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Characterisation of killer immunoglobulin-like receptors in rhesus macaques (Macaca mulatta)

Charakterisierung von killer immunoglobulin-like receptors von Rhesus Affen (Macaca mulatta)

Meike Hermes
Doctoral thesis
Date of Examination: 2012-07-13
Date of issue: 2012-10-22
Advisor: Walter, Lutz Prof. Dr.
Referee: Walter, Lutz Prof. Dr.
Referee: Lübke, Torben Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-000D-F0C1-A

 

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Abstract

English

Rhesus macaques are important animal models of human diseases in which NK cells play significant roles. Whereas data on KIR genetic variability were recently published, data on KIR protein expression was not available until now due to lack of specific (or cross‐reactive) antibodies. Therefore, mouse monoclonal antibodies against one activating and two inhibitory rhesus macaque KIR3D molecules were established and characterised. Specificity of the obtained antibodies was determined using various rhesus macaque KIR‐Fc fusion proteins (ELISA), cells transfected with single rhesus macaque KIR genes as well as lymphocytes of KIR‐typed rhesus macaques (flow cytometry). Besides broadly reacting ones, also antibodies with intermediate and with high specificity for single KIRs were obtained. Epitope mapping revealed a conformational epitope for all analysed antibodies. The antibodies were conjugated with suitable dyes and multicolour flow cytometry was performed with lymphocytes from different rhesus macaque individuals. The analysis revealed a clonal expression pattern of rhesus macaque KIR that is similar to human KIR. Differences were seen between individuals: 29‐78 % of NK cells were positive with a pan‐KIR antibody, whereas 2‐56 % of NK cells were positive with antibodies specific for single KIRs. For T cell subpopulations 12‐27 % of all CD8+ αβ T cells and 6‐58 % of all γδ T cells reacted specifically with the pan‐specific KIR antibody. Also T cells expressed KIR at a clonal expression pattern using antibodies specific for single KIRs. Similar results were obtained with lymphocytes from cynomolgus macaques, baboons and African green monkeys. Analysis of blood samples from SIV‐infected rhesus macaques identified changes in the number of KIR‐expressing cells during the acute phase of infection. KIR‐expressing NK cells were decreased in animals with low viral load and in elite controllers, whereas for γδ T cells an increase could be detected. In conclusion, the established monoclonal antibodies are important tools for future studies in which the role of NK cells in infectious and autoimmune diseases are studied in macaques or other Old World monkeys.
Keywords: killer cell immunoglobulin like receptors (KIR); rhesus macaque (Macaca mulatta); NK cell

Other Languages

Rhesusaffen stellen ein wichtiges Tiermodell für humane NK-Zell-assoziierte Krankheiten dar. Die genetische Variabilität von KIR ist bereits beschrieben, allerdings fehlen bisher jegliche Informationen über die Proteinexpression aufgrund fehlender Antikörper. Daher wurden im Rahmen dieser Arbeit monoklonale anti-Rhesusaffen KIR Antikörper etabliert und charakterisiert. Mit Hilfe verschiedener Methoden (ELISA, Durchflusszytometrie, Immunoblot) wurde die Spezifität verschiedener Antikörper bestimmt. Neben Antikörpern mit einer breiten Reaktivität für verschiedene KIR, konnten zudem Antikörper mit einer mittleren und hohen Spezifität für einzelne KIR Moleküle etabliert werden. Eine Epitopkartierung zeigte ein konformationales Epitop für alle analysierten Antikörper. Zur Durchführung von Vielfarben-Durchflusszytometrie wurden die Antikörper mit einem Fluoreszenzfarbstoff markiert und Lymphozyten verschiedener Rhesusaffen auf ihre KIR Expression untersucht. Es konnte ein klonales KIR-Expressionsmuster, ähnlich dem humaner Lymphozyten (NK und T Zellen), gezeigt werden. Zudem unterschieden sich verschiedene Individuen in der Anzahl KIR-positiver Zellen: 29-78 % aller NK Zellen waren KIR positiv unter der Verwendung eines breit reagierenden Antikörpers, 2-56 % aller NK Zellen unter Verwendung eines hoch spezifischen Antikörpers. 12-27 % aller CD8+ αβ T Zellen und 6-58 % aller γδ T Zellen zeigten KIR-Expression.
Schlagwörter: Killer cell immunoglobulin like Rezeptoren (KIR); Rhesusaffe (Macaca mulatta); NK Zelle
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