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Selected Publication:

Type of publication: PhD Thesis
Type of document:

Year: 2010

Authors: Käser, Tobias

Title: Phenotypic and functional characterisation of porcine regulatory T cells.

Source: PhD-Arbeit, Vet. Med. Univ. Wien, pp. 100.

Advisor(s):

Gerner Wilhelm
Saalmüller Armin

Vetmed Research Units:
Institute of Immunology


Abstract:
Regulatory T cells (Tregs) belong mainly to the CD4+CD25high T-cell subset and are defined by expression of the transcription factor forkhead box P3 (Foxp3) and their suppressive capacity. Their major function is to ensure immunological self-tolerance and immune homeostasis. Although many studies on Tregs in mice and humans led to a detailed knowledge about the phenotype and functionality of this immunosuppressive subset, no informations were available about Tregs in swine. Therefore, aim of this project was to proof the existence of porcine Tregs and to perform a basic phenotypic and functional characterisation. For detection of Tregs in swine, porcine CD4+CD25-defined T-cells (CD4+CD25–, CD4+CD25dim, and CD4+CD25high) were analysed regarding their Foxp3 expression and suppressive capacity. Reverse transcriptase polymerase chain reaction (RT-PCR) and subsequent Southern Blot hybridisation revealed an exclusive Foxp3-expression in the CD25+ subsets of CD4+ T cells which could be confirmed in flow cytometry (FCM) by detection of intracellular Foxp3 protein using monoclonal antibodies. Additionally, CD4+CD25high T cells had a suppressive effect on the proliferation of T-helper cells. Foxp3 expression in combination with the suppressive capacity in the CD4+CD25high T-cell subset demonstrated the existence of porcine Tregs. Phenotypic investigations of porcine Foxp3+ Tregs by multi-colour flow cytometry analysis revealed the existence of CD4+ and CD4– Tregs with a heterogeous expression of the activation markers CD8α and major histocompatibility complex-II (MHC-II) surface antigens in blood, spleen, and lymph nodes. This heterogeneity refers to several Treg subsets with regard to activation status and functional properties. Further functional analyses of porcine Tregs contained their activation requirements and mechanisms of suppression. It could be observed that porcine Tregs required exogenous interleukin-2 (IL-2) in addition to T-cell receptor (TCR) stimulation to become activated. After activation, porcine Tregs executed their suppressive activity by several mechanisms which can be divided into three main subcategories: i) cell-cell contact dependent mechanisms, ii) production of soluble immunosuppressive factors like interleukin-10 (IL-10), and iii) competition for growth factors like IL-2. In addition, the effect of this suppressive activity on proliferation of various T-cell subsets was analysed in FCM-based proliferation assays in six-colour FCM. These FCM data revealed that all analysed T-cell subsets – T-helper cells, cytotoxic T lymphocytes, and TCR-γδ-T cells – are susceptible to Treg suppression. In conclusion, this study provides first evidence for the existence of porcine Tregs and delivers insight into their phenotype and functions as well as their mechanisms and target cells of suppression. These basic informations on such a powerful immunoregulatory subset pave the way for more detailed investigations of Tregs in swine.


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