CD3ε+ Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS

Boettcher, Adeline N.; Cino‐Ozuna, Ada G.; Solanki, Yash; Wiarda, Jayne E; Putz, Ellie J.; Owens, Jeana Lee; Crane, Sara A.; Ahrens, A.; Loving, Crystal L.; Cunnick, Joan E.; Rowland, Raymond R. R.; Charley, Sara E.; Dekkers, Jack C. M.; Tuggle, Christopher K.

doi:10.3389/fimmu.2020.00510

Cited by 6 publications

(5 citation statements)

References 23 publications

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“…Flow cytometry staining with cell viability dye and antibodies reactive to extracellular epitopes was performed as previously described (Wiarda et al, 2020). For antibody panels with intracellular CD79α detection, intracellular staining was completed after staining for extracellular markers, using the True-Nuclear Transcription Factor Buffer Set (424401; BioLegend) according to manufacturer's instructions and as previously described (Boettcher et al, 2020a(Boettcher et al, , 2020b. Cell events were acquired on a FACSymphony A5 flow cytometer (BD Biosciences), and data were analyzed with FlowJo v10.6.1 software (FlowJo, LLC) as previously described (Wiarda et al, 2020).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Chromogenic RNA in situ hybridization FFPE tissues were obtained as described in IHC methods. Singlecolor chromogenic RNA ISH with Sus scrofa-specific channel 1 TRDC probe (553141; Advanced Cell Diagnostics [ACD]) was completed with the RNAscope 2.5 HD Reagent Kit-RED (322350; ACD) as previously described (Palmer et al, 2019) with the following modifications: (1) protease was applied for only 15 min at 40°C, and (2) slides were mounted and coverslipped as described elsewhere (Boettcher et al, 2020a). Dual chromogenic RNA ISH labeling with Sus scrofa-specific channel 1 (CD8B; ACD 815781) and channel 2 (CD4; 491891-C2; ACD) probes was completed with the RNAscope 2.5 HD Duplex Reagent Kit (322430; ACD) as previously described (Boettcher et al, 2020a).…”

Section: Chromogenic Immunohistochemistrymentioning

confidence: 99%

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Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells

et al. 2022

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Lymphocytes can heavily influence intestinal health, but resolving intestinal lymphocyte function is challenging as the intestine contains a vastly heterogeneous mixture of cells. Pigs are an advantageous biomedical model, but deeper understanding of intestinal lymphocytes is warranted to improve model utility. Twenty-six cell types were identified in the porcine ileum by single-cell RNA sequencing and further compared with cells in human and murine ileum. Though general consensus of cell subsets across species was revealed, some porcine-specific lymphocyte subsets were identified. Differential tissue dissection and in situ analyses conferred spatial context, revealing similar locations of lymphocyte subsets in Peyer’s patches and epithelium in pig-to-human comparisons. Like humans, activated and effector lymphocytes were abundant in the ileum but not periphery of pigs, suggesting tissue-specific and/or activation-associated gene expression. Gene signatures for peripheral and ileal innate lymphoid cells newly discovered in pigs were defined and highlighted similarities to human innate lymphoid cells. Overall, we reveal novel lymphocyte subsets in pigs and highlight utility of pigs for intestinal research applications.

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Section: Flow Cytometrymentioning

confidence: 99%

Section: Chromogenic Immunohistochemistrymentioning

confidence: 99%

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells

et al. 2022

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“…Flow cytometry staining with cell viability dye and antibodies reactive to extracellular epitopes was performed as previously described (Wiarda et al, 2020). For antibody panels with intracellular CD79α detection, intracellular staining was completed after staining for extracellular markers, using the True-Nuclear Transcription Factor Buffer Set (BioLegend 424401) according to manufacturer’s instructions and as previously described (Boettcher et al, 2020a; Boettcher et al, 2020b). Cell events were acquired on a FACSymphony A5 flow cytometer (BD Biosciences), and data were analyzed with FlowJo v10.6.1 software (FlowJo, LLC) as previously described (Wiarda et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

“…Chromogenic RNA in-situ hybridization FFPE tissues were obtained as described in IHC methods. Single-color chromogenic RNA ISH with Sus scrofa-specific channel 1 TRDC probe (Advanced Cell Diagnostics [ACD] 553141) was completed with the RNAscope 2.5 HD Reagent Kit-RED (ACD 322350) as previously described (Palmer et al, 2019) with the following modifications: (1) protease was applied for only 15 minutes at 40 ºC and (2) slides were mounted and coverslipped as described elsewhere (Boettcher et al, 2020a). Dual chromogenic RNA ISH labeling with Sus scrofaspecific channel 1 (CD8B; ACD 815781) and channel 2 (CD4; ACD 491891-C2) probes was completed with the RNAscope 2.5 HD Duplex Reagent Kit (ACD 322430) as previously described (Boettcher et al, 2020a).…”

Section: Chromogenic Immunohistochemistrymentioning

confidence: 99%

Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing

Wiarda

Trachsel

Sivasankaran

et al. 2022

Preprint

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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 the porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine ileum, including SELLhi γδ 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 αβ T cells, and cycling T/ILCs were enriched in ileum with Peyer's patches, while non-cycling γδ T, CD8 αβ T, and group 1 ILCs were enriched in ileum without Peyer's patches. scRNA-seq findings were leverages 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 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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“…This is the future of livestock farming and the immunological toolbox not only has a role to play in the identification of genes to be targeted, but it will also be important for defining subsequent immune function, including potential off-target effects. Genome editing is also creating the opportunities to engineer species to act as better models for human diseases alongside or in addition to genetically defined and tailored breeds, such as SCID pigs and MHC homozygous pigs (56,57). For example, pigs are emerging as a very powerful model to predict human influenza vaccine responses but to achieve the maximum benefit of such models a complete toolkit is required (58).…”

Section: The Futurementioning

confidence: 99%

The Veterinary Immunological Toolbox: Past, Present, and Future

et al. 2020

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CD3ε+ Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS

Cited by 6 publications

References 23 publications

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells

Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing

The Veterinary Immunological Toolbox: Past, Present, and Future

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