Single-cell transcriptomics reveals marker genes for immune cell subtypes in turkey peripheral blood

Authors: Monson, Melissa; SHARMA, SHARU - Iowa State University; Byrne, Kristen; Loving, Crystal

Submitted to: Conference Research Workers Disease Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 11/15/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: OBJECTIVE: Detecting and differentiating immune cell types is central to understanding the role each specific cell subtype plays in immune recognition and clearance or tolerance of different pathogens. However, distinguishing cell subtype-specific responses in turkeys is challenging due to limitations in species-compatible antibodies and other immune reagents. Single-cell RNA-sequencing (scRNA-seq) can capture the transcriptional profiles of individual cells and use them to separate cells into distinct populations without the need for turkey specific reagents. Therefore, this study used scRNA-seq to characterize the expression patterns of turkey leukocytes from peripheral blood. METHODS: Peripheral blood samples were collected from two turkeys, divided, and used to isolate peripheral blood mononuclear cells (PBMCs; n = 2) or all white blood cells (WBCs; n = 2). Samples were partitioned to single cells before library construction and sequencing. Gene count matrices were generated by mapping to the turkey genome and data were filtered, normalized, and used to cluster the cells (0.3 cluster resolution) with the Seurat package. PBMC and WBC datasets were clustered separately due to expected differences in cell type compositions. Major cell types were assigned to the initial clusters using canonical marker genes, then lymphocytes, monocytes, and heterophils were independently subclustered to refine predictions of specific cell subtypes and identify novel markers within their gene expression profiles. RESULTS: Gene expression was assayed within nearly 80,000 individual cells from turkey peripheral blood, with approximately 22,000 cells per PBMC library and 18,000 cells per WBC library. Initial clustering revealed 14 clusters of cells within PBMCs and 18 clusters in WBCs. The most abundant predicted cell type was thrombocytes, followed by heterophils (only present in WBCs) and T lymphocytes. Only a few subclusters of monocytes and B lymphocytes were identified in PBMCs and WBCs, with clear indications of plasma B cell and SOX5 expressing B cell populations. Heterophils and T lymphocytes were resolved into a larger number of subclusters with variable expression in both canonical and novel marker genes. For example, different granzymes, NK-lysin, and other effector genes were variably expressed across 4 subclusters of cytotoxic T cells in both PBMCs and WBCs, likely reflecting differences in their functional states. CONCLUSIONS: Single-cell transcriptomics in turkey peripheral blood identified cell subtype-specific gene markers and extended our knowledge of the expression patterns of turkey leukocyte subtypes. Future research could employ these markers and scRNA-seq to help disentangle subtype-specific responses to and control of the diverse pathogens seen in the turkey.