Solving technical issues in flow cytometry to characterize porcine CD8α/β expressing lymphocytes

Publikationen als Autor:in / Herausgeber:in der Vetmeduni

Mair, Kerstin; Stadler, Maria; Saalmüller, Armin

Abstrakt

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 alpha and the beta chain. In the pig, the CD8 receptor is expressed by several lymphocyte subsets, including Natural Killer cells, gamma delta T cells and antigen experienced CD4+ alpha beta T cells. On these cell populations CD8 is expressed as alpha alpha homodimers. Porcine cytolytic T cells on the other hand exclusively express CD8 alpha beta heterodimers. Several monoclonal antibodies (mAbs) for either of the two chains are available and are frequently used in flow cytometry. We observed that distinct combinations of mAb clones for CD8 alpha and CD8 beta chains can cause troubles in multi-color staining panels. Therefore, we aimed for an in-depth study of the usage of different CD8-specific mAb clones and optimizing co-staining strategies for flow cytometry. We tested mAb clones 11/295/33 and 76-2-11 for the detection of CD8 alpha and mAb clones PPT23 and PG164A for the detection of CD8 beta. The results indicate that the CD8 alpha clone 11/295/33 should not be used together with either of the two CD8 beta clones in the same incubation step, as co-staining led to a highly reduced ability of CD8 beta mAb binding and loss in signal in flow cytometry. This can lead to potential false results in detecting CD8 alpha beta cytolytic T cells. In case of the CD8 alpha mAb clone 76-2-11, no inhibition in binding of either CD8 beta mAb clones was observed, making it the preferred choice in multi-color staining panels. The obtained data will help in future panel designs for flow cytometry in the pig and therefore improving studies of porcine immune cells.

Dokumententyp

Originalarbeit

Open Access Type

Hybrid

DOI

10.1016/j.vetimm.2024.110853

PubMed ID

MEDLINE:39500097

Projekt

Christian Doppler Labor für eine optimierte Vorhersage des Impferfolges in Schweinen