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Phenotypic and functional differentiation of porcine CD8⁺ cytolytic T cells
Armin Saalmüller
Till Rümenapf
PhD thesis - University of Veterinary Medicine Vienna - 2023
The pig, with its anatomical, physiological, and metabolic similarities to humans, holds great potential as a research model for human diseases, pharmacology, and transplantation studies. However, a better understanding of the porcine immune system is essential. In this study, we investigated the transcriptomes of porcine CD8+ T-cell subsets and the response of CD8+ T cells after PRRSV infection. First, we characterized the gene expression profiles of three distinct subsets of porcine CD8+ T cells based on their CD11a/CD27 expression pattern: naïve (Tn; CD8β+CD27+CD11alow), intermediate (Tinter; CD8β+CD27dimCD11a+), and terminally differentiated cells (Tterm; CD8β+CD27-CD11ahigh). Our analysis revealed significant differences in gene expression between these subsets. Tn displayed a unique gene expression profile associated with early stages of T-cell differentiation, characterized by the upregulation of key transcription factors (LEF1, BACH2, TCF7), lymph node homing receptors (CCR7, SELL, CCR9), and genes involved in maintaining quiescence (SATB1, ZEB1, BCL2). In contrast, Tterm exhibited a distinct gene expression signature associated with late-stage differentiation, marked by the upregulation of cytolytic genes (GNLY, PRF1, GZMB, FASL, IFNG, TNF) and effector molecules. On the other hand, Tn did not exhibit expression of genes associated with cytolytic activity, even after in vitro stimulation. Furthermore, the Tinter showed a gene expression profile more closely resembling later stages of T-cell differentiation. In the second study, we investigated the immune response of porcine CD8+ T cells after PRRSV infection. CD8+ T cells exhibited a strong adaptive immune response, with the highest number of DEGs observed at 21 dpi. This response led to the formation of highly differentiated CD8+ T cells starting from 14 dpi. The gene expression pattern of CD8+ T cells revealed a distinctive profile characterized by the upregulation of effector and cytolytic genes (PRF1, GZMA, GZMB, GZMK, KLRK1, KLRD1, FASL, NKG7), indicating their pivotal role in the immune response after PRRSV infection. Temporal clustering analysis of DEGs in CD8+ T cells revealed four clusters, indicating tight transcriptional regulation of the adaptive immune response to PRRSV. The main clusters in CD8+ T cells represented the initial transformation and differentiation of these cells in response to PRRSV infection. GSEA further supported the effector state of CD8+ T cells at 21 dpi, with significant enrichment of gene sets associated with effector CD8+ T cells, IFN-α and IFN-γ responses, inflammatory response, T-cell activation and maintenance of effector CD8+ T cells during infection. Furthermore, the flow cytometry analysis of CD8+ T cells revealed a significant expansion of Tinter and Tterm subsets in the PRRSV-infected animals, indicating the impact of the infection on the CD8+ T-cell differentiation. These studies provide comprehensive insights into the gene expression profiles and immune responses of porcine CD8+ T cells during PRRSV infection. The findings highlight the dynamic nature of the immune response and provide potential biomarker targets for vaccine and therapeutic development. The combination of these studies expands our understanding of the porcine immune system and its interaction with pathogens, contributing to the advancement of porcine immunology research.
Englisch
2023
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