Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.
Cell Line, Tumor ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Genetic Engineering ; HLA-A2 Antigen ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; immunology

OBJECTIVETo study transfection efficiency of Ad5F11p-GFP and its influence on biological characteristics of CIK and NK-92 cells in order to predict the application of Ad5F11p vector in immunotherapy.

METHODSTwo kinds of immune cells, cytokine-induced killer (CIK) cells and natural-killer (NK) cell line NK-92 cells, were transfected by Ad5F11p-GFP at different multiplicity of transfection (MOI), and untransfected immune cells were used as negative control. GFP expression was determined by flow cytometry, the cell morphology was observed with microscope, the cell proliferation was analyzed by trypan blue staining, specific cytotoxicity of NK-92 cells was determined by LDH assay.

RESULTSAbout 90% of transfection efficiency for NK-92 cells could be achieved at a MOI of 25, while the transfection efficiency for CIK was less than 40% at a MOI of 200. In addition, the transfection efficiency basically unchanged at the same MOI for 48 h and 96 h, and the immune cells transfected with the virus trended to form agglomeration, displaying slower proliferation, increase of IFN-γ release and enhancement of tumor killing activity.

CONCLUSIONAd5F11p- modified NK-92 shows a good prospect for adoptive immunotherapy.

Adenoviridae ; Cell Line ; Cell Proliferation ; Cytokine-Induced Killer Cells ; cytology ; Cytotoxicity, Immunologic ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; cytology ; Neoplasms ; therapy ; Transfection

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.
Animals ; Body Weight ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; Interleukin-15 ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Neoplasm Transplantation ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

OBJECTIVETo assess the immunotoxicologic effects of genetically modified drought resistant wheat T349 with GmDREB1 gene.

METHODSA total of 250 female BALB/c mice (6-8 week-old, weight 18-22 g) were divided into five large groups (50 mice for each large group) by body weight randomly. In each large group, the mice were divided into five groups (10 mice for each group) by body weight randomly, which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, respectively. Mice in negative control and positive control group were fed with feedstuff AIN-93G, mice in common wheat group, non-genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat (proportion up to 76%) for 30 days, then body weight, organ coefficient of spleen and thymus, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell (PFC), serum 50% hemolytic value (HC50), mitogen-induced splenocyte proliferation, delayed-type hypersensitivity (DTH) reaction and phagocytic activities of phagocytes were detected respectively.

RESULTSAfter 30 days raise, among negative control group, common wheat group, non-genetically modified parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, mice body weight were (21.0±0.3), (20.4±0.7), (21.1±1.0), (21.1±1.0), (19.4±1.0) g, respectively (F=7.47, P<0.01); organ coefficient of spleen were (0.407±0.047)%, (0.390±0.028)%, (0.402±0.042)%, (0.421±0.041)%, (0.304±0.048)%, respectively (F=12.41, P<0.01); organ coefficient of thymus were (0.234±0.032)%, (0.246±0.028)%, (0.249±0.040)%, (0.234±0.034)%, (0.185±0.039)%, respectively (F=5.58, P<0.01); the percentage of T cell in peripheral blood were (70.43±4.44)%, (68.33±5.37)%, (73.04±2.68)%, (74.42±2.86)%, (90.42±1.66)%, respectively (F=57.51, P<0.01); the percentage of B cell were (13.89±3.19)%, (15.34±4.84)%, (13.06±4.22)%, (12.93±2.36)%, (3.01±0.96)%, respectively (F=12.79, P<0.01); the percentage of Th cell were (55.87±3.80)%, (55.24±4.60)%, (57.92±3.70)%, (59.57±2.54)%, (77.37±2.31)%, respectively (F=68.58, P<0.01);the Th/Ts ratio were 4.16±0.29, 4.73±0.96, 4.19±0.78, 4.52±0.40, 6.34±0.73, respectively (F=17.57, P<0.01);the serum IgG were (1046.38±210.67), (1065.49±297.22), (1517.73±299.52), (1576.67±241.92), (1155.88±167.05) µg/ml, respectively (F=10.53, P<0.01); the serum IgM were (333.83±18.97), (327.73±27.72), (367.47±27.18), (363.42±46.14), (278.71±24.42) µg/ml, respectively (F=12.11, P<0.01); the serum IgA were (51.69±10.10), (42.40 ± 8.35), (32.11±4.22), (37.12±4.90), (41.45±8.89) µg/ml, respectively (F=8.25, P<0.01); the PFC were (29.2±14.6), (28.0±20.0), (34.8±30.9), (33.2±25.1), (4.8±5.3) per 10(6) splenocyte, respectively (F=3.33, P<0.05); the HC50 were 82.3±6.5, 79.7±4.6, 75.8±4.1, 74.9±3.6, 70.8±2.1, respectively (F=9.99, P<0.01);the LPS-induced splenocyte proliferation were 0.21±0.10, 0.21±0.14, 0.26±0.12, 0.25±0.14, 0.07±0.06, respectively (F=4.18, P<0.05).

CONCLUSIONThe genetically modified drought-resistant wheat T349 was substantially equivalent to parental wheat in the effects on immune organs and immunologic functions of mice, and it didn't show immunotoxicity.

Animals ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; Droughts ; Female ; Mice ; Mice, Inbred BALB C ; Plants, Genetically Modified ; immunology ; toxicity ; Triticum ; genetics ; immunology ; toxicity

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration, and cellular transformation, and is one of the specific mRNA markers for circulating tumor cells in patients with gastrointestinal cancer. We investigated the feasibility of AGR2 as a potent antigen for tumor immunotherapy against colorectal cancer (CRC) cells using dendritic cells (DCs) transduced with a recombinant adenovirus harboring the AGR2 gene (AdAGR2). DCs transduced with a recombinant adenovirus encoding the AGR2 gene (AdAGR2/DCs) were characterized. These genetically-modified DCs expressed AGR2 mRNA as well as AGR2 protein at a multiplicity of infection of 1,000 without any significant alterations in DC viability and cytokine secretion (IL-10 and IL-12p70) compared with unmodified DCs as a control. In addition, AdAGR2 transduction did not impair DC maturation, but enhanced expression of HLA-DR, CD80, and CD86. AdAGR2/DCs augmented the number of IFN-gamma-secreting T-cells and elicited potent AGR2-specific cytotoxic T lymphocytes capable of lysing AGR2-expressing CRC cell lines. These results suggest that AGR2 act as a potentially important antigen for immunotherapy against CRC in clinical applications.
Adenoviridae ; Antigen Presentation/genetics ; Antigens, Neoplasm/immunology ; Carcinoma/*therapy ; Cell Line, Tumor ; Colorectal Neoplasms/*therapy ; Cytotoxicity, Immunologic/genetics ; Dendritic Cells/immunology ; Humans ; *Immunotherapy, Adoptive ; Interferon-gamma/secretion ; Lymphocyte Activation/genetics ; Proteins/genetics/*metabolism ; T-Lymphocytes, Cytotoxic/*immunology ; Transduction, Genetic ; Transgenes/genetics ; Tumor Markers, Biological/immunology

OBJECTIVETo establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.

METHODThree suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.

RESULTThe CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.

CONCLUSIONWe developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.

Case-Control Studies ; Cell Degranulation ; immunology ; Cell Membrane ; metabolism ; Chediak-Higashi Syndrome ; diagnosis ; genetics ; immunology ; metabolism ; Child, Preschool ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Interleukin-2 ; metabolism ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; genetics ; immunology ; metabolism ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Male ; Mutation ; Phytohemagglutinins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

OBJECTIVE: To investigate the effects and the related immunological mechanisms of dendritic cells (DCs) modified by SOCS1siRNA gene and IL-12 gene on activating and inducing cytotoxic T lymphocyte (CTL) as well as specific immune critically killing laryngocarcinoma in vitro. METHOD: DCs were derived from human peripheral blood mononuclear cells (hPBMC), modified by recombinant SOCSlsiRNA adenoviral and IL-12 adenoviral and then pulsed with tumor antigen of repeated freeze-thaw method. The IL-12 and IFN-y levels in culture supernatant of DCs and CTILs were examined by ELISA. RESULT: DC were cultivated successfully and had special morphologic haracteristicistics. The rate of Ad-GFP carrying fluorescent expression was over 90%. The expression of SOCS1 protein in DCs were effectively decreased by being modified SOCSlsiRNA and IL-12 genetic while the expression of IL-12 protein were increased. The secretion rate of IL-12 factor was higher than that of SOCSlsiRNA and IL-12 transfection of single gene respectively in modified DCs which could prompt T cell proliferation activation significantly as well. IFN-y was secreted constantly in DC and CTL, resulting in Hep-2. CONCLUSION DC modified by SOCSlsiRNA and IL-12 gene which pulsed with laryngeal carcinoma antigen could increased the production of IL-12 and IFN-y; DC modified by SOCSlsiRNA and IL-12 gene which pulsed with laryngeal carcinoma antigen could enhance the ability to stimulate proliferation of T cell, increase production of IFN-y, IL-12 by T cells and induce the stronger killing rate of CTL.
Adenoviridae ; genetics ; Antigens, Neoplasm ; immunology ; metabolism ; Cell Line ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Gene Silencing ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; genetics ; metabolism ; Laryngeal Neoplasms ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; RNA, Small Interfering ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

OBJECTIVETo prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.

METHODSA mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.

RESULTSMammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.

CONCLUSIONA vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

Antigens, Neoplasm ; metabolism ; Cancer Vaccines ; immunology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; secretion ; Interleukin-12 ; genetics ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

OBJECTIVEDendritic cell vaccines are one of the important active immunotherapies for neoplasms. The aim of this study was to observe the killing effect of specific cytotoxic T lymphocytes (CTL) on liver carcinoma HepG2 and SMMC-7721 cells in vitro. The CTL was induced by human peripheral blood mononuclear cells-originated dendritic cells (DC) transfected by recombinant adeno-associated virus (rAAV) with hAFP gene fragment (137-145).

METHODSImmature DCs were generated from peripheral blood mononuclear cells of healthy volunteers and then transfected by rAAV with AFP gene fragment. The CTL was thereafter induced. The activities of DC and CTL were measured and the killing effect of the CTL on HepG2 cells was detected using M1Tr assay.

RESULTSThe mature DC, transfected or not, highly expressed CD40, CD86 and IL-12. IFN-gamma was highly expressed in the CTL. The DC-induced CTL could effectively recognize and destroy the HepG2 and SMMC-7721 cells.

CONCLUSIONDC transfected by rAAV can stimulate the proliferation and differentiation of lymphocytes and also induce the proliferation of CTL, and their own phenotypes are not significantly altered. The DC vaccine can be effectively used as an adjuvant immunotherapy for patients with hepatocellular carcinoma.

B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Dependovirus ; genetics ; Hep G2 Cells ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; Peptide Fragments ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; pathology ; Transfection ; alpha-Fetoproteins ; genetics