Journal: bioRxiv

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

doi: 10.1101/2022.01.09.475571

Figure Lengend Snippet: (A) Cell compositions of scRNA-seq data from whole ileum (top), PP (middle), and non-PP (bottom) samples. Cells from each sample type (depicted on the far left) were combined from a total of two animals and overlaid onto t-SNE coordinates originally presented in . The total numbers of cells derived from the total of two animals for each sample type are listed on the far left. On the t-SNE plots, each point represents a single cell; the color of each point corresponds to cell lineage (left t-SNE), cell type (center t-SNE), or cell density (right t-SNE). (B) Pie charts showing proportions of cells from each annotated cell lineage within total cells derived from each sample type in A . The color of a pie slice indicates cell lineage. The total area of each pie chart is not proportional to the total number of cells derived from each sample type. Proportions were calculated from total cells derived from two pigs for each sample type. (C) Plot of the percentage of B cells (CD79α + ) within total leukocytes (CD45 + ; y-axis) from samples of whole ileum, PP, and non-PP (x-axis), as assessed by flow cytometry gating shown in Supplement to Figure 4 - Figure 2A . Measurements from different sample types derived from the same animal are connected by a light grey line. (D) IHC staining for B cell CD79α protein (brown) in a region of ileum with Peyer’s patches (left) or without Peyer’s patches (right). (E) Plot of the percentage of T cells (CD3ε + ) within total leukocytes (CD45 + ; y-axis) from samples of whole ileum, PP, and non-PP (x-axis), as assessed by flow cytometry gating shown in Supplement to Figure 4-Figure 2A . Measurements from different sample types derived from the same animal are connected by a light grey line. (F) IHC staining for T cell CD3ε protein (brown) in a region of ileum with Peyer’s patches (left) or without Peyer’s patches (right). (G) Plot of the percentage of CD4 αβ T cells (left), CD8 αβ T cells (center), or γδ T cells (right) within total T cells (y-axis) of the porcine ileum scRNA-seq dataset. Percentages from samples of whole ileum, PP, and non-PP are shown on the x-axis. CD4 αβ T cells included cells annotated as follicular CD4 αβ T cells, activated CD4 αβ T cells, or cycling CD4 αβ T cells and cells annotated as naïve CD4/CD8 αβ T cells with prediction probability to porcine PBMC CD4 + αβ T cells > prediction probability to porcine PBMC CD8αβ + αβ T cells. CD8 αβ T cells included cells annotated as activated CD8 αβ T cells, cytotoxic CD8 αβ T cells, or cycling CD8 αβ T cells and cells annotated as naïve CD4/CD8 αβ T cells with prediction probability to porcine PBMC CD8αβ + αβ T cells > prediction probability to porcine PBMC CD4 + αβ T cells. γδ T cells included cells annotated as activated γδ T cells, cytotoxic γδ T cells, cycling γδ T cells, SELL hi γδ T cells, and CD2 - γδ T cells. Measurements from different sample types derived from the same animal are connected by a light grey line. (H) Plot of the percentage of CD4 αβ T cells (γδTCR - CD4 + ; left), CD8 αβ T cells (γδTCR - CD8β + ; center), or γδ T cells (γδTCR + ; right) within total T cells (CD3ε + ; y-axis) from samples of whole ileum, PP, and non-PP (x-axis), as assessed by flow cytometry gating shown in Supplement to Figure 4-Figure 2B . Measurements from different sample types derived from the same animal are connected by a light grey line. (I) RNA ISH staining for TRDC (top, red), CD8B (bottom, green), or CD4 (bottom, red) transcripts in regions of ileum with Peyer’s patches (left) or regions of ileum without Peyer’s patches (right). (J) Differential abundance analysis of cell types from porcine ileum scRNA-seq PP versus non-PP samples. Annotated cell types are listed on the y-axis. Each point represents an individual cell neighborhood, where a neighborhood was assigned as a specific cell type if >70% of cells within the neighborhood belonged to the specified cell type annotation. Cell neighborhoods with <70% of cells belonging to a single cell type are not shown. Grey points indicate cell neighborhoods that were not significantly more abundant in a specific sample type. Non-grey points indicate cell neighborhoods exhibiting differential abundance (p<0.1), and red/blue fill of differentially abundant points corresponds to the magnitude and direction of logFC (also corresponding to values listed on the x-axis). Red indicates increased abundance in PP samples, while blue indicates increased abundance in non-PP samples. On the far right, counts of cell neighborhoods with increased abundance in PP samples/no differential abundance/increased abundance in non-PP samples are shown for each cell type. Cycling γδ T cells and cycling group 1 ILCs are not shown on the y-axis due to no cell neighborhoods being assigned to these cell types. scRNA-seq data shown in A-B, G, & J were derived from ileum of two seven-week-old pigs. Images shown in I were also taken from a seven-week-old pig used for ileum scRNA-seq. Flow cytometry and IHC experiments were not performed on animals used for scRNA-seq. Flow cytometry experiments shown in C & E were conducted using four six-week-old pigs. Flow cytometry data shown in H was performed using five nine-week-old pigs. IHC staining in D was completed on a six-week-old pig. IHC staining in F was completed on a nine-week-old pig. Abbreviations: IHC (immunohistochemistry); ILC (innate lymphoid cell); ISH ( in situ hybridization); logFC (log fold-change); No Sig (no significance); PBMC (peripheral blood mononuclear cell); PP (Peyer’s patch); scRNA-seq (single-cell RNA sequencing); t-SNE (t-distributed stochastic neighbor embedding); TCR (T cell receptor)

Article Snippet: A panel to identify γδ, CD4 αβ, and CD8 αβ T cells (gating strategy shown in Supplement to Figure 4-Figure 2B ) included Fixable Viability Dye-eFluor TM 780 (ThermoFisher 65-0865-14); mouse α-pig CD3ε-PE-Cy7 (BD 561477); mouse α-pig γδTCR-iFluor594 (primary antibody Washington State University PG2032; custom conjugation to iFluor594 performed by Caprico Biotechnologies); mouse α-pig CD4-PerCP-Cy5.5 (BD 561474); and mouse α-pig CD8β-PE (BioRad MCA5954PE).

Techniques: Derivative Assay, Flow Cytometry, Immunohistochemistry, Staining, In Situ Hybridization, RNA Sequencing Assay

Journal: bioRxiv

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

doi: 10.1101/2022.01.09.475571

Figure Lengend Snippet: (A) Flow cytometry gating strategy used to identify CD2 + Lin - (Lin - = CD172α - CD3ε - CD79α - ) within total viable cells of porcine ileal samples. Gating is shown for a whole ileum sample (containing both regions with and without Peyer’s patches) for pig A (corresponding to pig IDs in C ). (B) Flow cytometry forward- and side-scatter plots of total viable cells (left) and CD2 + Lin - cells (right) within a sample of porcine whole ileum shown in A . A gate identifying cells with scatter profiles consistent with lymphocytes is shown, with percentages of cells within the lymphocyte gate listed in the top right of each plot. (C) Histogram of the percentage of CD45 + cells within CD2 + Lin - cells identified from samples of porcine ileum using the flow cytometry gating strategy shown in A . A fluorescence-minus-one (FMO) sample lacking α-CD45 antibody staining was used as a negative control. (D) Dual fluorescent staining of CD3ε protein and ITGAE RNA in villi (epithelium + lamina propria) of porcine ileum. Left column: overlay of all stains, including CD3ε protein (green), ITGAE RNA (magenta), and nuclei (DAPI staining; blue). Additional columns show individual stain overlays in white, including (from left to right) nuclei, CD3ε protein, and ITGAE RNA. Panels of two separate villi are shown in each row. Panels were selected from larger stitched images as shown in Supplement to Figure 5-Figure 2A . Yellow arrows indicate location of ITGAE + CD3ε - cells. (E) Dual fluorescent staining of CD3ε protein and IL22 RNA in lamina propria/GALT of porcine ileum. Left column: overlay of all stains, including CD3ε protein (green), IL22 RNA (magenta), and nuclei (DAPI staining; blue). Additional columns show individual stain overlays in white, including (from left to right) nuclei, CD3ε protein, and IL22 RNA. Panels of four separate tissue locations are shown in each row. Panels were selected from larger stitched images as shown in Supplement to Figure 5-Figure 2B . Yellow arrows indicate location of IL22 + CD3ε - cells. Orange arrows indicate location of IL22 + CD3ε + cells. Flow cytometry experiments shown in A-C were conducted using three six-week-old pigs. Dual IF/ISH experiments shown in D-E were conducted using a seven-week-old pig used for ileum scRNA-seq. Abbreviations: FMO (fluorescence-minus-one); FSC-A (forward scatter area); FSC-H (forward scatter height); GALT (gut-associated lymphoid tissue); IF (immunofluorescence); ISH ( in situ hybridization); ILC (innate lymphoid cell); scRNA-seq (single-cell RNA sequencing); SSC-A (side scatter area)

Article Snippet: A panel to identify γδ, CD4 αβ, and CD8 αβ T cells (gating strategy shown in Supplement to Figure 4-Figure 2B ) included Fixable Viability Dye-eFluor TM 780 (ThermoFisher 65-0865-14); mouse α-pig CD3ε-PE-Cy7 (BD 561477); mouse α-pig γδTCR-iFluor594 (primary antibody Washington State University PG2032; custom conjugation to iFluor594 performed by Caprico Biotechnologies); mouse α-pig CD4-PerCP-Cy5.5 (BD 561474); and mouse α-pig CD8β-PE (BioRad MCA5954PE).

Techniques: Flow Cytometry, Fluorescence, Staining, Negative Control, Immunofluorescence, In Situ Hybridization, RNA Sequencing Assay

Journal: bioRxiv

Article Title: The actin-capping protein alpha-adducin is required for T-cell costimulation

doi: 10.1101/580191

Figure Lengend Snippet: (A) Expression of alpha-Adducin (Add1) was measured in CD4 T cells by western blot ex vivo and at d1, d4 and d6 after activation with anti-CD3 and anti-CD28. The two lower molecular weight bands are beta-and gamma-Adducin. (B) Phosphorylation of Add1 at Serine 726 was measured in naive CD4 T cells by western blot after anti-CD3 and anti-CD28 stimulation for the indicated times. (C) WT and Add1 KO OT-II CD4 T cells were labeled with CFSE and stimulated with LB27.4 B cells loaded with the indicated concentrations of Ova peptide. Cell division was measured on d3. Results are representative of two (A and B) or >5 experiments (C).

Article Snippet: Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

Techniques: Expressing, Western Blot, Ex Vivo, Activation Assay, Molecular Weight, Labeling

Journal: bioRxiv

Article Title: The actin-capping protein alpha-adducin is required for T-cell costimulation

doi: 10.1101/580191

Figure Lengend Snippet: (A) CFSE dilution of WT and KO CD4 T cells in response to the indicated concentrations of plate-bound anti-CD3 with or without soluble anti-CD28. Cell division was measured on d3. (B) Percent of cells that have divided at titrated doses of anti-CD3. (C) WT and Add1 KO OT-II CD4 T cells were stimulated with 2 μg/mL plate-bound anti-CD3 for 48 hr and assayed for CD25 expression. (D) Percent of cells positve for CD25 at titrated doses of anti-CD3. Results are representative of >5 (A and B) or two experiments (C and D).

Article Snippet: Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

Techniques: Expressing

Journal: bioRxiv

Article Title: The actin-capping protein alpha-adducin is required for T-cell costimulation

doi: 10.1101/580191

Figure Lengend Snippet: (A) IL-2 and (B) IFN-γ production by Add1 WT and KO CD4 T cells stimulated with titrated amounts of anti-CD3 with and without soluble anti-CD28. Cytokine concentrations were measured from supernatants on day 2 of activation by Cytokine Bead Array. Results are representative of two experiments.

Article Snippet: Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: The actin-capping protein alpha-adducin is required for T-cell costimulation

doi: 10.1101/580191

Figure Lengend Snippet: (A) CD8 T cells from WT and Add1 cKO mice were stimulated with the indicated concentrations of anti-CD3. (B and C) B6 CD45.1 recipient mice were infected with 3,000 CFU of Lm-Ova and 10 5 OT-I CD8 T cells from WT or KO mice. On d8, accumulation of donor CD45.2+ cells was measured (B) and quantified for multiple recipients (C) . (D-F) Among transferred cells, the percentage with a KLRG1+CD127-short-lived effector phenotype and KLRG1-CD127+ memory-precursor phenotype were measured (D) and quantified (E and F). (G-I) Splenocytes from infected mice were stimulated with Ova peptide and intracellular cytokine staining for IFN-γ and TNF-α was performed. The percentage of IFN-γ+ and TNF-α+ donor cells was measuredby FACS (G) and quantified (H and I). Results are representative of two experiments.

Article Snippet: Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

Techniques: Infection, Staining