monoclonal rat anti st2 il 33r  (R&D Systems)

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: Frequency and phenotype of ST2 + Tregs across tissues The frequency and phenotype of Tregs from Foxp3 YFP−cre mice were assessed by flow cytometry. (A) Representative flow plots showing ST2 vs CD4, gated on live singlets, CD45 + , TCRβ + CD4 + FOXP3 + cells in blood, spleen, lungs, VAT, colon, and skin. (B) Frequency of ST2-expressing Tregs across tissues, based on gating in Figure 1 A. Data are pooled from 7 experiments, each with n = 4–6 per cohort. (C) Representative overlays of ST2 + Tregs (red) on total Tregs (gray), showing CD62L vs CD44 expression in indicated tissues, pregated on live singlets, CD45 + , TCRβ + CD4 + FOXP3 + cells. (D–F) Left: representative flow plots and histograms showing staining of (D) KLRG1, (E) CD103, and (F) Ki-67 in selected tissues and populations (orange: ST2 + Tregs, blue: ST2 - Tregs, red: Foxp3 - CD4 + T cells), Right: frequency of (D) KLRG1, (E) CD103, and (F) Ki-67 on ST2 + Tregs, ST2 - Tregs, and Foxp3 - CD4 + T cells across tissues. D and F: Data are pooled from 3 experiments of n = 5 each. E: Data are pooled from 2 experiments of n = 5 each, except for VAT and colon with n = 4 in one of 2 experiments. Data are plotted as mean ± SD and indicated means were compared with two-way ANOVA with Sidak’s multiple comparisons test, ns p > 0.05, ∗∗∗∗p < 0.0001. Abbreviations: VAT: visceral adipose tissue, TCR: T cell receptor. See Figure S1 for flow cytometric gating and analysis of Tregs across tissues and in secondary lymphoid organs.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Flow Cytometry, Expressing, Staining

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: Correlation between Id2 + Tregs and ST2 + Tregs across tissues The frequency of Id2 + and ST2 + Tregs from Foxp3 mRFP Id2 YFP reporter mice was assessed by flow cytometry. (A) Relative distribution of Id2 and ST2 on Foxp3 + Tregs in indicated tissues, left: representative flow plots, right: quantification. (B) Frequency of ST2 + cells among Id2 + Tregs in indicated tissues. (C) Frequency of Id2 + cells among ST2 + Tregs in indicated tissues. (D) Frequency of KLRG1 on indicated Treg populations from Figure 2 A in blood, spleen, lungs, VAT, colon, and skin. A–D: Data are pooled from two independent experiments with n = 4 each; for D: data points were removed for populations that routinely had less than 100 cells. Data are plotted as mean ± SD. Abbreviations: VAT: visceral adipose tissue, TCR: T cell receptor. See Figure S2 for the correlation between Id3 - Tregs and ST2 + Tregs across tissues.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Flow Cytometry

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: RNAseq analysis of ST2 + Tregs and ST2 - Tregs across tissues RNAseq was performed on flow sorted ST2 + CD44 hi Tregs and ST2 - CD44 hi Tregs from 6 different tissue sites (blood, spleen, lungs, VAT, colon, and back skin) and ST2 - CD44 lo CD62L + Tregs from spleen and lungs. Data are from 5 male Foxp3 YFP−cre mice ages 16–19 weeks old. (A) PCA based on all genes from all populations and tissues. The percentages alongside the PC axes labels are the percentage of variance explained by each PC. (B) Dendrogram and correlation coefficient using Spearman’s ranked-based correlation analysis of all genes in libraries from ST2 + CD44 hi Tregs and ST2 - CD44 hi Tregs. (C) Heatmap of pooled tissue-specific DEGs, sorted by tissue. Data displayed as Z score. Abbreviations: VAT: visceral adipose tissue, PCA: principal component analysis, DEG: differentially expressed genes. See Figure S3 for the correlation of factors/categorical variables across PC coordinates and Venn diagrams identifying tissue-specific DEGs displayed in Figure 3 C.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques:

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: RNAseq analysis of DEGs between ST2 + and ST2 - Tregs (A) Volcano plots displaying DEGs between ST2 + CD44 hi Tregs versus ST2 - CD44 hi Tregs within each tissue. Genes with FC > 1.5 and adjusted p value <0.05 were considered significant. (B) Circos plot using DEGs between ST2 + versus ST2 - Tregs from individual tissues as assessed in Figure 4 A; arcs show DEGs shared between tissues; color code indicates the number of tissues sharing a given DEG. (C) Venn diagrams showing the overlap of DEGs between ST2 + versus ST2 - Tregs from individual tissues as assessed in Figure 4 A across all tissues excluding blood (left) and all tissues excluding skin (right). (D) Heatmap showing gene expression of DEGs between ST2 + versus ST2 - Tregs that are common to at least 5 of 6 tissues sampled. Three DEGs – Il1rl1 , Gata3 , and Rln3 – are common to all tissues. Data displayed as Z score. (E) Heatmap showing hierarchical clustering and gene expression of DEGs between ST2 + versus ST2 - Tregs that are common to at least 4 of 6 tissues sampled. Data displayed as Z score. (F) Heatmap of FDR (-log10) of top KEGG pathways across tissues. The KEGG pathways plotted represent the top 6 terms in each tissue based on FDR. Values in red represent significant pathways (-log10 FDR >1.3 or FDR <0.05); NA = pathways that are not represented in the gene list. Abbreviations: VAT: visceral adipose tissue, DEG: differentially expressed genes, KEGG: Kyoto Encyclopedia of Genes and Genomes Database. See Figure S4 for GATA3 flow cytometric analysis of ST2 + and ST2 - Tregs across tissues. Figure S5A for expression of core DEGs between ST2 + and ST2 - Tregs across tissues, Figure S5 B for Spearman’s correlation analysis of DEGs between ST2 + and ST2 - Tregs across tissues, Figure S6 for enriched KEGG pathways in DEGs between ST2 + and ST2 - Tregs in each tissue, and Table S1 for ST2 Treg RNAseq Datasets.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Expressing

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: Chemokine receptor expression and intravascular labeling of ST2 + and ST2 - Tregs (A) Heatmap showing gene expression of chemokine receptors in indicated populations and tissues. Data displayed as Z score. (B–E) Representative flow plots and relative frequency of (B) CCR7, (C) CCR3, (D) CCR4, and (E) CCR2 on ST2 + CD44 hi Tregs, ST2 - CD44 hi Tregs, and Foxp3 - CD4 + CD44 hi T cells across tissues as assessed by flow cytometry. Lymphocytes were pregated on live singlets, CD45 + , and TCRβ + CD4 + cells. Data are plotted as mean ± SD and pooled from 2 experiments with n = 4–5 for all tissues but colon. For colon, n = 5 in B and D and n = 4 in E, each from one experiment, and n = 7 pooled from 2 experiments in C. Indicated means were compared with two-way ANOVA with Sidak’s multiple comparisons test, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (F) Frequency of i.v.-labeled CD45 on ST2 + and ST2 - Tregs in indicated tissues. Mice received 3 μg of anti-CD45 i.v. prior to organ harvesting to evaluate the perivascular localization of cells. Data are plotted as mean ± SD and pooled from 2 experiments of n = 5 each. Indicated means were compared with two-way ANOVA with Sidak’s multiple comparisons test, ns p > 0.05, ∗∗∗∗p < 0.0001. Abbreviations: VAT: visceral adipose tissue, TCR: T cell receptor, CCR: CC chemokine receptor. See Figure S7 for chemokine receptor gene expression profiles of ST2 + and ST2 - Tregs across tissues.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Expressing, Labeling, Flow Cytometry

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: Adoptive transfer of ST2 + and ST2 - Tregs from spleen or lungs into sublethally irradiated WT hosts (A) Experimental setup: ST2 + CD44 hi Tregs and ST2 - CD44 hi Tregs were sorted from either the spleen or lungs of CD45.2 Foxp3 YFP−cre mice and transferred i.v. into separate sublethally irradiated CD45.1 hosts. Organs of host mice were harvested 4 weeks after transfer to assess recovery of transferred Tregs. Lymphocytes were gated on live singlets, CD4 + FOXP3 + CD45.2 + cells. (B) Total numbers of CD45.2 + Tregs recovered in the indicated tissues after transfer of splenic ST2 - CD44 hi Tregs or splenic ST2 + CD44 hi Tregs. (C) Total numbers of CD45.2 + Tregs recovered in the indicated tissues after transfer of pulmonary ST2 - CD44 hi Tregs or pulmonary ST2 + CD44 hi Tregs. B and C: counts are per whole organ, per 3 × 4 cm back skin or per 450–800 uL blood. (D) CD45.2 + Treg counts from B and C were normalized to 30,000 Tregs. (E) Gain of ST2 expression on transferred CD45.2 + ST2 - Tregs recovered from the indicated tissues. (F) Retention of ST2 expression on transferred CD45.2 + ST2 + Tregs recovered from the indicated tissues. B-F: Means were compared with unpaired two-tailed test, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data plotted as mean ± SD Data are pooled from 2 to 3 experiments of n = 2–5 mice each. For transferred splenic ST2 - Tregs, n = 12 for host blood, spleen, lungs, and colon; n = 10 for skin; n = 8 for VAT. For transferred splenic ST2 + Tregs, n = 10 for host blood, spleen, lungs, and colon; n = 8 for skin; n = 6 for VAT. For transferred pulmonary ST2 - Tregs, n = 9 for host blood, spleen, lungs, colon, and skin and n = 6 for VAT. And for transferred pulmonary ST2 + Tregs, n = 7 for host blood, spleen, lungs, colon, and skin and n = 4 for VAT. Abbreviations: VAT: visceral adipose tissue. See Figure S8 for adoptive transfer of 1∗10 6 of total ST2 - Tregs from the spleen into sublethally irradiated WT hosts.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Adoptive Transfer Assay, Irradiation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet: Adoptive transfer of ST2 + and ST2 - Tregs from spleen or lungs into sublethally irradiated IL-33 KO hosts (A) Experimental setup: ST2 + CD44 hi Tregs and ST2 - CD44 hi Tregs were sorted from either the spleen or lungs of CD45.2 Foxp3 YFP−cre mice and transferred i.v. into separate sublethally irradiated IL-33 KO CD45.1 hosts. Organs of host mice were harvested 4 weeks after transfer to assess recovery of transferred Tregs. Lymphocytes were gated on live singlets, CD4 + FOXP3 + CD45.2 + cells. (B) Total numbers of CD45.2 + Tregs recovered in the indicated IL-33 KO tissues after transfer of splenic ST2 - CD44 hi Tregs or splenic ST2 + CD44 hi Tregs. (C) Total numbers of CD45.2 + Tregs recovered in the indicated IL-33 KO tissues after transfer of pulmonary ST2 - CD44 hi Tregs or pulmonary ST2 + CD44 hi Tregs. B and C: counts are per whole organ, per 3 × 4 cm back skin or per 300–800 uL blood. (D) CD45.2 + Treg counts from B and C were normalized to 30,000 Tregs. (E) Gain of ST2 expression on transferred CD45.2 + ST2 - Tregs recovered from the indicated tissues. (F) Retention of ST2 expression on transferred CD45.2 + ST2 + Tregs recovered from the indicated tissues. B-F: Means were compared with unpaired two-tailed test, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data plotted as mean ± SD Data are pooled from 3 to 4 experiments. For transferred splenic ST2 - Tregs, n = 7 for host spleen; n = 8 for host VAT and skin; n = 9 for host blood, lung, and colon. For transferred splenic ST2 + Tregs, n = 9 for host blood, spleen, lungs, colon, and skin; n = 8 for VAT. For transferred pulmonary ST2 - Tregs, n = 8 for all host tissues. And for transferred pulmonary ST2 + Tregs, n = 7 for all host tissues. Abbreviations: VAT: visceral adipose tissue.

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Adoptive Transfer Assay, Irradiation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet:

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques: Recombinant, Staining, Cell Isolation, Sequencing, Sample Prep, Software

Journal: iScience

Article Title: Profiling of Tregs across tissues reveals plasticity in ST2 expression and hierarchies in tissue-specific phenotypes

doi: 10.1016/j.isci.2022.104998

Figure Lengend Snippet:

Article Snippet: BV421 Rat Anti-Mouse IL-33R (ST2) Clone U29-93 , BD Biosciences , Cat# 566309; RRID: AB_2744489.

Techniques:

Journal: Pathogens

Article Title: Eosinophils, but Not Type 2 Innate Lymphoid Cells, Are the Predominant Source of Interleukin 4 during the Innate Phase of Leishmania major Infection

doi: 10.3390/pathogens11080828

Figure Lengend Snippet: Detection of ILC2 in the ear skin of naïve C57BL/6 and BALB/c mice. Flow cytometry of ear skin cells from naïve 4get-C57BL/6 ( A ) and 4get-BALB/c mice ( B ). Lymphomononuclear cells were isolated from ear dermis and epidermis by enzymatic and mechanical dissociation. Single cells were pre-gated on viability and CD45 expression; eosinophils staining positively for SiglecF were excluded. ILC2 were defined as lineage marker-negative, CD90.2 + cells. For further characterization, the surface markers CD127, Sca-1, ICOS (CD278), PD1 and ST2 were used. Data are representative of 3 to 4 experiments ( A , B ) or 2 experiments ( C ) with 3 to 5 mice pooled per group. The lineage marker-mix included antibodies directed against CD2, CD3, CD5, CD11b, CD11c, CD19, FcεR1a, Ly6G, NKp46 and Ter119. The numbers within the histograms indicate the mean fluorescence intensity of the respective surface markers ( A – C ).

Article Snippet: Rat anti-mouse ST2 (IL-33R) , MD Bioproducts , 101001PE , DJ8 , PE , 1:100.

Techniques: Flow Cytometry, Isolation, Expressing, Staining, Marker, Fluorescence

Journal: Pathogens

Article Title: Eosinophils, but Not Type 2 Innate Lymphoid Cells, Are the Predominant Source of Interleukin 4 during the Innate Phase of Leishmania major Infection

doi: 10.3390/pathogens11080828

Figure Lengend Snippet: Antibodies and reagents used for flow cytometric analysis.

Article Snippet: Rat anti-mouse ST2 (IL-33R) , MD Bioproducts , 101001PE , DJ8 , PE , 1:100.

Techniques: Conjugation Assay

Journal: Frontiers in Immunology

Article Title: Gut Microbial Metabolite Pravastatin Attenuates Intestinal Ischemia/Reperfusion Injury Through Promoting IL-13 Release From Type II Innate Lymphoid Cells via IL−33/ST2 Signaling

doi: 10.3389/fimmu.2021.704836

Figure Lengend Snippet: PA protected against intestinal I/R injury via an IL-33/ST2 signal. (A) IL-33 immunohistochemical staining in the ileum from sham, I/R and I/R + PA mice, scale bar is 100 μm (n = 8). (B, C) Relative mRNA levels of IL-33 and IL-33 receptor (ST2) (n = 8). (D) HE staining, ZO-1, Occludin and Ki67 immunofluorescent staining and IL-33 immunohistochemical staining in WT mice and IL-33 -/- mice, scale bar is 100 μm (n = 8). (E) Pathological damage score in the ileum. (F–H) The relative fluorescence intensity quantification analysis of ZO-1, Occludin and Ki67 in the ileum. (I) The relative mRNA level of Lgr5 in the ileum was measured by quantitative PCR (n = 8). (J) The relative IL-33 intensity quantification analysis in the ileum. (K, L) Intestinal lamina propria cells were analyzed by flow cytometry for the ratio of ILC2/ILCs and IL-13 + ILC2/ILC2 in WT mice (n = 3-4). (M, N) IL-13 immunohistochemical staining and intensity quantification analysis in the ileum, scale bar is 100 μm (n = 8). The results are expressed as the mean ± SEM (B, C, E–J, N) . * p < 0.05, ** p < 0.01, *** p < 0.001, NS means No statistically significant difference by one-way ANOVA (Tukey’s test). PA, pravastatin; I/R, ischemia/reperfusion; ILC2, type II innate lymphoid cells.

Article Snippet: The cell surface was stained using APC-Cyanine7-conjugated CD45 Antibody (Cat# A15395, eBioscience), FITC-conjugated mouse Hematopoietic Lineage Antibody Cocktail (Cat# 22-7770-72, eBioscience), and BV421-conjugated Rat Anti-Mouse IL-33R (ST2) antibody (Cat# 566309, BD Biosciences, San Jose, CA, USA).

Techniques: Immunohistochemical staining, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Flow Cytometry