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

is online at: The Journal of ImmunologyInformation about subscribing to Permissions

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