Abstract:
BACKGROUND:Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS:DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS:The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION:Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Nguyen XD,Eichler H,Dugrillon A,Piechaczek C,Braun M,Klüter Hdoi
10.1016/s0022-1759(03)00002-4keywords:
subject
Has Abstractpub_date
2003-04-01 00:00:00pages
57-68issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175903000024journal_volume
275pub_type
杂志文章abstract::Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purp...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2010.09.001
更新日期:2011-01-05 00:00:00
abstract::The T cell receptor (TCR) determines the cellular response to antigens, which are presented on the surface of target cells in the form of a peptide bound to a product of the major histocompatibility complex (pepMHC). The response of the T cell depends on the affinity of the TCR for the pepMHC, yet many TCRs have been ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2008.09.016
更新日期:2008-12-31 00:00:00
abstract::Investigation of MHC class I endogenous antigen processing and presentation is important for understanding non-self immune recognition, vaccine design, immune evasion by pathogens and tumours, and elucidation of the mechanisms of certain auto-immune diseases. To study the MHC class I antigen-processing pathway, the pr...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2008.02.017
更新日期:2008-06-01 00:00:00
abstract::We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2004.10.006
更新日期:2004-12-01 00:00:00
abstract::Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monocl...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2007.05.001
更新日期:2007-07-31 00:00:00
abstract::A sensitive procedure based on the use of iodinated antibody allows the detection of antigenic material by double-diffusion analysis. An autoradiographed immunodiffusion pattern reveals distinct radio-labeled precipitation lines for an extract of yeast with wild type HIS4 protein and for extracts of mutants containin...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90048-0
更新日期:1978-01-01 00:00:00
abstract::One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(96)00074-9
更新日期:1996-08-14 00:00:00
abstract::E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2004.07.004
更新日期:2004-10-01 00:00:00
abstract::A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specific...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90070-v
更新日期:1991-04-08 00:00:00
abstract::The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90067-1
更新日期:1988-06-28 00:00:00
abstract::The ability to isolate antibodies against any antigen of interest has become increasingly important as antibodies have proved their utility both in antigen detection, quantification and as specific in vivo targeting agents. To this end, we have constructed a large antibody phage library in the single chain Fv (scFv) p...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2005.01.014
更新日期:2005-04-01 00:00:00
abstract::An Ia-rosette assay for the detection of Ia-like alloantigens on the chicken lymphoid cell surface is described. The method is based on the ability of cells treated with alloantiserum to Ia and then with rabbit antiserum against the Fc portion of chicken IgG, to form rosettes with sheep erythrocytes (SRBC) coated with...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(81)90161-7
更新日期:1981-01-01 00:00:00
abstract::The circumsporozoite (CS) protein is the major surface protein of Plasmodium sporozoites. Antibodies to the immunodominant repeat domain of CS immobilize sporozoites and prevent infection of hepatocytes. Plasmodium falciparum vaccines containing CS repeats are undergoing human trials in endemic areas, and proof of eff...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2004.06.017
更新日期:2004-09-01 00:00:00
abstract::A modification of the COS cell transient expression system for isolating cDNAs encoding antibody defined molecules was used to clone the carcinoma antigen defined by monoclonal antibody CORC-6. Bacteria were transformed with cDNA prepared from colon carcinoma cells and immediately divided into aliquots containing appr...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00155-4
更新日期:1995-10-26 00:00:00
abstract::We examined two ELISA methods for measuring antibodies to Streptococcus pneumoniae using tyraminated S. pneumoniae polysaccharide types 3, 7N, 9F and 14 as antigens. The ELISA has the usual format with a relatively long incubation time whereas the FAST-ELISA has a short incubation time and employs a different solid-ph...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90238-x
更新日期:1989-06-21 00:00:00
abstract::This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates down...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00103-h
更新日期:1995-09-11 00:00:00
abstract::Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assist...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2006.05.008
更新日期:2006-07-31 00:00:00
abstract::To date, in vivo apoptosis within the thymus has been assessed using morphological criteria and/or detection of a DNA ladder indicative of oligonucleosomal fragmentation of the DNA. Here, we have used a fluorometric method to investigate activation of the caspase protease family in the thymus following in vivo inducti...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00054-x
更新日期:1999-06-24 00:00:00
abstract::Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or c...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(01)00353-2
更新日期:2001-06-01 00:00:00
abstract::We have used a surface plasmon resonance biosensor (BIACORE 3000) to detect serum antibodies in chickens having current or recent infections. Three well-defined Salmonella flagellar recombinant DNA antigens reflecting Salmonella enteritidis (H:g,m flagellin) and Salmonella typhimurium (H:i and H:1,2 flagellins) expres...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(02)00102-3
更新日期:2002-08-01 00:00:00
abstract::A polymorphism at position 559 in the cDNA of the FCGR3A gene encoding the FcgammaRIIIa generates two allotypes with either a valine (Val) or a phenylalanine (Phe) at amino acid position 158. This polymorphism is of major importance in immunopathology and in pharmacogenetics, especially monoclonal antibody treatments....
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(03)00123-6
更新日期:2003-06-01 00:00:00
abstract::A lectin isolated from the tropical jackfruit, jacalin, previously reported to precipitate human immunoglobulin A (IgA), and conjugated to agarose was used to separate the two subclasses of IgA from secretions. Jacalin-agarose binds specifically to the D-galactose moiety of IgA1 but not to IgA2 which has a different c...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90037-8
更新日期:1987-05-04 00:00:00
abstract::Hybridoma cells producing antibody reactive with poly(A) were isolated following fusion of spleen cells from a BALB/c mouse immunized with poly(A).poly(dT) and myeloma cells. The antibody was purified from ascitic fluid by the formation of immune complexes with poly(A). The antibody reacted with poly(A) and some homop...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90269-d
更新日期:1993-08-26 00:00:00
abstract::A new method is described for demonstrating the individual subclasses of human IgG immunoglobulins by means of analytical isotachophoresis. Individual subclasses of IgG purified from sera of multiple myeloma patients have distinct mobility patterns. When a mixture with purified IgG for each of the 4 subclasses is anal...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(79)90163-7
更新日期:1979-01-01 00:00:00
abstract::The identification of distinct T helper lymphocyte subsets (Th1/2) with polarised cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high ...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/s0022-1759(00)00230-1
更新日期:2000-09-21 00:00:00
abstract::The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsial pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2007.12.018
更新日期:2008-03-20 00:00:00
abstract::Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.02.001
更新日期:2016-04-01 00:00:00
abstract::Biological matrix interference in detection and quantitation immunoassays remains a major challenge in the field of bioanalysis. For example, circulating drug may interfere with the detection of anti-drug antibodies (ADA) and drug target, or ADA may interfere with quantitation of drug levels in PK/TK analysis. Monoclo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2015.08.002
更新日期:2015-11-01 00:00:00
abstract::Using human heart-type fatty acid-binding protein (H-FABP) as an early cardiac marker to confirm or exclude a diagnosis of acute myocardial infarction (AMI) soon after admission requires a rapid assay system. Due to the requirement of skillful technicians and complicated assay procedures, most immunochemical assays fo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(03)00243-6
更新日期:2003-08-01 00:00:00
abstract::The identification of naturally processed peptides presented by molecules of the major histocompatibility complex (MHC) has progressed significantly over the past decade. The elution of peptides from immunoaffinity purified complexes of MHC class I or class II molecules has provided highly specific biochemical informa...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/s0022-1759(00)00361-6
更新日期:2001-03-01 00:00:00