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Results for “antigen expression and secretion”

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PubMed1987

Modulation of expression of class II histocompatibility antigens by secretion of a cellular inhibitor in K562 leukemic cells.

In this report we show that it is possible to induce the expression of HLA-DR antigens on K562 cells, previously reported to be unresponsive to interferon-gamma (IFN-gamma). However, only low cell concentrations and a high dose of IFN-gamma allowed the induction of HLA-DR antigens. Furthermore, the recombinant glycosylated IFN-gamma is 100-fold more efficient than the unglycosylated form. This induction of HLA-DR antigens on K562 was not related to a stage of differentiation or to the presence of cells subsets specifically sensitive to IFN-gamma, since repeated sorting of K562 HLA-DR-positive and negative cells did not lead to the selection of a cell subset with a different potential of induction for HLA-DR. The difficulty in obtaining induction is due to the production of a soluble endogenous inhibitor of proteic nature, whose action is not restricted to the K562 cell line since it operates also on both epithelial and fibroblastic cells. Treatment of normal human epithelial and fibroblastic cells with conditioned medium from K562 cultures caused a marked decrease in the expression of HLA class II antigens (DR and DP) induced by IFN-gamma (10,000 U/ml), but had no effect on cell growth; however, it also affected expression of HLA class I antigens. This inhibition is not mediated by prostaglandin or an IFN-alpha or IFN-beta-dependent mechanism. Production of this inhibitor by pluripotent human leukemic cells could cause an unbalance in the complex control exerted by the immunological system during hematopoietic differentiation or leukemic progression.

Antibodies, Monoclonal↗

PubMed1989

Antigen-stimulated human interferon-gamma generation: role of accessory cells and their expressed or secreted products.

In response to cytomegalovirus (CMV) and Toxoplasma gondii antigens, T4+ cells from seropositive donors produce interferon-gamma (IFN-gamma) by different mechanisms; one (T. gondii) dependent upon and the other (CMV) largely independent of interleukin-2 (IL-2) and its receptor. To determine whether IFN-gamma-generating mechanisms unrelated to IL-2 also differ, we examined the requirement for accessory cells and their expressed or secreted products. In response to both specific antigens, IFN-gamma secretion was strictly dependent upon the presence of accessory cells (monocytes), and was largely inhibited by monoclonal antibodies to class II (HLA-DR and -DQ) but not class I MHC antigens. Both CMV and T. gondii antigens stimulated monocytes to release interleukin-1 (IL-1), and IFN-gamma production in response to both antigens was abolished by pretreatment with anti-IL-1 antibody. In contrast, the secretion of tumour necrosis factor (TNF) was not stimulated by either antigen, and IFN-gamma production was not diminished by antisera directed at TNF-alpha or TNF-beta. We conclude that CMV and T. gondii antigen-induced IFN-gamma production requires a similar accessory cell mechanism, and that soluble antigen-stimulated IFN-gamma secretion by human T4+ cells is dependent on monocytes, expression of class II MHC antigens, and the presence of IL-1.

Animals↗

PubMed1993

Independent cell types are involved in the induction of antimelanoma responses in C57BL/6 mice immunized with interleukin-2-secreting allogeneic mouse fibroblasts expressing melanoma-associated antigens.

Cellular antimelanoma immune responses developed during the coincubation of spleen cells from naive C57BL/6 mice (H-2b) with LM mouse fibroblasts (H-2k) genetically modified to express melanoma-associated antigens (MAA) and to secrete interleukin (IL)-2 (RLBA-IL-2 cells). Antimelanoma responses also developed following coincubation of spleen cells from naive mice with allogeneic cells (H-2k) that expressed MAA, but did not secrete IL-2 (RLBA-ZipNeo cells), or allogeneic cells (H-2k) that secreted IL-2, but did not form MAA (LM-IL-2 cells). However, in these instances, the magnitude of the responses was significantly less (p < 0.01) than followed coincubation of spleen cells with the allogeneic cell construct (RLBA-IL-2) that combined IL-2 secretion with the expression of MAA. LM(TK-) cells (H-2k) or B16 cells (H-2b) failed to generate antimelanoma immune responses in populations of spleen cells from C57BL/6 mice. The cell types involved in the induction of the antimelanoma response by the genetically modified cells were determined by prior depletion of T-cell subsets with monoclonal antibodies before the addition of the cellular immunogens. Antimelanoma responses failed to develop in spleen cell populations depleted of T-helper cells if the cellular immunogen was non-IL-2 secreting (RLBA-ZipNeo cells). Prior depletion of T-helper cells did not affect the induction of an antimelanoma response in cell populations coincubated with constructs (RLBA-IL-2 or LM-IL-2) modified to secrete IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2001

Tumours can act as adjuvants for humoral immunity.

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.

Adjuvants, Immunologic↗

PubMed2006

ESTDAB: a collection of immunologically characterised melanoma cell lines and searchable databank.

Cancer immunologists working in humans often require panels of tumour cell lines expressing different combinations of HLA alleles and other characteristics. Sources of cell lines are usually large cell banks carrying little immunologically relevant information on the available cells and limited numbers of different lines from the same type of tumour. Access to cells with desired combinations of characteristics is, therefore, difficult. Here, we describe an interactive database of a large collection of melanoma cell lines which have been extensively characterised for HLA genotype and surface expression, oncogene and tumour antigen expression, cytokine secretion, surface molecule expression, adhesion to extracellular matrix components, cytokine gene polymorphisms and other factors of interest to immunologists. This enables investigators to search for cells with particular constellations of HLA alleles, tumour antigens, etc., and then request these from the cell bank. This European Searchable Tumour Cell Line and Data Bank (ESTDAB) was established as a Research Infrastructure of the European Commission. For access to the databank and further details, please go to http://www.ebi.ac.uk/ipd/estdab/ .

Animals↗

PubMed1994

Immunization of mice with allogeneic fibroblasts genetically modified for interleukin-2-secretion and expression of melanoma-associated antigens stimulate predetermined classes of anti-melanoma effector cells.

Immunization of C57BL/6 mice (H-2b) with a mouse fibroblast cell-line of C3H origin (H-2k) genetically modified for interleukin-2 (IL-2)-secretion and the expression of melanoma-associated antigens (MAAs) (RLBA-IL-2 cells) resulted in a systemic anti-melanoma cellular immune response that led to a prolongation of survival of mice with established melanoma. Here we report certain of the effector cell-types activated for anti-melanoma immunity in mice immunized with the modified cells and, for comparison, the anti-melanoma cell-types activated following immunization with IL-2-secreting, MAA-negative fibroblasts (LM-IL-2 cells) or with non-IL-2-secreting, MAA-positive fibroblasts (RLBA-ZipNeo cells). The data indicate that both Lyt-2.2+ (CD8+) and natural killer/lymphokine-activated killer (NK/LAK) cells with anti-melanoma cytotoxicity were predominant in mice immunized with RLBA-IL-2 cells. NK/LAK cells alone were predominant in mice immunized with LM-IL-2 cells, and Lyt-2.2+ cells were predominant in mice immunized with RLBA-ZipNeo cells. The involvement of L3T4+ (CD4+) cells in the effector phase of the response was not detected in mice immunized with the genetically modified cells. Immunization of mice with both LM-IL-2 cells and RLBA-ZipNeo cells resulted in an anti-melanoma response of greater magnitude than was present in mice immunized with either cell-construct alone. It was equivalent to the melanoma immunity in mice immunized with RLBA-IL-2 cells. These data indicate that the immunogenic properties of the modified cells determined the anti-melanoma effector cell-types and suggest that combination immunotherapy with cell-constructs that stimulate different classes of effector cells may be more effective in immune-mediated tumor regression than immunization with a construct that activates a single effector cell-type alone.

Animals↗

PubMed2002

Vascular endothelial growth factor production and regulation in human peritoneal mesothelial cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) was recently found in peritoneal effluents of peritoneal dialysis (PD) patients. It was suggested that human peritoneal mesothelial cells (HMC) contribute to the intraperitoneal production of VEGF, which may augment vascular permeability, vasodilation and neoangiogenesis in the peritoneal membrane. The present study was designed to assess the influence of proinflammatory cytokines, thrombin, d-glucose and glycated albumin in the regulation of VEGF synthesis in primary HMC cultures. METHODS: VEGF antigen concentrations were measured in the cell supernatant by ELISA and VEGF mRNA expression was evaluated by real time RT-PCR. RESULTS: Incubation of HMC with interleukin-1alpha (IL-1alpha; 10 to 100 U/mL), tumor necrosis factor-alpha (TNF-alpha; 500 to 1000 U/mL) or thrombin (1 to 10 U/mL) resulted in a time (24 to 72 hours) and concentration dependent increase in VEGF synthesis. In contrast, d-glucose (30 to 90 mmol/L), which is commonly used as an osmotic agent in peritoneal dialysis, was not able to up-regulate VEGF expression. High glucose levels even decreased VEGF production. However, exposure of HMC to Amadori-modified glycated albumin, which is generated in the peritoneal cavity in the presence of glucose-based dialysis solutions, resulted in a dose and time dependent increase in VEGF mRNA expression and antigen secretion. CONCLUSIONS: These results demonstrate, to our knowledge for the first time, that glycated serum albumin, not glucose, increases VEGF production in HMC. HMC play an important role as a source of intraperitoneal VEGF synthesis, and VEGF expression also is up-regulated in the presence of proinflammatory cytokines and thrombin. Additionally, these results confirm clinical data that the continuous exposure of the peritoneal membrane to glucose-based dialysis solutions is an important stimulus for VEGF expression. However, it is not glucose per se, but nonenzymatic glycation products like glycated albumin that up-regulate VEGF expression.

Antineoplastic Agents↗

PubMed1995

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale.

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.

Amino Acid Sequence↗

PubMed1999

D-glucose increases the synthesis of tissue-type plasminogen activator (t-PA) in human peritoneal mesothelial cells.

Physical and chemical irritation of the peritoneum through glucose-based hyperosmolar dialysis solutions results in a nonbacterial serositis with fibrinous exudation. Thereby, human peritoneal mesothelial cells (HMC) play an important role in maintaining the balance between the peritoneal generation and degradation of fibrin by expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) as well as the specific plasminogen activator inhibitor-1 (PAI-1). In this study, we analyzed the effect of D-glucose and metabolically inert monosaccharides on the synthesis of t-PA and PAI-1 in cultured HMC. Incubation of HMC with D-glucose or the metabolically inert monosaccharides mannitol and L-glucose (5-90 mM) resulted in a time- and concentration-dependent increase in t-PA mRNA expression and antigen secretion without affecting PAI-1 synthesis. A similar effect was evident when HMC were first exposed sequentially to pooled spent peritoneal dialysis effluent for up to 4 hours, and subsequently incubated for 20 hours in control medium. The stimulating effect of high D-glucose on t-PA expression in HMC was prevented by treating the cells with different protein kinase C (PKC) inhibitors (Ro 31-8220, Gö 6976), but could not be mimicked by the PKC-activating phorbol ester PMA, indicating that this effect of high glucose is dependent on PKC activity, but not mediated through PKC activation. Also, using specific inhibitors (PD 98059, SB 203580) and activators (PMA, anisomycin, IL-1alpha) of the major routes of the mitogen-activated protein kinases (MAPKs) cascade, we found no evidence for a role of this cascade in regulating t-PA expression in HMC. We conclude that hyperosmolarity induces t-PA (but not PAI-1) in HMC via a regulatory mechanism that requires active PKC, but that does not involve a major pathway in the MAPK cascade.

Cells, Cultured↗

PubMed2005

Sirolimus inhibits platelet-derived growth factor-induced collagen synthesis in rat vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation and extracellular matrix (ECM) accumulation play key roles in the development and the progression of vascular remodeling such as transplant arteriosclerosis and restenosis. The present study examined the effects of sirolimus (SRL) on platelet-derived growth factor (PDGF)-induced fibronectin secretion, collagen synthesis, and the related signaling pathways including reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) in rat VSMCs. Primary rat VSMCs were isolated from male Sprague-Dawley rats. Growth arrested, synchronized cells were treated with various concentrations of SRL before the addition of PDGF at 10 ng/mL. Proliferating cell nuclear antigen expression, fibronectin secretion, and the activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK were assessed by Western blot analysis, collagen synthesis by [(3)H]-proline incorporation, and cellular ROS by flow cytometry. PDGF (10 ng/mL) increased VSMC proliferation by 1.7-fold, fibronectin secretion by 1.5-fold, collagen synthesis by 2.1-fold, cellular ROS by 1.6-fold, and activation of ERK and p38 MAPK by 3.3- and 3.9-fold compared to controls. SRL above 1 nmol/L inhibited PDGF-induced VSMC proliferation and collagen synthesis but not PDGF-induced fibronectin secretion, cellular ROS, and activation of ERK and p38 MAPK. These data demonstrated that PDGF increased ECM synthesis as well as proliferation through cellular ROS and subsequent MAPK activation and that SRL inhibited PDGF-induced VSMC proliferation and collagen synthesis in a cellular ROS- and MAPK activation-independent way.

Animals↗

PubMed2002

Astrocytes are less efficient in the removal of apoptotic lymphocytes than microglia cells: implications for the role of glial cells in the inflamed central nervous system.

Apoptosis of T lymphocytes is a common pathway to terminate autoimmune inflammation in the brain as shown in experimental autoimmune encephalomyelitis (EAE) and in the autoimmune inflamed human brain. To date it is unclear to what extent different glial cells are involved in the removal of apoptotic cells. In an in vitro phagocytosis assay we compared the phagocytic capacity of rat microglia cells to remove apoptotic lymphocytes with that of astrocytes. Apoptosis was induced in autologous thymocytes and myelin basic protein (MBP)-specific T-cells by methylprednisolone (MP) or by irradiation. Apoptotic cells were then added to glial cells that were untreated or prestimulated with interferon-gamma (IFN-gamma), interleukin-4 (IL-4), transforming growth factor-beta (TGF-beta), or tumor necrosis factor-a (TNF-a). Supernatants were collected from cell cultures to measure their cytokine secretion. Surface antigen expression was analyzed by flow cytometry. Both cell types significantly increased their phagocytic activity in response to the addition of apoptotic lymphocytes when compared to non-apoptotic cells (p < 0.0001). Astrocytes removed only up to one third of the number of apoptotic lymphocytes ingested by microglia cells (p < 0.0001). Microglia cells significantly increased their phagocytosis rate after IFN-gamma stimulation and decreased it in response to IL-4. In contrast, astrocyte phagocytosis was almost unresponsive to cytokine stimulation. After interaction with apoptotic cells, microglia secreted significantly less TNF-alpha. Astrocytic TNF-alpha production was also decreased but not to a statistically significant extent. MHC-class II expression after phagocytosis was increased on microglia cells but not on astrocytes. Both microglia cells and astrocytes are capable of ingesting apoptotic cells, but microglia cells are much more efficient phagocytes. Their phagocytic capacity is modulated by the local microenvironment and microglial immune function is downregulated after phagocytosis. We suggest that in vivo astrocytes might be activated as phagocytes once the limit of microglial phagocytic capacity has been reached.

Animals↗

PubMed1993

Development of second generation monoclonal antibodies recognising Lewisy/b antigen by anti-idiotypic immunisation.

Five new monoclonal antibodies (Mabs) recognising the Lewisy/b hapten of the IgG isotype have been produced following immunisation with rat anti-idiotypic antiserum to C14, an IgM Lewisy/b hapten Mab and boosting with C14gp200 antigen. They have the same fine specificity as the original Mab binding to a cell surface and secreted antigen preferentially expressed by colorectal tumour cells.

Animals↗

PubMed2000

Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis.

In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world. In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guérin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria. In this study, we have cloned and overexpressed three immunodominant secretory antigens of M. tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex. All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength. The expression was analysed in the fast-growing strain M. smegmatis and the slow-growing vaccine strain M. bovis BCG. The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium. These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.

Antigens, Bacterial↗

PubMed2001

Molecular basis of the adult i phenotype and the gene responsible for the expression of the human blood group I antigen.

The human blood group i and I antigens are characterized as linear and branched repeats of N-acetyllactosamine, respectively. Conversion of the i to the I structure requires the activity of I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT). Thus the blood group I gene is assigned to encode a beta-1,6-N-acetylglucosaminyltransferase; however, its identity has not been confirmed. The null phenotype of I, the adult i phenotype, provides a means to identify the I gene. Interestingly, the adult i phenotype has been noted to be associated with congenital cataracts in Asians. Molecular genetic studies of 3 adult i pedigrees are reported here. The results obtained on mutation detection within the 2 I-branching enzyme encoding genes, segregation analyses, and enzyme function assays identify molecular changes associated with the adult i phenotype. The adult i phenotype in 2 of the pedigrees studied resulted from 1043G-->A and 1148G-->A mutations, which predict Gly348Glu and Arg383His alterations, respectively, in the IGnT gene. These amino acid changes abolished the original GlcNAc-transferase activity. Deletion of the IGnT gene was observed in the person with adult i phenotype in the third pedigree. These findings suggest that the IGnT gene, first reported in 1993, is the candidate for the blood group I gene. Confirmation of the blood group I gene will further assist in the investigations of the molecular genetics that control I antigen expression in secretions and the molecular basis for the association of the adult i phenotype with congenital cataracts in Asians.

Adult↗

PubMedDate not supplied

Early glycated albumin, but not advanced glycated albumin, methylglyoxal, or 3-deoxyglucosone increases the expression of PAI-1 in human peritoneal mesothelial cells.

OBJECTIVE: The continuous contact of glucose-containing peritoneal dialysis (PD) fluids with the peritoneum results in the intraperitoneal formation of early and advanced glycation end-products. This nonenzymatic glycation of proteins may cause morphological and functional alterations to the peritoneum, which may contribute to patient dropout from PD therapy. Because fibrinolytic system components have been demonstrated to play an important role in the balance of intraperitoneal generation and degradation of fibrin, we studied the effect of early and advanced glycated human serum albumin, methylglyoxal, and 3-deoxyglucosone on the synthesis of tissue-type plasminogen activator (tPA), as well as its specific inhibitor (PAI-1), in human peritoneal mesothelial cells (HPMC). METHODS: Antigen concentrations in the supernatants of cultured HPMC were measured by ELISA. Northern blot analysis was conducted for mRNA expression. Electrophoretic mobility shift assays were applied to demonstrate the involvement of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in signal transduction. RESULTS: Incubation of HPMC with early glycated albumin (GHSA) resulted in a time- and concentration-dependent increase in PAI-1 mRNA expression and antigen secretion. In contrast, no changes in PAI-1 synthesis occurred after stimulation with either the 1,2-dicarbonyl compounds methylglyoxal and 3-deoxyglucosone, or with late advanced glycation end-products. tPA synthesis was not affected by any of the tested components. Furthermore, HPMC exposed to GHSA induced NF-kappaB and AP-1 DNA binding activity, suggesting that GHSA-induced overexpression of PAI-1 is transcriptionally regulated by both transcription factors. CONCLUSIONS: We conclude that Amadori modified glycated albumin upregulates PAI-1 synthesis in HPMC, possibly mediated through the activation of the transcription factors NF-kappaB and AP-1. The present data support the clinical relevance of the formation of glycated proteins and their involvement in pathological processes in PD patients. Thus, glycated albumin may contribute to an imbalance between intraperitoneal formation and degradation of fibrin that causes peritoneal structural alterations, with subsequent membrane failure.

Albumins↗

PubMed1997

The versatility of Helicobacter pylori in the adaptation to the human stomach.

A growing body of data indicates that H. pylori colonization of human is ancient, which is consistent with its high prevalence, chronicity of carriage, and generally low level of disease, which, when it occurs has only marginal or no effects on host reproductive capacity. All of these phenomena are markers for a relatively benign co-existence, which may include all of the entire spectrum of interactions from parasitism, through commensalism, to symbiosis. Recent studies suggest the emergence of "quasispecies" during prolonged colonization, and the presence of multiple strains colonizing individual hosts. Such observations suggest that concepts of competition between strains and mutualism will be important in understanding the ecology of colonization and its effects on hosts. The presence of particular pathologies in the host may in part be a function of the characteristics of the bacterial population present. At a genomic level, H. pylori appears to adapt to changing conditions by point mutation, genomic rearrangement, and horizontal gene transfer, the latter is favored by its natural competence. The ability of H. pylori to alter phenotypic properties including superficial Lewis antigen expression and secretion of proinflammatory molecules is evidence of its sensitivity to environmental signals from the host. In such a universe, disease outcomes such as ulceration or neoplasia may be considered as accidents secondary to microbial persistence.

Biological Evolution↗

PubMed2002

Induction of MUC1-specific cellular immunity by a recombinant BCG expressing human MUC1 and secreting IL2.

MUC1 mucin is aberrantly expressed in many epithelial malignancies and is a promising tumor antigen for target-directed immunotherapy against human breast cancer. Mycobacterium BCG is an effective immunoadjuvant which is known to induce Th1 immune response. Recombinant BCG expressing tumor antigen and secreting cytokine may therefore potentiate the tumor antigen-specific immune responses. In this study, we constructed a recombinant BCG-MUC1-IL2, which expresses a high level of human MUC1 VNTR core protein and secretes functional interleukin 2 (IL2). The immune responses induced by BCG-MUC1-IL2 were examined using a SCID mouse model reconstituted with immunologically competent human lymphocytes, SCID/hu-PBL. The mucin-specific IFN-gamma was secreted only by the lymphocytes derived from animals immunized with BCG-MUC1-IL2, but not with BCG-vector or purified mucin protein for the vaccination. In contrast, in vitro secretion of IL4 by the immunized lymphocytes was only seen in the group of animals which received native MUC1 protein, but not BCG-MUC1-IL2 and BCG-vector. Minimal MUC1-specific IgG and IgM were detected in SCID/hu-PBL mice vaccinated with BCG-MUC1-IL2. These results suggest that BCG-MUC1-IL2 preferentially induces MUC1-specific cellular immune responses and it may serve as a vaccine for breast cancer prevention and treatment.

Animals↗

PubMed1992

Immunity to B16 melanoma in mice immunized with IL-2-secreting allogeneic mouse fibroblasts expressing melanoma-associated antigens.

Co-presentation of weak tumour-associated antigens along with strongly immunogenic determinants leads to the development of an anti-tumour immune response in recipients syngeneic with the tumour. Tumour immunity develops in mice immunized with tumour cells modified by the introduction of cDNA for interleukin-2 (IL-2). Here, we report the anti-tumour response following immunization with an IL-2-secreting cell construct that expresses tumour-associated antigens, along with allogeneic major histocompatibility antigens. The construct was prepared by transfecting LM(TK-) mouse fibroblasts (H-2k) with genomic DNA from B16 melanoma cells syngeneic in C57BL/6J mice (H-2b). Transfectants expressing melanoma-associated antigens (MAA) were then infected with an expression-competent retroviral vector containing a cDNA specifying human IL-2. Cytotoxicity toward B16 cells was detected for as long as 5 months in both spleen and macrophage cell populations in C57BL/6J mice immunized with the IL-2-secreting cells. Mice immunized with non-IL-2-secreting, MAA-positive allogeneic cells developed melanoma immunity as well, but to a lesser extent. Immunity to 2 tumour-cell lines expressing the H-2d haplotype and to YAC-1 cells was detected in peritoneal macrophages, but not in spleen cells from C57BL/6J mice immunized with the cell construct, indicating that the response to B16 cells was only partially specific. C57BL/6J mice immunized with the IL-2-secreting cell construct survived significantly longer, following an injection of viable B16 cells, than mice in various control groups. The contribution of allogeneic antigens to the melanoma immunity was indicated by the failure of mice syngeneic with LM(TK-) cells to develop melanoma immunity following immunization with non-IL-2-secreting, MAA-positive cell constructs. The formation of IL-2 partially compensated for the lack of allogeneic antigens.

Animals↗