BACKGROUND: Asbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8 METHODS: For MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8 RESULTS: IL-15 addition partially reversed the decrease in CD3 CONCLUSION These findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.
Asbestos/adverse effects* ; CD8-Positive T-Lymphocytes/metabolism* ; Humans ; Interleukin-15/pharmacology* ; Lymphocyte Activation/immunology* ; T-Lymphocytes, Cytotoxic/metabolism*

OBJECTIVETo report the diagnosis, differential diagnosis and treatment of a rare case of primary bone marrow CD8+ cytotoxic T-cell lymphoma coexpressed CD20.

METHODSThe clinical characteristics, therapeutic course and the outcome of this patient were reviewed. Meanwhile, a series of examinations including morphology, flow cytometry, immunohistochemistry and molecular biology of bone marrow and skin samples were also performed.

RESULTSBone marrow biopsy showed an extensive involvement by abnormal T lymphocytes. Flow cytometry and immunohistochemistry showed weakly positive CD20, CD8(+), CD2(+), CD3(+), CD5(+), TIA(+), PAX-5(-), CD4(-), CD56(-), CD57(-), CD30(-), ALK-1(-), P53(-), TdT(-), Ki-67≈5%. A final diagnosis of primary bone marrow CD8+ cytotoxic T-cell lymphoma coexpressed CD20 was made. The patient initially presented a relatively indolent course was, but he was expired in the end 3 years later due to extensive involvements of skin and other organs though timely therapy was administrated.

CONCLUSIONPrimary bone marrow CD8 cytotoxic T-cell lymphoma coexpressed CD20 was encountered rarely in clinical practice, which might be a challenging in terms of diagnosis and differential diagnosis. Further investigation of pathogenesis and therapeutic strategies of this rare disease was warranted.

Antigens, CD20 ; metabolism ; CD8-Positive T-Lymphocytes ; metabolism ; pathology ; Humans ; Lymphoma, T-Cell ; diagnosis ; pathology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; metabolism ; pathology

OBJECTIVETo explore the specific anti-leukemia immune response of CD8+ cytotoxic T lymphocyte (CTL) derived from cord blood (CB) ex vivo and evaluate the feasibilities and values of the CTL for specific immunotherapy.

METHODSDendritic cells (DC) were induced from mononuclear cells (MNC) by combination cytokines in 10 CB samples. Loading U937 cell lysate antigen on the mature DC, they could stimulate the lymphocytes of the same origin to generate CTL. MidiMACS was used to isolate CD8+ CTL. Analysis of DC was performed by inverted microscopy, scanning electron microscopy and flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of the CTL.

RESULTSCocultured with GM-CSF, IL-4, TNF-alpha and PGE2, CB-MNC could be induced into functional DC with typical morphology. The mean cytotoxicity of CD8+ CTL to U937 cells was significant stronger than that of CD8- CTL and TL at the same E: T ratios. The mean cytotoxicity rate of CD8+ CTL to U937 cells was higher than that to K562 cells [(66.36 +/- 12.43)% vs (41.97 +/- 14.24)%] at E: T ratio of 40: 1 (P < 0.05). The cytotoxicity of CD8- CTL to K562 cells showed no difference from that to U937 cells (P > 0.05).

CONCLUSIONMature CB-DC loading U937 cell antigens could induce CB-T lymphocytes to generate leukemia-specific CD8+ CTL. The cytotoxicity of the CD8+ CTL is specific against U937 cells and is more potent than that of CD8- CTL.

CD8-Positive T-Lymphocytes ; cytology ; immunology ; Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; In Vitro Techniques ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

OBJECTIVETo investigate the effect of adenovirus vector encoding human vascular endothelial growth factor receptor-2 (hVEGFR-2 or hKDR) on breaking the immune tolerance and inducing immunity against murine hepatocellular carcinomas.

METHODSHuman and mouse KDR cDNA were cloned from human umbilical vein endothelial cells (HUVEC) and C57BL/6 mouse embryo cells respectively using RT-PCR, and then Ad hKDR and Ad mKDR were constructed. Seven days after immunization of the mice with Ad hKDR or Ad mKDR, an analysis of cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTL) was made by lactate dehydrogenase (LDH) release assay, in which splenocytes of the immunized mice acted as effectors and Hepa 1-6/mKDR cells as the targets. In addition, the survival of the mice immunized with Hepa 1-6 hepatoma cells was checked.

RESULTSSeven days after immunization, the 6 h killing activities of CTL elicited by the Ad hKDR were 84.3%+/-6.7%, 71.5%+/-5.2%, and 44.6%+/-4.7% at the ratio of the effectors:targets (E:T) of 100:1, 50:1, and 25:1, respectively. Correspondingly, the CTL activities by Ad mKDR were 65.2%+/-6.1%, 46.7%+/-5.0%, and 22.6%+/-3.7%. Sixty percent of the Ad hKDR-immunized mice with 5*10(6) Hepa 1-6 hepatoma cells were still alive two months after the inoculation, whereas just 40% of the Ad mKDR-immunized mice with 2*10(6) Hepa 1-6 cells survived two months. When CD8+ or CD4+ T lymphocytes were deleted in the mice the above mentioned CTL activities and protection of the mice from tumors disappeared.

CONCLUSIONAdenovirus vector-mediated xenogeneic KDR can effectively break the immune tolerance to hepatocellular carcinomas in an animal model and induce a strong antigen-specific T cell response, which is dependent on CD8+ and CD4+ T cells.

Adenoviruses, Human ; genetics ; Animals ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Carcinoma, Hepatocellular ; genetics ; immunology ; Cell Line, Tumor ; Female ; Humans ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

OBJECTIVETo investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells.

METHODSHLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay.

RESULTSThe WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402.

CONCLUSIONThese data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.

CD8-Positive T-Lymphocytes ; cytology ; Cell Line, Tumor ; Genes, T-Cell Receptor ; Humans ; Immunotherapy, Adoptive ; Lung Neoplasms ; pathology ; Peptides ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Retroviridae ; T-Lymphocytes, Cytotoxic ; cytology ; Transduction, Genetic ; WT1 Proteins ; genetics

Minimal residual disease (MRD) is the principal root of relapsed leukemia. Application of specific immunotherapy is effective in eradication of MRD and one of the immunotherapeutic strategies is induction and re-transfusion of leukemia-specific cytotoxic T lymphocytes (CTLs). This study was aimed to investigate the possibility of ex vivo induction and generation of CD8 positive CTLs of umbilical cord blood cell origin and to explore their potential of specific anti-leukemia cytotoxicity so as to evaluate the feasibility of application of cord blood cell derived lymphocytes to specific immunotherapy. Dendritic cells (DCs) were induced and generated ex vivo from cord blood mononuclear cells (MNC) with a cytokine cocktail, and loaded with frozen and thawed U937 leukemia antigen. The matured DCs were used to stimulate T lymphocytes derived from the same cord blood cell sample into CTLs. CD8 positive CTLs were then isolated by magnetic activated cell sorting (MACS). Inverted microscopy, scanning electronic microscopy and flow cytometry were used to detect DCs and methyl thiazolyl tetrazolium (MTT) cytotoxicity method was used to assay the killing activity. The results showed that DCs with typical morphology and mature function were cultured from 10 human cord blood cell samples. The cytotoxicities of CD8 positive CTLs, CD8 negative CTLs and T lymphocytes (TLs) to U937 cells were (66.36 +/- 12.43)%, (34.47 +/- 8.19)% and (15.79 +/- 4.64)% respectively under the same effector target ratio (40:1). Among them, the anti-leukemia cytotoxicity of the CD8 positive group was highest. At effctor target ratio of 40 to 1, the cytotoxicity of CD8 positive CTLs to U937 cells (66.36%) was higher than that to K562 cells (41.97%) (P < 0.05), whereas the cytotoxicity of CD8 negative CTLs to U937 cells was not significantly different from that to K562 cells (P > 0.05). It is concluded that specific CD8 positive CTLs can be generated from cord blood lymphocytes by induction of mature cord blood DCs loaded with U937 leukemia antigen. The cytotoxicity of CD8 positive CTLs against U937 cell is more potent than CD8 negative CTLs, and is strain specific.
Antibody Specificity ; Antigens, Neoplasm ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunotherapy ; T-Cell Antigen Receptor Specificity ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

OBJECTIVETo study the improvement of CTL effect by CTL epitopes which are modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2.

METHODSObservations were made on the specific immune response induced by the CD8+ CTL epitopes(SSIEFARL, S1), the CD8+ CTL epitopes modified by KDEL(SSIEFARL-KDEL, S1-KDEL), the tandem four copies CD8+ CTL epitopes[(SSIEFARL)4, S4] and the tandem four copies CD8+ CTL epitopes modified by KDEL[(SSIEFARL)4-KDEL, S4-KDEL], 25 male C57BL/6 mouse were randomly divided into 5 group, 5 mice per group, respectively immunized with istonic Na chloride, S1, S1-KDEL, S4 and S4-KDEL. Lymphocyte proliferation was detected by 3H-TdR and the CTL effect induced by CTL epitopes in vivo was detected by 51Cr.

RESULTSIn the 3H-TdR test, compared with the control, the group S1 and group S1-KDEL, the cpm values of Group S4 and Group S4-KDEL were markedly higher (P < 0.05) and the cpm value of Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the cpm values of Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that of Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells sensitized by S1 were attacked as target cells, the CTL activities induced in Group S4 and Group S4-KDEL were markedly higher (P < 0.05) compared with the control, Group S1 and Group S1-KDEL, and that induced in Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the CTL activity induced in Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that induced in Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells were attacked as target cells, the kill rate was below 10% in every group, not significantly different from the control.

CONCLUSIONThe tandem four copies CD8+ CTL epitopes, modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2, can improve the CTL effect.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cell Line ; Epitopes, T-Lymphocyte ; immunology ; Herpesvirus 2, Human ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Oligopeptides ; immunology ; Protein Sorting Signals ; Random Allocation ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo construct the eukaryotic expression vector harboring Wilms' tumor gene (WT1y) and assess the cytotoxic T lymphocyte (CTL) response specific to the DNA vaccine of WT1y product in Balb/c mice and in vitro cultured cells.

METHODSSynthesized WT1y gene (321 bp including HLA-A*2402 anchoring residue) was cloned into the eukaryotic vector to construct the plasmid pCDNA3.1(+)/WT1y. WT1-specific antibody was detected in mouse serum by ELISA, and T lymphocyte proliferation was detected by flow cytometry. The CTL response was detected by co-culture of the murine spleen cells with the tumor cells.

RESULTSThe recombinant plasmid was confirmed using DNA sequencing. WT1-specific antibodies were detected in the serum of immunized mice, which showed significantly greater T lymphocyte proliferation than the control group (Plt;0.01). The CTLs resulted in specific lysis of WT1-expressing murine tumor cells.

CONCLUSIONUsing HBVC as the vector for WT1y gene, wenave obtained the virus-like particles that induce high cellular immune response, which shed light on a new approach for treatment of Wilms' tumor.

Animals ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Female ; Genes, Wilms Tumor ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology ; Xenograft Model Antitumor Assays

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

BACKGROUNDS/AIMS: Hepatitis B virus(HBV) specific cytotoxic T lymphocyte (CTL) response is believed to play a major role in virus control and liver damage in chronic hepatitis B(CHB). We performed this study to evaluate whether HBV specific CTL could be visualized directly by tetrameric HLA-A2/core 18-27 complex(T c18-27) in the peripheral blood and liver of patients with CHB. On the basis of our results we clarified patients intrahepatic compartmentalization and correlation with HBV specific CTL and viral replication or liver damage. METHODS: We stained peripheral blood mononuclear cells of 33 HLA-A2 + and 8 HLA-A2 patients with CHB with cychrome conjugated anti-CD8 mAb and phycoerythrin conjugated T c18-27. Among these we analysed intrahepatic lymphocyte of 11 HLA-A2 + patients. We compared the frequency of T c18-27 specific CD8+ cells with serum HBV-DNA levels or alanine aminotransferase(ALT) levels. RESULTS: The frequency of circulating T c18-27 specific CD8+ cell was higher(9-101 cells per 50,000 CD8+ cells) than background level in 14 among 33 patients. The frequency of intrahepatic T c18-27 specific CD8+ cells was 12-2100 cells per 50,000 CD8+ cells in 8 out of 11 patients whose liver was obtained This was 17.4-150 times higher than circulating T c18-27 specific CD8+ cells. The frequency of circulating T c18-27 specific CD8+ cells was increased in 10 out of 18 patients with serum HBV DNA level <0.5 pg/mL and ALT < 40 IU/L. It was increased in just 4 out of 15 patients with HBV DNA level > 800 pg/mL and ALT >70 IU/L. The frequency of intrahepatic T c18-27 CTL tended to be lower in high levels of serum HBV DNA and was not correlated with liver inflammation. CONCLUSION: This study provess that if HBV-specific CTLs are barely detectable in the peripheral blood of CHB, much more HBV-specific CTLs are in the liver and most HBV-specific CTLs are infiltrated in the liver. Also, in the presence of an effective HBV specific CD8 response the inhibition of viral replication can be independent of liver damage.
CD8-Positive T-Lymphocytes/immunology ; DNA, Viral/analysis ; English Abstract ; HLA-A2 Antigen/analysis/*immunology ; Hepatitis B Virus/genetics/*immunology ; Hepatitis B, Chronic/*immunology/virology ; Human ; T-Lymphocytes, Cytotoxic/*immunology ; Viral Core Proteins/*immunology