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*

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.
Animals ; Antibodies, Bispecific ; immunology ; Antibodies, Monoclonal ; immunology ; pharmacokinetics ; CD3 Complex ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; ErbB Receptors ; metabolism ; Female ; Humans ; Lymphocyte Activation ; immunology ; Mice, SCID ; Neoplasms ; immunology ; pathology ; Rats, Sprague-Dawley ; T-Lymphocytes, Cytotoxic ; immunology

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

OBJECTIVETo find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic.

METHODSOnline biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice.

RESULTSFour natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner.

CONCLUSIONNatural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.

Adaptor Proteins, Signal Transducing ; immunology ; Animals ; Epitopes, T-Lymphocyte ; metabolism ; HLA-A2 Antigen ; metabolism ; Humans ; Mice ; Mice, Transgenic ; T-Lymphocytes, Cytotoxic

PURPOSE: Acute hepatitis A (AHA) and acute hepatitis B (AHB) are caused by an acute infection of the hepatitis A virus and the hepatitis B virus, respectively. In both AHA and AHB, liver injury is known to be mediated by immune cells and cytokines. In this study, we measured serum levels of various cytokines and T-cell cytotoxic proteins in patients with AHA or AHB to identify liver injury-associated cytokines. MATERIALS AND METHODS: Forty-six patients with AHA, 16 patients with AHB, and 14 healthy adults were enrolled in the study. Serum levels of 17 cytokines and T-cell cytotoxic proteins were measured by enzyme-linked immunosorbent assays or cytometric bead arrays and analyzed for correlation with serum alanine aminotransferase (ALT) levels. RESULTS: Interleukin (IL)-18, IL-8, CXCL9, and CXCL10 were significantly elevated in both AHA and AHB. IL-6, IL-22, granzyme B, and soluble Fas ligand (sFasL) were elevated in AHA but not in AHB. In both AHA and AHB, the serum level of CXCL10 significantly correlated with the peak ALT level. Additionally, the serum level of granzyme B in AHA and the serum level of sFasL in AHB correlated with the peak ALT level. CONCLUSION: We identified cytokines and T-cell cytotoxic proteins associated with liver injury in AHA and AHB. These findings deepen the existing understanding of immunological mechanisms responsible for liver injury in acute viral hepatitis.
Acute Disease ; Adult ; Alanine Transaminase/blood ; Biomarkers/blood ; Cytokines/*blood ; Enzyme-Linked Immunosorbent Assay ; Fas Ligand Protein/blood ; Female ; Hepatitis A/blood/virology ; Hepatitis A virus/*genetics/immunology ; Hepatitis B/blood/virology ; Hepatitis B virus/*genetics/immunology ; Humans ; Interleukin-6/blood ; Interleukin-8/blood ; Interleukins/blood ; Liver Failure/immunology/metabolism/*pathology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic/immunology/*metabolism

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.
Adult ; Aged ; CD8-Positive T-Lymphocytes/drug effects/*immunology ; Case-Control Studies ; Cell Proliferation ; Cell Separation ; Dermatitis, Atopic/*immunology/pathology ; Female ; Granzymes/metabolism ; Humans ; Interleukin-10/metabolism ; Lymphocyte Count ; Male ; Perforin/metabolism ; Skin/*immunology/pathology ; T-Lymphocytes, Cytotoxic/drug effects/immunology ; T-Lymphocytes, Regulatory/drug effects/*immunology ; Transforming Growth Factor beta1/pharmacology

PURPOSE: This study aimed to compare the regulatory T cells in cord blood of appropriate for gestational age (AGA) neonates with those of small for gestational age (SGA) neonates. MATERIALS AND METHODS: Umbilical cord blood was collected upon labor in 108 healthy full-term (between 37 and 41 gestational weeks) neonates, who were born between November 2010 and April 2012. Among them, 77 samples were obtained from AGA neonates, and 31 samples were obtained from SGA neonates. Regulatory T cells and lymphocyte subsets were determined using a flow cytometer. Student's t-test for independent samples was used to compare differences between AGA and SGA neonates. RESULTS: Regulatory T cells in cord blood were increased in the SGA group compared with normal controls (p=0.041). However, cytotoxic T cells in cord blood were significantly decreased in the SGA group compared with normal controls (p=0.007). CONCLUSION: This is the first study to compare the distribution of lymphocyte subsets including regulatory T cells in cord blood between AGA neonates and SGA neonates.
Biological Markers/metabolism ; Female ; Fetal Blood/*immunology ; Gestational Age ; Humans ; Infant, Newborn/*blood ; Infant, Small for Gestational Age/*blood ; Lymphocyte Count ; T-Lymphocytes, Cytotoxic/metabolism ; T-Lymphocytes, Regulatory/*metabolism

BACKGROUND/AIMS: This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. METHODS: The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. RESULTS: When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D+ NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. CONCLUSIONS: The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.
Carcinoma, Hepatocellular/*physiopathology ; Case-Control Studies ; Female ; Humans ; K562 Cells ; Killer Cells, Natural/*physiology ; Liver Neoplasms/*physiopathology ; Lymphocyte Subsets/physiology ; Lymphopenia/physiopathology ; Male ; Middle Aged ; NK Cell Lectin-Like Receptor Subfamily K/metabolism ; T-Lymphocytes, Cytotoxic/physiology

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
Adult ; Amino Acid Sequence ; Animals ; Binding Sites ; Epitopes ; chemistry ; immunology ; metabolism ; Female ; Genotype ; HEK293 Cells ; HLA-A2 Antigen ; metabolism ; Hepatitis B Core Antigens ; chemistry ; immunology ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Hydrogen Bonding ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Middle Aged ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

BACKGROUNDCancer testis antigens (CTAs) are a novel group of tumor associated antigens. Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism, thus enhance the immunogenicity of leukemia cells. However, few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo, and if so, whether this effect contributes to disease control. In this study, we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.

METHODSSeveral mouse CTAs were screened by RT-PCR. CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry. The activity of specific CTLs was measured by real time RT-PCR.

RESULTSWe firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs. Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment. Finally, we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.

CONCLUSIONSOur study showed the autologous immune response induced by decitabine in vivo. And more importantly, we firstly proved that this response may contribute to disease control. We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine, and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.

Animals ; Antigens, Neoplasm ; metabolism ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Cytotoxic ; drug effects ; metabolism