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Xenotransplantation and Immune Response / Atherosclerosis and Cardiovascular Diseases · Journal article
Scientific Reports · August 1, 2026
Early or partial results. Treat as a signal, not a conclusion.
This is an early-stage validation of a novel ankyrin repeat protein (Ankyron EG40360) as a reagent for detecting porcine CD69 on T cells. The protein successfully bound CD69 in transfected cells and detected CD69 upregulation on PBMCs following mitogenic and antigen-specific stimulation, confirming CD69 as an early lymphocyte activation marker in pigs. However, the study is limited to in vitro proof-of-concept and lacks formal comparison to established methods, standardization data, or clinical utility evidence.
In vitro reagent validation with proof-of-concept immunology. Porcine PBMCs from pigs that received a protein subunit vaccine; HEK293T cells transfected with porcine CD69.. Intervention: Ankyron EG40360 (ankyrin repeat protein) binding assay; PMA/ionomycin stimulation; in vitro vaccine antigen restimulation.. Compared with: Non-stimulated control cells; unstimulated PBMCs..
CD69 was basically absent in non-stimulated control cells but highly expressed upon PMA/ionomycin stimulation as early as 4 h post-stimulation Following in vitro restimulation with vaccine antigen, the frequency of CD4 and CD8 T cells expressing CD69 and CD25 increased, indicating detection of antigen-specific T cells
Safety was not reported in the material analysed. Check the source before drawing any conclusion about harm.
If validated in vivo and across multiple animal sources, Ankyron EG40360 could enable more comprehensive T-cell phenotyping in large animal immunology and vaccine studies where porcine-specific CD69 reagents have been unavailable. Current evidence is insufficient to support adoption without comparative and standardization studies.
Novel reagent validation in porcine cells and PBMCs showing CD69 detection capacity and early activation kinetics, but lacks clinical efficacy data, comparative validation against gold standards, or multi-site reproducibility.
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If validated in vivo and across multiple animal sources, Ankyron EG40360 could enable more comprehensive T-cell phenotyping in large animal immunology and vaccine studies where porcine-specific CD69 reagents have been unavailable. Current evidence is insufficient to support adoption without comparative and standardization studies.
Graded across the dimensions that decide whether you should act, each from what the source actually supports. There is no single score, and where a dimension was not assessed it says so.
Abstract 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 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 h 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, the frequency of CD4 and CD8 T cells expressing CD69 and CD25 increased indicating detection of antigen-specific T cells and making CD69 a potential target for activation-induced marker assays in pigs. 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.
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