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Butyrophilin-like 2 (Btnl2) regulates site-specific adaptations of tissue-resident gd intraepithelial lymphocytes

ABSTRACT: Background: Tissue-resident gd intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vg TCR+ IELs, however, the mechanisms that control gd IEL maintenance within discrete intestinal segments are unclear. Btnl2, a member of Btnl family, has been shown to induce Treg differentiation and suppress T cell activation and proliferation in vitro. Btnl2 is highly enriched in villous IECs across different intestinal compartments and its expression is reported to be altered by inflammatory cues such as epithelial injury and tumor burden. We sought to understand the role of Btnl2 in regulating intestinal immune responses during homeostasis and inflammation and, particularly, in the induction and maintenance of intestinal gd IELs. Methods: We surveyed the gut immune system of in-house generated Btnl2-KO and WT mice within discrete intestinal segments (duodenum, jejunum, ileum, colon) during homeostasis and DSS-induced epithelial injury using flow cytometry, primary cell assays, immunoassays, and gene expression profiling. In addition, we performed high throughput transcriptomic (bulk RNAseq and scRNAseq) and TCR repertoire (scTCRseq) characterization of segment-specific Btnl2-KO and WT gd IELs at steady-state. Results: We characterized newly generated Btnl2 knockout mice and identify a role for Btnl2 in regulating the frequencies and phenotype of gd IELs preferentially in the ileum at steady state. We found that gd IELs derived from the ileum, but not duodenum, of Btnl2-KO mice possess a dysregulated antibacterial response module. By integrating RNA and single-cell TCR expression data we identified distinct transcriptional signatures and greater TCR repertoire diversity in ileal Btnl2-KO gd IELs. Upon DSS challenge, Btnl2-KO mice displayed enhanced colonic, but not ileal, intestinal inflammation and delayed mucosal repair. Conclusions: Collectively, our findings suggest that context-dependent Btnl2 expression fine-tunes intestinal immune responses to protect against epithelial injury. Overall, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.

ORGANISM(S): Mus musculus

PROVIDER: GSE178273 | GEO | 2021/06/16

REPOSITORIES: GEO

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Project description:Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine intestine, including SELLhi gd T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular CD4 ab T cells, and cycling T/ILCs were enriched in ileum with Peyer’s patches, while non-cycling gd T, CD8 ab T, and group 1 ILCs were enriched in ileum without Peyer’s patches. scRNA-seq findings were leveraged to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral porcine ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.

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Butyrophilin-like 2 (Btnl2) regulates site-specific adaptations of tissue-resident gd intraepithelial lymphocytes

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