Solving technical issues to characterize porcine CD8alpha/beta expressing lymphocytes

The CD8 molecule is a cell surface receptor and well described as co-receptor on T cells binding directly to the major histocompatibility complex class I on antigen presenting cells. CD8 antigens are comprised of two distinct polypeptide chains, the α and the β chain. In the pig, the CD8 receptor is expressed by several lymphocyte subsets, including Natural Killer cells, γδ T cells and antigen experienced CD4+ αβ T cells. On these cell populations CD8 is expressed as αα homodimer. Porcine cytolytic T cells on the other hand exclusively express CD8 αβ heterodimers. Several monoclonal antibodies (mAbs) for either of the two chains are available and are frequently used in flow cytometry. Previous results indicate that distinct combinations of mAb clones for CD8α and CD8β chains can cause troubles in multi-colour staining panels, possibly due to steric inhibition of the antibodies binding to spacial closely located epitopes. Therefore, aim of this project was the in-depth study of the usage of different CD8-specific mAb clones and optimizing co-staining strategies. For the experiments, peripheral blood mononuclear cells were isolated by gradient centrifugation from blood of healthy pigs obtained from a conventional slaughterhouse in Lower Austria. For flow cytometric analysis mAb clones 11/295/33 and 76-2-11 were used for the detection of CD8α and clones PPT23 and PG164A for the detection of CD8β. The results indicate that the CD8α clone 11/295/33 should not be used together with either of the two CD8β clones. It can be assumed, that the antibodies bind to spatially close epitopes on the CD8α and the CD8β chains. As a result, steric hindrance occurs, leading to a highly reduced ability of CD8β mAbs in binding their specific epitopes and loss in signal in flow cytometry. In case of the CD8α mAb clone 76-2-11, no inhibition of CD8β binding was observed. Therefore, we assumed that CD8α mAb clones 11/295/33 and 76-2-11 bind to different epitopes on the CD8α chain, that are distant from each other. This was confirmed in blocking experiments using both CD8α-specific mAb clones in combination where no effects on the staining intensities were observed. The obtained data will help in future panel designs for multi-colour flow cytometry in the pig and therefore improving studies of porcine immune cells.