Preferential differential gene expression within the WC1.1+γδ T cell compartment in cattle naturally infected withMycobacterium bovis

Abstract

ABSTRACTBovine tuberculosis (bTB), caused by infection withMycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterize differential gene expression in γδ T cells – a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known asWorkshop Cluster 1(WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturallyM. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n= 10–12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1-and γδ-) was used to generate transcriptomes using bulk RNA-seq (n= 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in theM. bovis-infected compared to the control groups for the WC1.1+γδ T cell compartment (absolute log2FC ≥ 1.5 and FDRPadj.≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21andEOMES), chemokine receptors (CCR5andCCR7), granzymes (GZMA, GZMM, andGZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, WC1.1+γδ T cells have been proposed as major regulatory cell subset in cattle, and we provide evidence for preferential differential activation of this specific subset in cattle naturally infected withM. bovis.