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1.
The use of clonal mRNA as an antigenic format for the detection of antigen-specific T lymphocytes in IFN-gamma ELISPOT assays 总被引:2,自引:0,他引:2
Britten CM Meyer RG Frankenberg N Huber C Wölfel T 《Journal of immunological methods》2004,287(1-2):125-136
Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8(+) lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8(+) T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8(+) T-cell responses restricted by single HLA class I molecules. 相似文献
2.
CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines. 相似文献
3.
Z. Sun P. Zhu L. Li Z. Wan Z. Zhao R. Li 《European journal of clinical microbiology & infectious diseases》2012,31(11):3089-3096
Aspergillus fumigatus (A. fumigatus) is the most common pathogen of invasive aspergillosis (IA), a life-threatening infection in immunocompromised patients. Recent findings revealed that CD8+ T cells can mediate cytotoxic activity against A. fumigatus. Here, we bioinformatically identified three HLA-A*0201-restricted peptides from Asp f16, an A. fumigatus antigen which was previously shown to be involved in T cell immunity. Our immunological results demonstrated that these peptides can potently induce cytotoxic T lymphocyte (CTL) response in CD8+ T cells, thus, damaging the conidia and hyphae of A. fumigatus. Moreover, the Asp f16 peptides can also raise Th1 cell-like response, as measured by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT). Furthermore, we established an invasive pulmonary aspergillosis model in HLA-A*0201 transgenic mice. Adoptive transfer of Asp f16 peptides-specific CTL significantly extended the overall survival time in the A. fumigatus-infected immunocompromised mice. In conclusion, our results demonstrate that the Asp f16 peptides might provide immunity against invasive A. fumigatus infection. 相似文献
4.
Carralot JP Dumrese C Wessel R Riessen R Autenrieth I Walter S Schoor O Stevanovic S Rammensee HG Pascolo S 《International immunology》2005,17(5):591-597
Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8(+) T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunity. 相似文献
5.
Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism. 相似文献
6.
目的:构建增强型绿色荧光蛋白(EGFP)与HLA-A*0201(A*0201)融合蛋白哺乳动物细胞表达载体,分析其在K562细胞中的表达和亚细胞定位。方法:以RT-PCR方法克隆A*0201cDNA,构建A*0201-EGFP融合蛋白表达载体,转染K562细胞,以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果:从两个HLA-A2阳性供者外周血细胞中克隆到A*0201cDNA编码区全长序列。通过PCR方法在起始位点前加入Kozak序列并删除终止密码,成功构建A*0201-EGFP融合蛋白表达载体。以该质粒转染K562细胞,5h后A*0201和EGFP的表达百分率分别为25.12±2.26、27.37±3.59,24h后表达水平无明显提高。表达的融合蛋白主要分布于细胞膜上,胞内分布较少。相反,转染空载体的细胞不表达A*0201分子,仅表达EGFP,且其在细胞内呈弥散样分布。结论:成功构建A*0201-EGFP融合蛋白表达载体,并在K562细胞中得到表达,表达产物主要分布于细胞膜表面,提示表达该融合蛋白的K562细胞是潜在的人工抗原提呈细胞。 相似文献
7.
Christian Guelly Zaruhi Küpcü Doris Zalusky Margarete Karner Margit Zehetner Tamás Schweighoffer 《European journal of immunology》2002,32(1):182-192
We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines. 相似文献
8.
Jonathan W. Lewis Andrew Sewell David Price Tim Elliott 《European journal of immunology》1998,28(10):3214-3220
Tapasin is a 48-kDa endoplasmic reticulum (ER)-resident glycoprotein that binds to the transporter associated with antigen processing (TAP) and mediates an interaction between TAP and newly synthesized MHC class I molecules. It is also essential for the proper antigen presenting function of HLA-A*0101 (HLA-A1), HLA-A*0801 (HLA-B8) and HLA-B*4402 (HLA-B4402). We show here that while tapasin is required for HLA-A*0201 (HLA-A2) molecules to bind to TAP, its absence does not block the presentation of HLA-A2-restricted TAP-dependent epitopes to cytotoxic T lymphocytes indicating that, unlike HLA-A1, HLA-B8 and HLA-B4402, HLA-A2 has access to the TAP-dependent peptide pool even in the absence of tapasin. Nevertheless, the overall efficiency with which HLA-A2 was loaded with optimal, stabilizing peptides was impaired in the cell line .220, resulting in a significant increase in the fraction of HLA-A2 molecules being released from the ER in a “peptide-receptive” state. 相似文献
9.
10.
To study the relative roles of CD4(+)and CD8(+)T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4(+)and CD8(+)T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4(+)T cells (by anti-CD2 mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8(+)T cells (by anti-CD8 mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4(+)or CD8(+)T cells completely prevented IFN-gamma mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10 mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4(+)or CD8(+)T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-alpha) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-alpha mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4(+)and CD8(+)T cells is required for IFN-gamma and IL-10 production in islets and beta-cell destruction. Depletion of either CD4(+)or CD8(+)T cells may prevent beta-cell destruction by decreasing IFN-gamma and IL-10 production in islets and increasing IL-4 and TNF-alpha production systemically. 相似文献
11.
For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients. 相似文献
12.
Smith CM Wilson NS Waithman J Villadangos JA Carbone FR Heath WR Belz GT 《Nature immunology》2004,5(11):1143-1148
Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing. 相似文献
13.
Nam K Akari H Terao K Shibata H Kawamura S Yoshikawa Y 《International immunology》2000,12(7):1095-1103
We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys. 相似文献
14.
15.
S Ishihara M Nieda J Kitayama T Osada T Yabe Y Ishikawa H Nagawa T Muto T Juji 《European journal of immunology》1999,29(8):2406-2413
A unique subset of T cells that co-express NKR-P1, which is a lectin type of NK receptor and is thought to have a major role in triggering NK activity, has been identified. In mice, NK1.1 (mouse NKR-P1C)(+) T cells, called NKT cells, preferentially accumulate in the liver and bone marrow. They predominantly use invariant Valpha14 chain TCR and phenotypically are CD4(+)CD8(-) or CD4(-)CD8(-) T cells. In this study, we analyzed, phenotypically and functionally, the NKR-P1A (analogue of murine NKR-P1C)(+) T cells resident in the human liver. Here, we show that in complete contrast to the NKT cells in the mouse liver, the majority of NKR-P1A(+) T cells in the human liver are CD8(+) and their TCR repertoire is not skewed to Valpha24 TCR, the homologue of murine Valpha14 TCR. Almost all of the NKR-P1A(+) T cells in the human liver expressed CD69, suggesting that they were activated. Furthermore, the NKR-P1A(+) T cells in the human liver exhibited strong cytotoxicity against a variety of tumor cell lines including K562, Molt4 and some colonic adenocarcinoma cell lines. 相似文献
16.
Y M Mueller S C De Rosa J A Hutton J Witek M Roederer J D Altman P D Katsikis 《Immunity》2001,15(6):871-882
Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells. 相似文献
17.
Rosenplänter C Sommer F Kleemann P Belkovets A Schmidt A Lohoff M 《European journal of immunology》2007,37(7):1905-1915
Helicobacter pylori is a Gram-negative bacterium that causes a variety of gastrointestinal diseases, such as duodenal ulcer and gastric carcinoma. The T cell response against H. pylori is thought to contribute to the pathogenesis of these diseases. Here, we show that mouse-adapted H. pylori is able to polyclonally activate murine CD4(+) T lymphocytes, irrespective of their antigen specificity. Murine T helper cell clones as well as short-term cultured, polyclonal Th1 and Th2 cell lines and a human T cell clone, but not naive CD4(+) T cells, could be activated in this manner. The effect was independent of antigen-presenting cells and required direct contact between H. pylori and T cells. Only whole cells of H. pylori, but not lysates or sonicates were able to activate T cells. The activity was lost after long-term culture of H. pylori on agar-plates. Degradation of H. pylori proteins with specific peptidases dramatically reduced the stimulating ability, implicating that the responsible molecule is likely to be a protein. This unexpected polyclonal T cell stimulatory mechanism may contribute to the T cell-mediated pathogenicity characteristic for H. pylori-mediated diseases. 相似文献
18.
In mice Listeria monocytogenes infection induces a strong T cell response. In an attempt to quantitatively analyze the magnitude and kinetics of the CD4(+) and CD8(+) T cell response during L. monocytogenes infection in vivo we used a T cell transfer system that is independent of in vitro cell culture techniques and information about the identity of immunogenic T cell epitopes. Our results demonstrate substantial expansion of the in vivo primed and transferred T cell populations in response to L. monocytogenes. At the peak of response, transferred T cells represented more than one third of the total CD4(+) and CD8(+) T cell populations in blood and spleen of recipient mice. After stimulation in vitro, 40 % of these CD4(+) T cells responded to heat-killed listeriae with the production of IFN-gamma. Thus, our results reveal that in addition to the large CD8(+) T cell population an almost equally large population of Listeria-reactive CD4(+) T cells is generated in response to L. monocytogenes infection. 相似文献
19.
Kronin V Fitzmaurice CJ Caminschi I Shortman K Jackson DC Brown LE 《International immunology》2001,13(4):465-473
Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation. 相似文献
20.
Jun Kai Kao Yin Tzu Hsue Ching Yuang Lin 《Journal of microbiology, immunology, and infection》2007,40(5):419-427
BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a CD4(+)-dependent chronic systemic inflammatory disease with autoimmune features. Autoreactive CD4(+) T-cell activation can result in autoimmune diseases. One of the key regulators is the CD4(+)CD25(high) regulatory T (Treg) cell. In an animal arthritis model, CD11c(+)CD8(+) T cells were found to be elevated, and could suppress pathogenic CD4(+) T cells after cross-linking with CD137. The purpose of this study was to compare the expression of CD137, CD4(+)CD25(high) Treg cells, and CD11c(+)CD8(+) in the peripheral blood T lymphocytes of RA patients during active and remissive states, and evaluate the correlation with disease activity. METHODS: Thirty nine RA patients treated at the rheumatology outpatient clinic at the Changhua Christian Hospital were assessed clinically for disease activity and classified as either highly active or remissive by the Disease Activity Score 28. Peripheral blood mononuclear cells were isolated from these patients and compared against normal controls. RESULTS: The presence of CD11c(+)CD8(+) T cells or the expression of CD137 molecules in peripheral blood cells was not related to disease activity. In contrast, CD4(+)CD25(high) Treg cell levels were increased significantly in patients with active RA compared with patients with remissive RA or controls (p<0.05). These lymphocytes were intact, without evidence of apoptosis. CONCLUSIONS: Our results indicate that CD4(+)CD25(high) Treg cells play an important role in modulating RA disease activity and can serve as a parameter of disease activity. 相似文献