9 research outputs found
Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis
Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8(+ )T cells in HLA-B27(+)-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8(+ )T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8(+ )T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27(+ )Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8(+ )T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8(+ )T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8(+ )T cells could be increased by stimulating CD8(+ )T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8(+ )T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27(+ )patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional
New insights into the immunopathology of spondylarthropathies
Die ankylosierende Spondylitis (AS) und reaktive Arthritis (ReA) sind eng mit
dem humanen Leukozytenantigen (HLA) B27 assoziiert. Im Rahmen dieser Arbeit
durchgeführte histologische Untersuchungen zeigten an Femurköpfen von AS
Patienten Ansammlungen von T Zellen, auch CD8+ T Zellen, die das Knorpelgewebe
vom subchondralen Knochenmark aus infiltrierten. Das Ausmaß dieser T Zell-
Infiltrationen korrelierte mit dem Vorhandensein von Knorpel auf der
Gelenkoberfläche. Eine erhöhte Dichte von Blutgefäßen in Bereichen mit akuter
Entzündung am angrenzenden Knorpel deutete auf die Möglichkeit einer
erleichterten Einwanderung von T Zellen. Diese Beobachtungen warfen die Frage
auf, auf welchen Ebenen der Beitrag von CD8+ T Zellen auf lokale Entzündungen
bei AS reguliert wird. Besonderes Augenmerk galt den über HLA B27 vermittelten
Antigen-spezifischen Mechanismen. Zunächst wurden Peptide aus Proteinen von
Chlamydia trachomatis in Patienten mit Chlamydia-getriggerter ReA und AS,
sowie Peptide aus Proteinen der extrazellulären Knorpelmatrix bei AS Patienten
gesucht, die T Zellen HLA B27-restringiert stimulieren. Das wurde
durchflusszytometrisch in CD8+ T Zellen über die Peptid-abhängige Induktion
der Produktion von Interferon (IFN)γ sowie über den Nachweis spezifischer T
Zellrezeptoren mittels selbst hergestellter HLA B27/Peptid-Tetramere geprüft.
Chlamydia Peptid-spezifische CD8+ T Zellen waren nur in geringen Frequenzen in
der Synovialflüssigkeit und nicht im peripheren Blut von ReA- und AS Patienten
nachweisbar. Vier im Modell mit HLA B27-transgenen Mäusen identifizierte
immundominante Aggrecanpeptide bestätigten Aggrecan als mögliches Autoantigen
und aus dem Screening weiterer Knorpelproteine ergaben sich 8/97 Peptide, die
HLA B27-restringiert CD8+ T Zellen aktivierten. Um diese systemischen
Beobachtungen im zellulären Kontext des lokalen Entzündungsgeschehens weiter
beurteilen zu können, wurden Methoden zum Nachweis Peptid-spezifischer CD8+ T
Zellen in situ etabliert. Die Befunde, dass artikuläre Chondrozyten in situ
konstitutiv kostimulatorisches CD80 exprimieren und dass proinflammatorisches
IFNγ die Expression von HLA B27 induzierte, führte zu der Hypothese, dass
Chondrozyten in einer Entzündung im Sinne nicht-professioneller Antigen-
präsentierender Zellen lokale CD8+ T Zell-spezifische Immunantworten
induzieren und/oder aufrechterhalten können. In einem Modellsystem mit
EBNA258-266, einem HLA B27-restringierten immundominanten Peptid aus dem
Epstein Barr Virus, wurden Interaktionen von autologen artikulären
Chondrozyten mit CD8+ T Zellen in vitro untersucht. In Gegenwart HLA B27+
EBNA258-266 beladener Chondrozyten produzierten HLA
B27/EBNA258-266-spezifische CD8+ T Zellen IFN. Die konfokale Laserscanning-
Mikroskopie zeigte bei direktem Chondrozyt/T Zell-Kontakt eine Anreicherung
von zytolytischem Perforin und Granzym B in den CD8+ T Zellen sowie durch sie
lysierte benachbarte Chondrozyten. Die Aktivierung zytotoxischer CD8+ T Zellen
in Gegenwart eines immunogenen HLA B27-restringierten Peptids legt nahe, dass
Chondrozyten im Gelenkknorpel über eine entzündungsabhängige
Antigenpräsentation an der Aufrechterhaltung der Entzündung bei AS beteiligt
sein könnten. Damit liefern die im Rahmen dieser Arbeit gezeigten Antigen-
spezifischen Mechanismen Anhaltspunkte, wie die knorpelabhängige Sukzession
der Infiltration von T Zellen in Knochenendplatte und Knorpelgewebe zum
Verlauf der Hüftgelenksarthritis von AS Patienten beitragen könnte.Ankylosing spondylitis (AS) and reactive arthritis (ReA) are strongly
associated with the class I human leukocyte antigen (HLA) B27. Histopathology
of hip joints from AS patients as done for this study revealed an accumulation
of infiltrating mononuclear cells, including CD8+ T cells, within the bone
endplate and the hyaline articular cartilage. The extent of T cell aggregates
was significantly reduced at bone sites devoid of cartilage. Enhanced
microvessel densities in acute inflammation at the bone–cartilage interface
pointed to a facilitated access of immunocompetent cells to the cartilage.
These observations raised the question on the levels of involvement of CD8+ T
cells to local inflmmations in AS. This work mainly focussed on antigen-
specific mechanisms mediated by HLA B27. As for the role of exo- and
endogeneous antigens, HLA–B27–restricted CD8+ T cell responses were
investigated using peptides derived from proteins of Chlamydia trachomatis and
of the extracellular matrix from human cartilage and fibrocartilage.
Phenotypic and functional analysis of CD8+ T cells was performed by flow
cytometry of suspended cells and in situ by immunohistochemistry and
immunofluorescence. Asking for frequencies of antigen-specific CD8+ T cells,
cytokine expression was studied and self manufactured HLA B27 tetramers were
used. Chlamydia-specific CD8+ T cells were found at low frequencies in the
synovial fluid and not in the peripheral blood of ReA and AS patients. Four
HLA B27-restricted immunodominant peptides derived from human aggrecan
recently described in BALB/c-B27 transgenic mice confirmed aggrecan as an
auto-antigen in AS. Screening of additional human cartilage proteins
identified 8/97 peptides to stimulate HLA B27-restricted CD8+ T cells. To
study the cellular context during hip joint inflammation, methods for the
detection of peptide-specific CD8+ T cells in situ were established. Articular
chondrocytes prepared from the femoral head joint cartilage constitutively
expressed costimulatory CD80 and upregulated HLA B27 expression after
incubation with proinflammatory interferon (IFN) γ. This raised the question
whether chondrocytes have the potential to directly trigger local CD8+ T cell
dependent inflammation as non-professional antigen-presenting cells. Human T
cell lines from an HLA B27+ AS patient and autologous chondrocytes were
available. These cells were used in a model system with an Epstein Barr Virus-
derived peptide EBNA258 266 to study peptide specificity and functionality of
cytotoxic CD8+ T cells and to assess cellular interactions of T cells and
chondrocytes by fluorescence confocal microscopy. Pulsed with the EBNA258 266
peptide, autologous chondrocytes specifically induced IFNγ production in CD8+
T cells. In mixed chondrocyte-T cell cultures cell-cell contacts were strictly
EBNA258 266 peptide-dependent. T cells adjacent to chondrocytes produced
perforin and granzyme B. Both molecules were found in focal aggregates, a
prerequisite for an antigen-specific lysis of target cells. In summary, the
finding that human articular chondrocytes attract and stimulate antigen-
specific cytotoxic CD8+ T cells in an HLA B27-restricted manner suggests that
chondrocytes might contribute to maintenance of inflammation in AS. Antigenes
might derive from exo- or endogenous sources. This antigen-presenting capacity
of human articular chondrocytes might explain a facilitated invasion of
antigen-specific CD8+ T cell into the bone end-plate and cartilage observed in
hip arthritis of AS patients
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is published twice each month byThe Journal of Immunolog
Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies
The
conventional hybridoma screening and subcloning process is
generally considered to be one of the most critical steps in hapten-specific
antibody production. It is time-consuming, monoclonality is not guaranteed,
and the number of clones that can be screened is limited. Our approach
employs a novel hapten-specific labeling technique of hybridoma cells.
This allows for fluorescence-activated cell sorting (FACS) and single-cell
deposition and thereby eliminates the above-mentioned problems. A
two-step staining approach is used to detect antigen specificity and
antibody expression: in order to detect antigen specificity, hybridoma
cells are incubated with a hapten–horseradish peroxidase conjugate
(hapten–HRP), which is subsequently incubated with a fluorophore-labeled
polyclonal anti-peroxidase antibody (anti-HRP–Alexa Fluor 488).
To characterize the expression of membrane-bound immunoglobulin G
(IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG–Alexa
Fluor 647) is used. Hundreds of labeled hybridoma cells producing
monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated
and deposited from a fusion mixture as single-cell clones via FACS.
Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants
of the sorted hybridoma clones revealed that all hapten-specific hybridoma
clones secrete antibodies against the target. There are significant
improvements using this high-throughput technique for the generation
of mAbs including increased yield of antibody-producing hybridoma
clones, ensured monoclonality of sorted cells, and reduced development
times