimen in the haploidentical hematopoietic stem cell transplantation (haploidentical HSCT). F1 derived-NK cells were enriched with MACS magnetic separation system, in which the proportions of the Ly49C+ and Ly49A+ cells were detected by flow cytometry and the alloreactivity was measured by LDH method. The haploidentical HSCT models were constructed, and the myeloablativity in vivo, donor engraftment and the intensity of GVHD were compared between different myeloablative and nonmyeloablative conditioning regimens, including 9 Gy TBI, 6.5 Gy TBI, flu + cy, and flu + cy + allo-NK. The results showed that the flu + cy + allo-NK conditioning was nonmyeloablative, but the rate of donor chimerism after haploidentical HSCT was significantly higher as compared with other nonmyeloablative methods, which were (28.70 +/- 5.90)% in bone marrow and (46.40 +/- 5.00)% in spleen at day 21 post-transplantation. When compared with the flu + cy conditioning, the intensity of GVHD was slight in the flu + cy + allo-NK group, in which only a half of C57BL/6 recipients experienced weight loss, and no distinct pathological damages observed in the liver, intestine, kidney and skin samples. It is concluded donor derived-alloreactive NK cells can facilitate engraftment of the haploidentical hematopoietic stem cells and mitigate GVHD. The flu + cy + allo-NK conditioning provides a new method for those elder patients with high-risk solid tumor undergoing haploidentical-HSCT.

CD4 +CD25 + regulatory T cells decreased the antitumor activity of cytokine-induced killer (CIK) cells of lung cancer patients.

: CD(4) (+)CD(25) (+) regulatory T cells (Tregs) have been shown to inhibit cytotoxic lymphocytes-mediated immune responses. Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches. Therefore, the purpose of this report was to determine the effect of Tregs on CIK cell growth and CIK-induced cytotoxicity for inhibition of tumor growth in vivo as well as in vitro. After depletion of CD(4) (+)CD(25) (+) cells before culture, the proliferation and cytotoxicity of CIK cells, which indicated in bromodeoxyuridine (BrdU) and lactic dehydrogenase (LDH) assays, were significantly increased. Depletion of CD(4) (+)CD(25) (+) cells preculture also enhanced the suppression effect on the lung cancer cells inoculated in experimental animals. Blockage of glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor beta1 (TGF-beta1) by antibodies partially abrogated the suppressive effect of CD(4) (+)CD(25) (+) cells on CIK. These results indicated that Tregs could inhibit the antitumor activity of CIK cells. The molecules TGF-beta and GITR may contribute to the suppressive function of CD(4) (+)CD(25) (+) cells.

Th1 bias in PBMC induced by multicycles of auto-CIKs infusion in malignant solid tumor patients.

OBJECTIVE: The aim of this paper was to verify the feasibility and validity of autologous cytokine-induced killer cells (Auto-CIKs) treatments in solid malignancy patients. METHODS: Amplification, phenotypic characteristics, antitumor cytotoxicity, and clinical and immunological response of Auto-CIKs derived from 66 cases of patients with solid tumor of different pathological types and at different clinical stages were examined in a large-scale clinical trail. RESULTS: We found that seriousness of disease and metastasis status has no influence on effective components and antitumor activity of Auto-CIKs. Comparing the cytotoxicity against various tumor cells with LAKs, CIKs showed more effective cytotoxicity against NK sensitive and nonsensitive solid tumor cell lines, even at a low E/T ratio (6:1). In 20 patients receiving multicycles of Auto-CIKs infusions, 3 reached partial response (PR), 14 obtained stable disease (SD), and 3 died. Th1-kind cytokines secretion in peripheral blood mononuclear cells (PBMCs increased significantly, according with the enhanced cytolytic activity against K562 cells of them after multicycles of Auto- CIKs infusions. CONCLUSIONS: Induction of special "Th1 bias" in PBMCs after multicycles of Auto-CIKs treatments suggested an immunological promoting effect of Auto-CIKs, which seems to be a suitable immunotherapy for those solid-malignance patients who are at high risk of relapse to prevent recurrence.

[Dendritic cells reduce the number and function of CD4+CD25+ cells in cytokine-induced killer cells].

OBJECTIVE: To investigate the influence of dendritic cells (DCs) on the prevalence and function of regulatory T cells (Tregs) in cytokine induced killer (CIK) cells. METHODS: The blood samples of 20 patients with solid tumors were collected. The peripheral mononuclear cells (PBMCs) were isolated. CIK cells were added into the culture fluid without CD(4)(+)CD(25)(+)T cells (CIK-Treg(del) cells) and the culture fluid of regular PBMCs respectively, and the proliferation and cytotoxicity of the CIK cells were detected by BrdU method and with the cells of human lung carcinoma, breast carcinoma, colon carcinoma, and lymphoma as target cells respectively. CD(4)(+)CD(25)(+)T cells were added into another culture fluid of CIK-Treg(del) cells at the proportions of 20:1, 10:1, and 5:1 respectively, then the proliferation and cytotoxicity of the CIK cells were detected as described above. Flow cytometry was used to detect the surface markers of CIK cells. To identify the influence of DCs on the anti-tumor activity of CIK, PBMCs were isolated from the patients with solid tumor to culture the DCs and CIK cells. Dendritic cells were harvested on day 7 and co-cultured with the CIK cells (DC+CIK cells). The frequency of Tregs in CIK was determined by flow cytometry. The cytotoxicity was examined by LDH assay. The levels of TGF-beta, IL-10, IFN-gamma, IL-2, and IL-6 were analyzed by ELISA. RESULTS: The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells was higher than that of the regular CIK cells (P < 0.05), however, after the addition of selected cells, the cytotoxicity of the CIK-Treg(del) cells decreased. Flow cytometry showed that the proportions of CD(4)(+)CD(25)(+) Treg cells in the CIK cells and DC-CIK cells were 13% +/- 5% and 10% +/- 4% respectively (t = 3.977, P = 0.001). After the DC induction the cytotoxicity of CIK cells was significantly higher than that of the regular CIK cells. ELISA showed that after DC induction the levels of TGF-beta and IL-10 of the DC+CIK group were significantly lower than those of the regular CIK cells (t = 2.136, P = 0.046; and t = 2.965, P = 0.008), and the level of IFN-gamma was significantly higher in the DC+CIK group (t = 2.220, P = 0.039). CONCLUSION: CD(4)(+)CD(25)(+) regulatory T cells inhibit the anti-tumor activity of CIK cells. The interaction between CIK cells and DCs is sufficient for the blockage of the properties of regulatory T cells. CIK cells have the desirable properties for immunotherapy approaches, especially after co-culture with DCs.

[Study on large-scale CD34+ cell enrichment using immunomagnetic isolation apparatus].

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.

Induction of specific CTL by MAGE-3/CEA peptide-pulsed dendritic cells from HLA-A2/A24(+) gastrointestinal cancer patients.

PURPOSE: In this study, we aimed to investigate whether MAGE3/CEA peptide-pulsed dendritic cells could induce specific cytotoxic T lymphocytes (CTL). METHODS: In this pilot study, we selected 25 patients expressing MAGE-3-HLA-A2/A24 or CEA-HLA-A24. Patients' dendritic cells (DCs) were expanded in vitro in the presence of recombined-human granular macrophage colony stimulating factor (rhGM-CSF) and recombined-human interleukin 4 (rhIL-4), pulsed with MAGE-3/CEA (HLA-A2/A24) peptide. The cytolytic cells' activity, induced by peptide-pulsed DCs and unpurified T cells as effector cells, and with Mel526, 803, Raji, and K562 as target cells, were measured using LDH-releasing assay. RESULTS: DCs were obtained by in vitro expansion in all cases although DC harvest rates varied among different patients (7.1+/-3.2%). Compared with T-IL-2 (IL-2-induced T cells), T-DC-P - which resulted from T-IL-2 co-cultured with DCs pulsed by MAGE3 or CEA peptides - exhibited an increase in cytolytic activity against Mel526 (expressing MAGE-3-HLA-A2) and 803 (expressing CEA-HLA-A24) cell lines by about 25-30% ( P<0.01). In contrast, there was no significant difference between the activity against Raji and K562 cells, which are negative for both peptides. CONCLUSIONS: This study showed that combined usage of rhGM-CSF and rhIL-4 in vitro could expand DCs, and that the DCs pulsed with specific peptides could induce MAGE- and CEA-specific CTL responses. The DC-based vaccine may provide an important method for the immunological treatment of gastrointestinal cancers.