Journal: Stem Cell Research & Therapy

Article Title: Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses

doi: 10.1186/s13287-015-0050-0

Figure Lengend Snippet: Multiple allogeneic mesenchymal stem cell (MSC) injections result in changes in splenic regulatory T cell percentages. (A-D) There were no significant changes in splenic CD21 + B-cell (A) , CD4 + T-cell (B) , or CD8 + T-cell percentages (C) or CD4/CD8 ratios (D) following multiple MSC injections. (E) There were no significant changes in activated (CD25 + ) lymphocyte proportions. (F) There were significantly higher percentages of splenic FoxP3 + regulatory T cells in the horses injected with bone marrow (BM)-derived MSCs compared with horses injected with adipose tissue (AT)-derived MSCs. Data are presented as mean ± standard error of the mean. * P <0.05.

Article Snippet: The following antibodies were used: mouse-anti-equine CD3 (clone UC F6G 1:250; Jeffery Stott, University of California, Davis, CA, USA) [ ], mouse-anti-human CD21 (clone B-ly4 1:20; BD Pharmingen, San Jose, CA, USA) [ , ], polyclonal goat-anti-human CD25 (clone AF-223; R&D Systems) [ ], rat-anti-mouse/human FoxP3 (clone PCH101; ebioscience, San Diego, CA, USA) [ ], mouse-anti-CD4 (clone HB61A 1:133; VMRD, Pullman, WA, USA) [ ], mouse-anti-equine CD8 (clone F18P 1:500; J. Stott) [ ], and a donkey-anti-mouse secondary when necessary (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).

Techniques: Injection, Derivative Assay

Journal: Frontiers in Microbiology

Article Title: Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1

doi: 10.3389/fmicb.2016.00832

Figure Lengend Snippet: Table of antibodies.

Article Snippet: CD25 , Mouse , Pig , K231.3B2 , IgG1 , Unconjugated , AbD Serotec.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Identification of CD25− Tfr cells. Mice were vaccinated s.c. with 100 μg of NP-Ova (Biosearch) in alum, and draining LNs (dLNs) or Peyer’s patches were taken at day 7 or the indicated time. (A) Gating strategy of CD25+ and CD25− Tfr cells. Cells were first gated as CD3+CD4+B220−CD11c−CD11b−Live/Dead-dye− before the start of the shown gating. The fluorescence minus one (FMO) control is CD44+CD62L− Treg cells with anti-CXCR5 omitted. Data are representative of >10 separate experiments. Max, maximum. (B) Proportion of CD25− cells in gated CXCR5 and PD-1 low to high populations in Peyer’s patch CD44+CD62L− Treg cells. Data are representative of >10 separate experiments. (C) Time course of GC, Tfh, CD25+ Tfr, and CD25− Tfr proportions in dLNs following vaccination at day 0. (D) Tfr cells in BCL6flox/flox and BCL6wt/wt CD4-Cre mice, with Peyer’s patch T cells pregated on CD44+CD62L− Treg cells. (C and D) Data are pooled from three mice, representative of two separate experiments.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Fluorescence, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Identification of CD25− Tfr cells. Mice were vaccinated s.c. and i.p. with 100 μg of NP-Ova in alum, and dLNs, spleens (Spl), or Peyer’s patches (PP) were taken at day 7 or the indicated time. (A) Percentage of CD25− within total Tfr cells from indicated organs. Data are pooled from 10 mice, representative of three separate experiments. (B) ACCENSE analysis of PP CD4+ T cells. PD1, BCL6, CXCR5, Foxp3, and CD25 were used for mapping. CD44 and CD62L clustered on the basis of other parameters. Data are pooled from three mice concatenated into a single flow cytometry standard file, representative of two separate experiments. Scales are Z-scores (±SD from mean). (C) Time course of GC, Tfh, CD25+ Tfr, and CD25− Tfr cell numbers per 1 × 105 lymphocytes in dLNs following vaccination at day 0.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Protein expression profile of CD25− Tfr cells. Mice were vaccinated s.c. with 100 μg of NP-Ova in alum, and dLNs were taken at day 7 or day 14. (A, Left) Heat map of geometric mean fluorescence intensity (gMFI; unless indicated otherwise) or percent positive (CD103, Ki-67, IL-21, BLIMP-1, KLRG1) of indicated markers. (A, Right) Significant differences between CD25− Tfr and CD25+ Tfr cells. The scale is the Z-score of mean. Data are pooled from three mice, representative of two to four separate experiments. (B) Expression of indicated markers by gMFI or percent positive of indicated markers. Mean ± SEM. Data are pooled from three mice, representative of two to four separate experiments. (C) Contour plots of KLRG1 and CD103 expression by indicated Treg populations. Data are representative of two separate experiments (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Expressing, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Phenotyping of CD25− Tfr cells. Mice were vaccinated s.c. with 100 μg of NP-Ova in alum, and dLNs were taken at day 7 or day 14. Expression of indicated markers by geometric mean fluorescence intensity (gMFI) or percent positive as assessed by flow cytometry. Mean ± SEM. Data are pooled from three mice, representative of two to four separate experiments (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Expressing, Fluorescence, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Localization of CD25− Tfr cells. Mice were/were not vaccinated s.c. and i.p. with 100 μg of NP-Ova in alum and then killed 14 d later. Spleens and dLNs were taken and fixed in 2% paraformaldehyde. Sections were then stained, and expression of Foxp3, CD25, and IgD or Foxp3, CD25, and GL7 was assessed by immunohistochemistry. (A) Foxp3 and CD25 expression by Treg cells in the follicle and T-cell zones from the spleens of unvaccinated mice. (B) Foxp3 and CD25 expression of Treg cells in the follicle, T-cell zones, and GC from spleens of vaccinated mice. CD25, Foxp3, and IgD expression (Upper) and CD25, Foxp3, and GL7 expression (Lower) are shown. White arrows denote Foxp3+CD25− cells, and the red arrow denotes a Foxp3+CD25+ cell. (C) Proportion of CD25 expression within Foxp3+ cells in the follicle, T-cell zone, and GC from spleens (Spl) (Left) and dLNs (Right). Each data point represents a separate field. Data are pooled from three mice, representative of two separate experiments (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001).

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Staining, Expressing, Immunohistochemistry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: RNA-Seq of CD25− Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 104 cells were sorted by Becton Dickinson (BD) FACSAria-SORP before RNA-Seq. RNA was extracted using RLT buffer (Qiagen), and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton (Life Technologies). Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of <0.01 and a fold change of ≥2 were considered DE. Z-scored heat maps of the top 25 up-regulated (A) and top 25 down-regulated (B) Tfh genes are shown. (C) GSEA of CD25− Tfr cells vs. CD25+ Tfr cells with a Tfh vs. eTreg DE gene list. Positive enrichment shows enrichment in CD25− Tfr cells, and negative enrichment shows enrichment in CD25+ Tfr cells. (D) Z-scored heat map of selected Treg suppressor genes. (E) Principal component (PC) analysis (top 500 most variable genes). (F) Euclidean distance to Tfh or eTreg gene expression profile.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: RNA Sequencing, Produced, Software, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: RNA-Seq of CD25− Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 104 cells were sorted by FACS before RNA-Seq. RNA was extracted using RLT buffer, and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton. Heat maps, hierarchical clustering, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of <0.01 and a fold change of ≥2 were considered DE. (A) Z-scored heat map of a full list of Tfh vs. eTreg DE genes, with columns and rows hierarchically clustered. (B) Z-scored heat map of selected Tfh-related genes. (C) GSEA of CD25− Tfr vs. CD25+ Tfr cells with the BCL6hi Tfh vs. BCL6lo Tfh gene list from ref. 22 (GEO accession no. GSE24574). Positive enrichment shows enrichment in CD25− Tfr cells, and negative enrichment shows enrichment in Tfr cells. (D) Euclidean distance analysis of gene expression.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: RNA Sequencing, Produced, Software, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Suppressive function and stability of CD25− Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. Cells were sorted by BD FACSAria-SORP (Becton Dickinson) after negative selection of CD4 by magnetic beads. (A) Total of 1 × 104 B cells were cultured in the presence of 0.5 μg/mL anti-CD3, 10 μg/mL anti-IgM, and 5 × 103 Tfh cells with/without 5 × 103 of the indicated Treg population for 6 d. Intracellular IgG1 expression is shown. Data are representative of two separate experiments. (B) Total of 5 × 103 nTconv cells were stained with CTV and incubated for 3 d with 5 × 103 B cells; 0.5 μg/mL anti-CD3; and 5 × 103, 2.5 × 103, or 1.25 × 103 suppressor cells. Data are representative of two separate experiments. (C) Total of 2.5 × 103 purified cells were stained with CTV and incubated with Dynabeads with/without the indicated cytokines (100 units of IL-2 + 20 ng of IL-6 or 20 ng of IL-4 + 20 ng of IL-6) for 3 d. Foxp3 and CD25 expression is shown. Data are representative of two separate experiments. (D) CD4-enriched T cells from dLNs before and after sorting by BD FACSAria-SORP. (E and F) Total of 1 × 105 CD45.1 nTconv cells and 1 × 104 CD45.2 eFox CD25− Tfr cells were transferred i.v. into nude mice. One day later, mice were vaccinated, and dLNs were collected at day 14. (E) CD25 and CXCR5 expression by Foxp3− CD45.1 cells (Right) and Foxp3-GFP+ CD45.2 cells (Left). (F) Percentage of CD45.2−Foxp3+Foxp3−GFP− and CD45.2+Foxp3−GFP+ cells in eTreg, Tfr, and CD25− Tfr populations. Data are representative of two separate experiments.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Selection, Magnetic Beads, Cell Culture, Expressing, Staining, Incubation, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Suppressive function and stability of CD25− Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. Cells were sorted by BD FACSAria-SORP after negative selection of CD4 by magnetic beads. (A) Total of 1 × 104 B cells were cultured in the presence of 0.5 μg/mL anti-CD3 and 10 μg/mL anti-IgM, and 5 × 103 Tfh cells were cultured with/without 5 × 103 of the indicated Treg population for 6 d. (A) Supernatant IgG1 concentrations were determined by ELISA. Mean ± SEM of duplicates. Data are representative of two separate experiments. (B) Total of 1 × 105 nTreg cells were transfected with retrovirus containing EV or ASCL2 by spinfection with viral supernatants, and then cultured in the presence of anti-CD3, anti-CD28 Dynabeads, and 100 U/mL IL-2 for 72 h. CXCR5 expression (Upper) and CD25 expression (Lower) are shown. The filled histogram is NGFR−, and the clear histogram is NGFR+. Data are representative of three separate experiments. (C) Total of 2.5 × 103 purified cells were stained with CellTrace Violet (CTV) and incubated with anti-CD3, anti-CD28 Dynabeads alone (top row), Dynabeads with 100 U/mL IL-2 (middle row), or 20 ng IL-4 (bottom row) for 3 d. (Left) Foxp3-GFP and CD25 expression. (Right) Proliferation and total cell number (in parentheses) of indicated populations. Data are representative of two separate experiments. (D) Total of 1 × 104 cells were assessed for DNA demethylation by bisulfide sequencing. Data are representative of two separate experiments. (E) Total of 1 × 106 CD62L+CD25− nTconv cells from CD45.1 BALB/c mice and 1 × 105 CD62L+CD25+Foxp3−GFP+ nTreg cells from CD45.2 BALB/c eFox mice were transferred i.v. into athymic BALB/c nude mice. One day later, mice were vaccinated, and dLNs, spleens, and Peyer’s patches were collected at day 14. CD25 and CXCR5 expression by eTreg cells (Foxp3+CD44+CD62L−CXCR5−) and Tfr cells (Foxp3+CD44+CD62L−CXCR5+PD1+) is shown. Data are pooled from three mice, representative of two separate experiments.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Selection, Magnetic Beads, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Purification, Staining, Incubation, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Effect of IL-2 KO and antibody blockade on CD25− Tfr cells. (A–D) Four-week-old IL-2 KO homozygous (−/−), heterozygous (−/+), or wild-type (+/+) littermates were killed. (A) Foxp3 and CD25 expression by CD4+ cells in LNs. (B) Summary data of GC and Tfh cells from LNs of indicated mice. (C) Summary data of Treg, Tfr, and CD25− Tfr cells within Tfr cells from LNs of indicated mice. (D) Summary data of Treg, Tfr, and CD25− Tfr cells within Tfr cells from Peyer’s patches of indicated mice. Mean ± SEM. Data are pooled from three mice, representative of two separate experiments. (E–G) Mice were given 1 mg of anti–IL-2 (S4B6) or isotype control i.p. at day −1. Mice were then vaccinated s.c. with 100 μg of NP-Ova in alum at day 0. (E) Expression of CD25 and CXCR5 by CD44+CD62L−Foxp3+ Treg cells. (F) Summary data of GC and Tfh cells from LNs of indicated mice. (G) Summary data of Treg, Tfr, and CD25− Tfr cells within Tfr cells or CD4 from LNs of indicated mice. Mean ± SEM. Data are pooled from four mice, representative of two separate experiments (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Effect of IL-2 complex on CD25− Tfr cells. (A–D) Mice were treated at days −1, 1, 2, 3, 4, and 5 with IL-2 complex at a 1:5 or 2:10 ratio of 1 μg of murine IL-2/5 μg of anti–IL-2 (JES6-1A12), or with 5 μg of isotype (Iso) control alone. Mice were vaccinated with NP-Ova in alum at day 0, and spleens taken at day 7. (A) Zebra plots and histograms of Foxp3 and CD25 expression by CD4+ cells (Left), CXCR5 and PD1 expression by Foxp3+ Treg cells (Center), and CD25 expression by Tfr cells (Right). (B) Summary data of Treg, Tfr, and CD25− Tfr cells. Data are pooled from seven mice, representative of two separate experiments. The line indicates mean. (C) Summary data of GC-B cells, Tfh cells, total number of CD25− Tfr cells, and gMFI of BCL6 expression by Tfr cells in spleens (n = 3–7 as indicated). (D) Summary data of Tfr, CD25− Tfr, and CD25− Tfr cells as a proportion of CD4 in Peyer’s patches (one outlier removed from 2:10 group as outlined in Materials and Methods). (E) GC-B cells and Tfh cells in Peyer’s patches (n = 7, line denotes mean). (F) Z-score heat map and clustering dendrogram of microarray gene expression by CD25hi or CD25lo Treg cells from naive mice or Treg cells from IL-2−/− mice with/without peritoneal injections of recombinant IL-2 every 8 h for the 24 h before euthanasia. Microarray data are from Fontenot et al. (12) (GEO accession no. GSE4179). (G) GSEA of CD25− Tfr vs. CD25+ Tfr cells with the gene list from IL-2β KO Treg vs. wild-type Treg cells. Data are from Yu et al. (34), (GEO accession no. GSE14350). Positive enrichment shows enrichment in CD25− Tfr cells, and negative enrichment shows enrichment in CD25+ Tfr cells (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Control, Expressing, Microarray, Gene Expression, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Human circulating Tfr cells. PBMCs were purified from the blood of healthy donors. (A) Zebra plot of Foxp3 and CXCR5 expression by CD4+CD3+CD45RA− T cells (Left) and histograms of CD127, CD25, and Helios expression by the indicated populations (Right), representative of more than five (Left) and two separate (Right) experiments, respectively. (B) Total of 1 × 104 cells were sorted by BD FACSAria-SORP after negative selection of CD4 by magnetic beads as CD45RA+CD127+CD25− nTconv cells, CD45RA−CD127+CD25−CXCR5+ cTfh cells, CD45RA+CD127loCD25+ nTreg cells, CD45RA−CD127loCD25+CXCR5− eTreg cells, and CD45RA−CD127loCD25+CXCR5+ cTfr cells, and methylation status was assessed by bisulfide sequencing. Data are representative of two separate experiments. (C) Summary data of protein expression by indicated populations. Data are pooled from four to nine mice, representative of two to four separate experiments. (D) Total of 5 × 104 B cells were cultured in the presence of anti-CD3 and anti-CD28 Dynabeads with/without 1 × 104 cTfh cells and/or 1 × 104 eTreg cells or cTfr cells for 6 d. CD20loCD38hi plasma cell formation (Upper) and IgG concentration in supernatants determined by ELISA (Lower) are shown (mean ± SEM of duplicates). Data are representative of two separate experiments. (E–G) Total of 1 × 104 cells were sorted by BD FACSAria-SORP as in B before RNA-Seq. Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of <0.05 and a fold change of ≥2 were considered DE. (E) Euclidean distance analysis of whole-gene expression profile. (F) Z-scored heat maps of the top 25 up-regulated (Left) and the top 25 down-regulated (Right) Tfh genes. (G) Plots of log2-fold change (M) and gene expression (A) between cTfr and Tfh cells (Left) or cTfr and eTreg cells (Right). DE genes are highlighted in red (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Purification, Expressing, Selection, Magnetic Beads, Methylation, Sequencing, Cell Culture, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Produced, Software, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Function and phenotype of Tfr cells in blood and tonsils. (A–D) PBMCs were purified from the blood of healthy donors. (A) Expression of HLA-DR and Ki-67 by nTreg (Left), cTfr (Center), and eTreg (Right) cells. Data are representative of three separate experiments. (B and C) Total of 5 × 104 B cells were cultured in the presence of anti-CD3 and anti-CD28 Dynabeads at a 1:32 Dynabead/T-cell ratio (Thermo Fisher Scientific) with/without 1 × 104 Tfh cells and/or 1 × 104 eTreg cells or indicated Tfr cells for 6 d. (B) CD20loCD38hi plasma cell formation. (C) Total number of CD20loCD38hi plasma cells. Mean ± SEM of duplicates. Data are representative of two separate experiments (*P ≤ 0.05; ns, not significant). (D) Total of 1 × 104 nTconv cells from PBMCs were stained with CTV cultured for 6 d with irradiated CD4− PBMCs with 1 μg of anti-CD3 with/without 5 × 103 of the indicated suppressor cells. Data are representative of two separate experiments. (E and F) Fresh human tonsils were obtained from the National Disease Resource Interchange. (E) BCL6 and CXCR5 expression by Foxp3+Helios− cells. (F) ACCENSE analysis of gated CD45RA−Foxp3+Helios+ Treg cells. Cells were mapped by CD25, BCL6, CXCR5, and PD1. K-means were used to identify separate regions (Upper Left). (G) Histograms of ACCENSE-identified populations. Population 2, CD25− Tfr cells; population 8, Tfr cells; population 1, 3–7, eTreg cells. Population 1, 3–7 was recombined by concatenation of flow cytometry standard files.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Purification, Expressing, Cell Culture, Clinical Proteomics, Staining, Irradiation, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Human tonsillar Tfr cells. (A–C) Fresh human tonsils were obtained from the National Disease Resource Interchange. (A) CD45RA−CD4+CD3+B220− gated cells were then further dissected to CXCR5−BCL6− Foxp3+ as eTreg cells, CXCR5+BCL6loFoxp3− as Tfh cells, CXCR5+BCL6loFoxp3+Helios+ as BCL6lo Tfr cells or Helios− Tfr cells (when Helios−), and CXCR5+BCL6hi as GC-Tfh (when Foxp3−) or BCL6hi Tfr cells (when Foxp3+). (Upper Right) Foxp3 FMO staining is shown. (B) Expression of PD1, CD25, and CXCR5 by indicated populations. (C) Summary data of indicated marker expression. Data are pooled from six mice, representative of two separate experiments (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001). ns, not significant.

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques: Staining, Expressing, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A distinct subpopulation of CD25 − T-follicular regulatory cells localizes in the germinal centers

doi: 10.1073/pnas.1705551114

Figure Lengend Snippet: Primary and secondary antibodies for immunohistochemistry

Article Snippet: Antibodies used for microscopy are shown in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Primary Secondary Foxp3 Rat anti-mouse Foxp3 Biotin (clone FJK-16s; eBioscience) Donkey anti-rat Alexa Fluor 488 or 594 CD25 Goat anti-mouse CD25 antibody (AF2438, Accession no. Q544l2; R&D Systems) Donkey anti-goat Alexa Fluor 568 IgD Rat anti-mouse IgD AF647 (clone 11-26c.2a; Biolegend) — GL7 Rat anti-human/mouse GL7, eFluor660 (clone GL-7; eBioscience) — Open in a separate window Primary and secondary antibodies for immunohistochemistry Flow Cytometry and Antibodies.

Techniques:

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included . (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in and the other experiments are in .

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Labeling

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking promotes Foxp3 expression in an IL-2-independent manner . (a) Fold Increase (FI) in GFP/FoxP3 MFI for Treg activated with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab with or without anti-IL-2 Ab (Anti-IL-2), recombinant CD25 (rCD25), or IL-2 ( n = 7). (b) Representative histograms demonstrating GFP/Foxp3 expression by Treg isolated from GFP/Foxp3 knock-in mice on a conventional B6 background mice or on a CD25 −/− background (B6 GFP/Foxp3.CD25 −/− mice) following 3 days of culture with anti-CD3 and anti-28 alone or in conjunction with plate-bound anti-CD44. (c) Representative histograms illustrating GFP/FoxP3 expression of Treg following 3 days of culture with anti-CD3 and anti-CD28 alone, or in conjunction with plate-bound CD44 Ab, and with or without varying doses of IL-2. Data are representative of two experiments.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Recombinant, Isolation, Knock-In

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking . (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Inhibition, Expressing, Isolation, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 1. Experimental strategy and background. (A) To assess the effect of PDZ proteins on GluN1-3 exit from the ER, the C-terminus was appended to VE (VE1-3). (B) The last two exons coding for the C-terminus GluN1 are alternatively spliced, and have either the C1 splice cassette, or not, joined to the C2 or C2′ splice cassette. The possible sequence determinants affecting ER retention and ER exit are shown for the different GluN1 C-termini. The PDZ-binding domains are underlined. The ER retention motif is indicated by three asterisks (***). PKC and PKA phosphorylation sites are shown as double-daggers (‡‡). GluN1-3 and GluN1-4 have a nested di-valine exit signal in the PDZ-binding motif, which has been shown to speed forward trafficking (++). (C) When cells are maintained at 40 °C, VE remains unfolded and chaperone-bound in the ER, but mobile [19], and when they are shifted to 32 °C, VE rapidly folds correctly and exits the ER in a wave, which can be assessed by: 1) changes in co-localization with subcellular markers, 2) Endo H sensitivity of two N-linked glycans on VE, and 3) antibody labeling to cell surface VE. Since the rate of ER exit and traversal of the secretory pathway can be accurately assessed, appending the GluN1-3 C-terminus to VE and co-transfecting with prospective interacting proteins were used to identify interacting proteins that could enhance ER-exit. (D) IL-2α has been used extensively as a trafficking reporter for analysis of trafficking determinants. An excellent monoclonal antibody hybridoma is available (7G7B6; ATCC) for immunofluorescence, ELISAs, and immunoprecipitation. For analysis of secretory trafficking by Western blot, IL2RA is extensively glycosylated such that the cell-surface form is about 25 kD larger than the immature form localized early in the secretory pathway.

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Sequencing, Binding Assay, Phospho-proteomics, Antibody Labeling, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 3. An SH3–GuK domain interaction is necessary for GluN1-3 ER exit. (A) Surface versus total ELISAs were performed at 3 h after switching cells to 32 °C with co-transfected VE1-3 and SAP97, or VE1-3 and cotransfected truncated segments of SAP97. Co-transfections of PDZ1-3 domains resulted in diminished surface expression. Successive removal of the GuK domain, and SH3–GuK domains of SAP97 resulted in a significant loss of cell-surface VE1-3 (**p b 0.05, ANOVA, post hoc comparisons), indicating that the SH3–GuK domains may be necessary to suppress ER retention of GluN1-3. (B) Expression levels of SAP97 and SAP97ΔSH3–GuK were assessed by immunoblotting transfections with anti-SAP97 antibody and anti-β-actin to control for load. No difference was observed. (C) Tac1-3Δ4 co-localized with GuK and SH3. Immunofluorescence micrographs of Tac1-3Δ4 co-expressed with EGFP control showed no shared edges and minimal co-localization (top row; scale bars: 10 μm). Tac1-3Δ4 co-expressed with EGFP–GuK showed striking co-localization (middle row). Tac1-3Δ4 showed co- localization with the SH3–GFP domain as well (bottom row; yellow arrows indicate shared edges). (D) Co-immunoprecipitation experiments (repeated 4 times with similar results) indicated that the SH3 and GuK domains interacted with the GluN1-3 C-terminus. Cells co-transfected with Tac1-3Δ4 and EGFP, EGFP–GuK, or SH3–EGFP were lysed, prepared, and subjected to immunoprecipitation with 7G7B6 antibody and SDS PAGE. Blots on the left were probed with anti-human IL2RA antibody to confirm immunoprecipitation. Blots on the right were probed with anti-GFP antibody. Panels show input (5%; In), I.P. (25%), and unbound (Un).

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Transfection, Expressing, Western Blot, Control, Immunoprecipitation, SDS Page

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 4. The SAP97 SH3 domain is sufficient to block ER retention. (A) Cell surface immunofluorescence immunolabeling of cells transfected with Tac1-3Δ4 and GFP (top row), or GFP–GuK (middle row), or SH3–GFP (bottom row) was performed as described in Materials and methods. Images were taken at low magnification (10×; scale bar: 100 μm) with the same settings for comparison. Left column shows GFP fluorescence, and the right column shows surface labeling of Tac1-3Δ4. Tac1-3Δ4 + GFP surface labeling showed background immunofluorescence (upper right picture), +GFP–GuK showed modest surface labeling coincident with GFP fluorescence (middle right; see arrows), and Tac1-3Δ4 showed saturating fluorescence on some cells that co-expressed SH3–GFP (bottom right; top two white arrows). This experiment was repeated more than 3 times with similar results. (B) Western blots of Tac1-3Δ4 alone or co-transfected with SH3–GFP, or GFP–GuK, full length SAP97, or full-length Tac1-3 probed with anti-IL2RA antibody (lanes indicated above blot) demonstrated that Tac1-3Δ4 alone showed only the immature, ER-localized band, whereas SH3–GFP co-expression caused a shift to mature Tac1-3Δ4 that was similar to the full length C-terminus, Tac1-3. Tac1-3Δ4 co-expression with full-length SAP97 showed only a small amount of mature form. (C) Surface versus total ELISAs with Tac1-3, and mutants, co-transfected with segments or chimeras of SAP97. Tac1-3 surface expression was significantly greater than Tac1-3Δ4 (††p b 0.01, one-way ANOVA, post hoc comparisons; N = number of independent transfections). Co-expression of Tac1-3Δ4 with the GFP–GuK domain and SH3–GuK–GFP domains resulted in no significant change in surface expression. Fusion of the N-terminal 120 amino acids with the SH3–GuK domain of SAP97 (Wu–GFP) co-expressed with Tac1-3Δ4 resulted in significant suppression of ER retention (†p b 0.05, ANOVA, post hoc comparisons). The SH3–GFP domain of SAP97 alone was sufficient to block ER retention of Tac1-3Δ4 (††p b 0.01, ANOVA, post hoc comparisons), confirming the results in (A) and (B). Co-expression of full-length SAP97 with Tac1-3Δ4 resulted in only a small and insignificant increase in surface expression. (D) Deletions, segments, and fusions of βSAP97 used in this study.

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Blocking Assay, Immunolabeling, Transfection, Comparison, Labeling, Western Blot, Expressing