Human antigen-presenting cell/tumour cell hybrids stimulate strong allogeneic responses and present tumour-associated antigens to cytotoxic T cells in vitro.
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Most tumours do not stimulate effective antitumour immune responses in vivo. In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein-Barr virus transformed B-lymphoblastoid cell line (EBV B-LCL) in vitro, to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T-cell responses in vitro, as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+) as well as memory (CD45RO+) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T-cell co-stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T-cell responses. Fusion of the EBVB-LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma-associated antigens MAGE-1 and MAGE-3, and presented these antigens to antigen-specific, HLA class I-restricted cytotoxic T-lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen-presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy. (+info)
Differential effects of cytokines and immunosuppressive drugs on CD40, B7-1, and B7-2 expression on purified epidermal Langerhans cells1.
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Langerhans cells are MHC class II antigen-positive antigen-presenting cells in the epidermis. Recent studies have revealed that Langerhans cells express costimulatory molecules like B7-1 and B7-2 and the accessory molecule CD40. Although these molecules are important for the antigen-presenting function of Langerhans cells, little is known about the precise regulation of their expression on purified Langerhans cells. Using a panning technique, we purified epidermal Langerhans cells to around 95% purity. Freshly prepared Langerhans cells (fLC) expressed the mRNA for receptors for M-CSF (cfms), GM-CSF (GM-CSFR), and TNF-alpha (TNFRII). TNF-alpha markedly upregulated CD40 and B7-1 expression on Langerhans cells, but not B7-2 expression. GM-CSF moderately upregulated B7-1 and B7-2 expression, and slightly upregulated CD40 expression. M-CSF moderately upregulated B7-1 expression, but did not modulate CD40 or B7-2 expression. Dexamethasone (DEX) markedly inhibited CD40, B7-1, and B7-2 expression on Langerhans cells. Cyclosporin A (CsA) and FK506 slightly inhibited CD40 and B7-1 expression on Langerhans cells, but not B7-2. Furthermore, TNF-alpha restored the DEX-induced inhibition of CD40 expression on Langerhans cells, but not the inhibition of B7-1 or B7-2 expression. GM-CSF restored DEX-induced inhibition of CD40, B7-1, and B7-2 expression. M-CSF did not affect the DEX-induced inhibition of these molecule expressions. These data provide a better understanding of the role of selective cytokines and immunosupressive drugs in the modulation of the antigen-presenting capacity of Langerhans cells. (+info)
Heterogeneous reactivity of murine epidermal Langerhans cells after application of FITC: a histochemical evaluation.
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To elucidate the detailed kinetics of epidermal Langerhans cells after topical contact sensitizer stimulation, we examined ATPase or Ia positive epidermal cells of BALB/c mice in a time-spaced manner after the topical application of fluorescein isothiocyanate (FITC). We also performed double labeling of Langerhans cells in epidermal sheets with ATPase activity and Ia antigen or costimulatory molecules (B7-1 and B7-2) after the same stimulation. Observations showed that the density of ATPase positive cells and Ia positive cells decreased following a different time course; the former reached a nadir (77.4% of control) at 4 h but the latter reached a minimum (82.8% of control) at 16 h after the application of FITC. A double labeling technique revealed an increase in Ia single positive cells at 4 h as opposed to that of ATPase single positive cells at 16 h after application. Both costimulatory molecules were expressed on the dendritic processes of many Langerhans cells as a dotty pattern at 4 h after application; B7 positive and ATPase negative areas were observed at this time. On electron microscopic observation, a few activated Langerhans cells found in the dermis at 4 h after application had distinctive profiles compared with residual Langerhans cells in the epidermis. These findings suggest that there is a heterogeneity of reactivity to FITC in epidermal Langerhans cells, and that only a small portion of them migrates from the epidermis during sensitization. The findings also indicate the importance of the interaction between the Langerhans cell and its surrounding microenvironment in the epidermis for its activation. In addition, the results indicate that the enzymatic and the phenotypic markers do not definitively reflect the presence (or absence) of Langerhans cells. (+info)
This study determined the effects of exercise on the ability of macrophages (Mphi) to present antigen to T cells. Pathogen-free male Balb/c mice (8 +/- 2 wk of age) were randomly assigned to either home cage control, moderate exercise (Mod; 18 m/min, 5% grade, 0.5 h/day), exhaustive exercise (Exh, 18-30 m/min, 3 h/day), or treadmill control groups. The mice underwent treatments for 4 days during peritoneal thioglycolate inflammation. Peritoneal Mphi were harvested, purified, and incubated with chicken ovalbumin (C-OVA; 0-10 mg/ml) for 18 h. Mphi were then cocultured with C-OVA-specific T cells for 48 h, and the supernatants were analyzed via ELISA for interleukin-2 as an indication of Mphi antigen presentation (AP). Exh exhibited suppressed ( approximately 25-34%) Mphi AP across a wide range of C-OVA doses when measured immediately, 3, and 24 h postexercise. In contrast, Mod had reduced Mphi AP only at 3 h postexercise. Mphi AP was also lower in the treadmill control (4-27%) compared with the home cage control group, but was significantly higher than Exh. The reduction in Mphi AP was not due to exercise-induced differences in Mphi number, percentage, or expression of intercellular adhesion molecule-1, B7-2, or major histocompatability complex II, molecules important in AP. In conclusion, our data lend evidence that may help explain the increased incidence of infection observed after prolonged exhaustive exercise or overtraining. (+info)
Differential requirement for CD80 and CD80/CD86-dependent costimulation in the lung immune response to an influenza virus infection.
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The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-gamma production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-gamma by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86. (+info)
A critical role for B7/CD28 costimulation in experimental autoimmune encephalomyelitis: a comparative study using costimulatory molecule-deficient mice and monoclonal antibody blockade.
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The B7/CD28 pathway provides critical costimulatory signals required for complete T cell activation and has served as a potential target for immunotherapeutic strategies designed to regulate autoimmune diseases. This study was designed to examine the roles of CD28 and its individual ligands, B7-1 and B7-2, in experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the CNS. EAE induction in CD28- or B7-deficient nonobese diabetic (NOD) mice was compared with the effects of B7/CD28 blockade using Abs in wild-type NOD mice. Disease severity was significantly reduced in CD28-deficient as well as anti-B7-1/B7-2-treated NOD mice. B7-2 appeared to play the more dominant role as there was a moderate decrease in disease incidence and severity in B7-2-deficient animals. EAE resistance was not due to the lack of effective priming of the myelin peptide-specific T cells in vivo. T cells isolated from CD28-deficient animals produced equivalent amounts of IFN-gamma and TNF-alpha in response to the immunogen, proteolipid protein 56-70. In fact, IFN-gamma and TNF-alpha production by Ag-specific T cells was enhanced in both the B7-1 and B7-2-deficient NOD mice. In contrast, peptide-specific delayed-type hypersensitivity responses in these animals were significantly decreased, suggesting a critical role for CD28 costimulation in in vivo trafficking and systemic immunity. Collectively, these results support a critical role for CD28 costimulation in EAE induction. (+info)
Expression of costimulatory molecules CD80 and CD86 and their receptors CD28, CTLA-4 on malignant ascites CD3+ tumour-infiltrating lymphocytes (TIL) from patients with ovarian and other types of peritoneal carcinomatosis.
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Costimulation of T lymphocytes by the leucocyte surface molecules CD80 and CD86 expressed on antigen-presenting cells (APC) is required for the development of T cell responses. The CD28 and CTLA-4 molecules on T cells serve as receptors for the CD80 and CD86 costimulatory antigens. We have examined the frequency of expression of CD80 (B7.1), CD86 (B7.2), CD28 and CTLA-4 surface antigens on TIL isolated from malignant ascites or peritoneal washings of 26 patients with ovarian carcinoma and five patients with non-ovarian peritoneal carcinomatosis. Expression of CD80 and CD86 antigen was detected by reverse transcription-polymerase chain reaction (RT-PCR), and by FACS analysis. Significantly higher proportions of intraperitoneal CD3+ cells expressed CD86 antigen than the CD80 antigen (14 +/- 9% versus 3 +/- 3%, P < 0.05). Moreover, CD3+CD86+ cells were significantly more frequent in the peritoneal fluid (14 +/- 9%) than in the peripheral blood (3 +/- 0.4%, P < 0.05) of ovarian patients or normal controls (3 +/- 1%). CTLA-4 and CD28 antigen were expressed, respectively, on 9 +/- 4% and 86 +/- 14% of ascitic CD3+ cells of ovarian cancer patients. Both CD80 and CD86 antigens were expressed primarily on HLA-DR+ ascites TIL and were present in a very low proportion of HLA-DR- ascites TIL. These HLA-DR+ cells may represent a population of lymphocytes that have been activated in vivo, and function as APC. An anti-CD86 MoAb or a combination of anti-CD86 and anti-CD80 MoAbs significantly inhibited the proliferation of cultured intraperitoneal TIL. We have shown that in addition to CD28 and CTLA-4, CD3+ intraperitoneal TIL express the costimulatory molecules CD80 and CD86. The expression of these molecules on T cells could be dependent upon certain factors in the tumour microenvironment that could determine the outcome of in vivo immune responses. (+info)
Human polymorphonuclear neutrophils express a B7-1-like molecule.
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Polymorphonuclear neutrophils (PMN) are part of the innate immune system and are first-line effector cells in acute inflammatory responses. On activation PMNs secrete cytokines and oxygen metabolites that might be involved in the regulation of the acquired immune response. We show here that peripheral blood PMNs constitutively express a B7-1-like molecule as detected by immunostaining with several B7-1 antibodies. Reverse transcriptase-polymerase chain reaction using three sets of primers spanning different regions of B7-1 indicate dissimilarities at the mRNA level. B7-1 mRNA is expressed in bone marrow cells and lipopolysaccharide (LPS)-stimulated but not in unstimulated PMNs. The B7-1-like molecule is localized to the cytoplasmic granules and translocated to the cell surface after stimulation with LPS or interleukin-12 in some donors. Binding of CTLA4-Ig suggests that the B7-1-like molecule can interact with functional B7 ligand and might be important in the immunobiology of PMNs. (+info)