A novel CD69 binding ankyrin repeat protein (AnkyronTM ) enables detection of early activated and antigen-specific T cells in pigs

T cells are essential for a wide range of protective immune responses, but significant gaps remain in our understanding of their phenotypic and functional diversity, especially in large animal models, like the pig. This is partly due to the limited availability of porcine-specific reagents and validated markers to study T-cell activation and differentiation. One marker involved in both of these processes is CD69: In mice and humans, it serves as an early activation marker and is used to define tissue-resident memory T cells. However, its use for the identification and functional assessment of T-cell subsets in pigs has been previously restricted by the lack of monoclonal antibodies. To address this limitation, we evaluated an alternative approach to antibodies: A novel ankyrin repeat protein, Ankyron™ EG40360, was used as a tool to detect porcine CD69. Ankyrin repeat proteins offer advantages over conventional antibodies, including their small size, improved tissue penetration, and simplified integration into flow cytometry panels due to the absence of isotype restrictions. We confirmed specific binding of Ankyron™ EG40360 to porcine CD69 using both flow cytometry and microscopy in porcine CD69-transfected HEK293T cells. Thereafter, we studied CD69 expression in PBMCs stimulated with PMA/ionomycin. While CD69 was basically absent in non-stimulated control cells, CD69 was highly expressed upon PMA/ionomycin stimulation as early as 4 hours post stimulation. Thereby, we could confirm CD69 as an early marker of lymphocyte activation in pigs. To further assess the expression of CD69 with the Ankyron™ EG40360 in a more natural setting, we studied CD69 expression in PBMCs from pigs that received a protein subunit vaccine. Following in vitro restimulation with the vaccine antigen, CD4 and CD8 T cells upregulated CD69, which makes CD69 not only a suitable marker for primed T cells but also for activation-induced marker (AIM) assays. Thus, Ankyron™ EG40360 expands the porcine immunology toolkit and will facilitate a more comprehensive characterization of activated T cells in vaccine development and infection studies.