article Cytotoxic-T; Effector; Memory; Expression; Receptor; Subsets; Virus; Lymphocytes; Infection; Responses Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8β+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8β+CD27+CD11alow), intermediate differentiated (CD8β+CD27dimCD11a+), and terminally differentiated cells (CD8β+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail. Lagumdzic, Emil (University of Veterinary Medicine Vienna) Mair, Kerstin H. (University of Veterinary Medicine Vienna) Saalmüller, Armin (University of Veterinary Medicine Vienna) Schmitt, Michael W. (Merck Healthcare KGaA) Olgiati, Simone (Istituto di Ricerche Biomediche "A. Marxer" RBM S.p.A.) Pernold, Clara (University of Veterinary Medicine Vienna) Viano, Marta (Istituto di Ricerche Biomediche "A. Marxer" RBM S.p.A.) doi:10.3389/fimmu.2022.849922 https://phaidra.vetmeduni.ac.at/o:1806 eng CC BY 4.0 International http://creativecommons.org/licenses/by/4.0/ 2022 Frontiers in Immunology 13 (2022) Frontiers Media Sa application/pdf