anti plzf Search Results


antibody anti plzf  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology antibody anti plzf
Antibody Anti Plzf, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/PLZF+Antibody/pm24554733-157-11-15
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anti plzf for if  (R&D Systems)
93
R&D Systems anti plzf for if
MBZ leads to a loss of germ cells and impaired function of Sertoli cells. ( A ) Co-IF staining of <t>Plzf</t> <t>and</t> <t>γH2Ax</t> in the testes of MBZ-treated and control mice on the 7th day post-injection; scale bars = 50 and 10 μm. ( B ) The average number of Plzf and γH2Ax positive cells, per tubule, in the testes of mice exposed to MBZ after the 7th day; ( C ) IF staining of Sox9 in the MBZ-treated and control testes. Cell nuclei were stained with Hoechst (grey); red arrow: Sertoli cell; scale bars = 50 and 10 μm. ( D , E ) Western blot analysis of Sox9 in the analyzed group of animals. GAPDH served as a normalization control. WB results were visualized by Image J. Data are MEAN ± SEM, n = 3. Three sets of biological replicates were performed. The three values are indicated as three triangles on each bar. The obtained results were statistically evaluated by the student’s t -test, where p values < 0.05 (*) and <0.01 (**) were considered statistically significant.
Anti Plzf For If, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti plzf  (R&D Systems)
94
R&D Systems anti plzf
MBZ leads to a loss of germ cells and impaired function of Sertoli cells. ( A ) Co-IF staining of <t>Plzf</t> <t>and</t> <t>γH2Ax</t> in the testes of MBZ-treated and control mice on the 7th day post-injection; scale bars = 50 and 10 μm. ( B ) The average number of Plzf and γH2Ax positive cells, per tubule, in the testes of mice exposed to MBZ after the 7th day; ( C ) IF staining of Sox9 in the MBZ-treated and control testes. Cell nuclei were stained with Hoechst (grey); red arrow: Sertoli cell; scale bars = 50 and 10 μm. ( D , E ) Western blot analysis of Sox9 in the analyzed group of animals. GAPDH served as a normalization control. WB results were visualized by Image J. Data are MEAN ± SEM, n = 3. Three sets of biological replicates were performed. The three values are indicated as three triangles on each bar. The obtained results were statistically evaluated by the student’s t -test, where p values < 0.05 (*) and <0.01 (**) were considered statistically significant.
Anti Plzf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/Human+PLZF+Antibody/pm37004113-74-14-20
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plzf apc  (R&D Systems)
93
R&D Systems plzf apc
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Plzf Apc, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/Human+PLZF+APC-conjugated+Antibody/pm37856221-226-50-54
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plzf  (Proteintech)
93
Proteintech plzf
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Plzf, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/PLZF+Antibody/pmc09458860-53-21-23
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anti plzf  (Novus Biologicals)
92
Novus Biologicals anti plzf
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Anti Plzf, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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monoclonal antibody mab2944  (R&D Systems)
93
R&D Systems monoclonal antibody mab2944
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Monoclonal Antibody Mab2944, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/Human+PLZF+Antibody/pm29458530-178-9-12
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anti plzf allophycocyanin  (R&D Systems)
94
R&D Systems anti plzf allophycocyanin
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Anti Plzf Allophycocyanin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti zbtb16  (R&D Systems)
94
R&D Systems anti zbtb16
Figure 1. Prenatal <t>PLZF+CD4+</t> T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.
Anti Zbtb16, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti promyelocytic leukemia zinc finger plzf  (Novus Biologicals)
92
Novus Biologicals anti promyelocytic leukemia zinc finger plzf
Primer sequence for real-time quantitative PCR
Anti Promyelocytic Leukemia Zinc Finger Plzf, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plzf antibody  (R&D Systems)
94
R&D Systems plzf antibody
Figure <t>3</t> <t>DBCP</t> and 2-BP reduce <t>PLZF+</t> spermatogonia and HILI+ spermatocytes, respectively, in in vitro spermatogenic cultures. (A) Graphical representation showing that 100 μM DBCP and 2-BP reduce overall colony size (left graph) and PLZF expression intensity (right graph) compared to controls. DBCP significantly reduces mean colony size area, whereas 2-BP and DBCP significantly reduce PLZF staining intensity. p b 0.01. (B) Representative 5X images obtained by the Cellomics ArrayScan VT1 of PLZF+ colonies used for analyses in (A). All images are taken under the same imaging conditions and parameters. Scale bar, 5000 μm. Second row depicts zoomed up images of representative individual colonies. Scale bar, 100 μm. (C) 2-BP reduces the number of HILI+ cells in culture. Representative images of HILI staining (red). Scale bar, 5000 μm. Second row shows zoomed representative images of individual colonies. Scale bar, 100 μm. Secondary-only control image is shown in Supplemental Fig. 2.
Plzf Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+plzf/PLZF+Antibody/pm25863443-145-23-25
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anti scp3  (Atlas Antibodies)
92
Atlas Antibodies anti scp3
Figure <t>3</t> <t>DBCP</t> and 2-BP reduce <t>PLZF+</t> spermatogonia and HILI+ spermatocytes, respectively, in in vitro spermatogenic cultures. (A) Graphical representation showing that 100 μM DBCP and 2-BP reduce overall colony size (left graph) and PLZF expression intensity (right graph) compared to controls. DBCP significantly reduces mean colony size area, whereas 2-BP and DBCP significantly reduce PLZF staining intensity. p b 0.01. (B) Representative 5X images obtained by the Cellomics ArrayScan VT1 of PLZF+ colonies used for analyses in (A). All images are taken under the same imaging conditions and parameters. Scale bar, 5000 μm. Second row depicts zoomed up images of representative individual colonies. Scale bar, 100 μm. (C) 2-BP reduces the number of HILI+ cells in culture. Representative images of HILI staining (red). Scale bar, 5000 μm. Second row shows zoomed representative images of individual colonies. Scale bar, 100 μm. Secondary-only control image is shown in Supplemental Fig. 2.
Anti Scp3, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


MBZ leads to a loss of germ cells and impaired function of Sertoli cells. ( A ) Co-IF staining of Plzf and γH2Ax in the testes of MBZ-treated and control mice on the 7th day post-injection; scale bars = 50 and 10 μm. ( B ) The average number of Plzf and γH2Ax positive cells, per tubule, in the testes of mice exposed to MBZ after the 7th day; ( C ) IF staining of Sox9 in the MBZ-treated and control testes. Cell nuclei were stained with Hoechst (grey); red arrow: Sertoli cell; scale bars = 50 and 10 μm. ( D , E ) Western blot analysis of Sox9 in the analyzed group of animals. GAPDH served as a normalization control. WB results were visualized by Image J. Data are MEAN ± SEM, n = 3. Three sets of biological replicates were performed. The three values are indicated as three triangles on each bar. The obtained results were statistically evaluated by the student’s t -test, where p values < 0.05 (*) and <0.01 (**) were considered statistically significant.

Journal: International Journal of Molecular Sciences

Article Title: Mebendazole-Induced Blood-Testis Barrier Injury in Mice Testes by Disrupting Microtubules in Addition to Triggering Programmed Cell Death

doi: 10.3390/ijms23084220

Figure Lengend Snippet: MBZ leads to a loss of germ cells and impaired function of Sertoli cells. ( A ) Co-IF staining of Plzf and γH2Ax in the testes of MBZ-treated and control mice on the 7th day post-injection; scale bars = 50 and 10 μm. ( B ) The average number of Plzf and γH2Ax positive cells, per tubule, in the testes of mice exposed to MBZ after the 7th day; ( C ) IF staining of Sox9 in the MBZ-treated and control testes. Cell nuclei were stained with Hoechst (grey); red arrow: Sertoli cell; scale bars = 50 and 10 μm. ( D , E ) Western blot analysis of Sox9 in the analyzed group of animals. GAPDH served as a normalization control. WB results were visualized by Image J. Data are MEAN ± SEM, n = 3. Three sets of biological replicates were performed. The three values are indicated as three triangles on each bar. The obtained results were statistically evaluated by the student’s t -test, where p values < 0.05 (*) and <0.01 (**) were considered statistically significant.

Article Snippet: The first antibody against Sox9 for WB and IF was purchased from Invitrogen (AB5535, Millipore, Billerica, MA); anti-espin for WB and IF (611656, BD Biosciences, New York City, NY, USA); anti-β-tubulin for WB and IF (AB0039, Abways, Beijing, China); anti-E-cad for WB (3195, CST, Boston, MA, USA); anti-N-cad for WB (NBP1-48309,Novus Biologicals, Littleton, CO, USA); anti-β-catenin for WB (610153, BD Biosciences, New York City, NY, USA); anti-VIM for WB (AF2105, RD,Minneapolis, MN, USA); anti-β-actin for WB (ab8226, Abcam, Cambridge, UK); anti- GAPDH for WB (Abcam, ab9485, Cambridge, UK); anti-Ddx4 for IF (ab13840, Abcam, Cambridge, UK); anti-Plzf for IF (AF2944, RD, Minneapolis, MN, USA); anti-γH2AX for IF (ab26350, Abcam, Cambridge, UK).

Techniques: Staining, Injection, Western Blot

Figure 1. Prenatal PLZF+CD4+ T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 1. Prenatal PLZF+CD4+ T cells are a heterogeneous population. (A) Population-wide RNA-Seq of prenatal small intestine (SI) PLZF+CD4+ T cells (n = 5) identifies transcriptional expression of numerous cytokines after PMA/ionomycin stimulation. Volcano plot of enriched (orange) and depleted (blue) differentially expressed (DE) gene transcripts (log2 fold-change > 1, FDR ≤ 0.05) within stimulated SI PLZF+CD4+ T cells. (B) Single-cell transcriptional analysis of memory Vα7.2–PLZF+CD4+TCRαβ+ T cells from the prenatal SI of 3 donors identifies 7 distinct clusters. Uniform manifold approximation and projection (UMAP) visualization of identified clusters within SI PLZF+CD4+ T cells in which each cluster is surrounded by a median-centered ellipse and labeled according to its functional annotation. ISG, IFN-stimulated gene. (C) Distinct transcriptional signatures within clusters of SI PLZF+CD4+ T cells. Heatmap of unsupervised hierarchical clustering of DE genes by indicated cluster. Labels indicate genes utilized to determine functional annotation. (D) Pathway enrichment analysis within clusters of SI PLZF+CD4+ T cells. Dot plot of selected Gene Ontology terms, Reactome Gene Sets, and Kyoto Ency- clopedia of Genes and Genomes pathways enriched within genes DE in each cluster by Metascape analysis. Downregulated pathways are indicated with negative number of genes and outliers indicated with text overlay.

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: RNA Sequencing, Expressing, Labeling, Functional Assay

Figure 2. Prenatal PLZF+CD4+ T cells display spatially compartmentalized effector function. (A) Representative flow plot of the gating strategy used for the identification of cytokine-producing cells within memory PLZF+ and PLZF– CD4+ T cells in the prenatal SI and MLNs. All PLZF+ and PLZF– populations were pregated on TCRαβ+CD4+CD45RO+ T cells. (B–I) Distinct patterns of cytokine production within memory CD4+ T cells from the prenatal SI and MLNs. Quantification (B, D, E, G, and H) and representative flow plots (C, F, and I) of indicated intracellular cytokine staining after stimulation within PLZF– (gray) and PLZF+ (orange) CD45RO+CD4+ T cells. Cells were stimulated as indicated for 16 hours, and Brefeldin A was added in the last 4 hours. Flow plots indicate

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 2. Prenatal PLZF+CD4+ T cells display spatially compartmentalized effector function. (A) Representative flow plot of the gating strategy used for the identification of cytokine-producing cells within memory PLZF+ and PLZF– CD4+ T cells in the prenatal SI and MLNs. All PLZF+ and PLZF– populations were pregated on TCRαβ+CD4+CD45RO+ T cells. (B–I) Distinct patterns of cytokine production within memory CD4+ T cells from the prenatal SI and MLNs. Quantification (B, D, E, G, and H) and representative flow plots (C, F, and I) of indicated intracellular cytokine staining after stimulation within PLZF– (gray) and PLZF+ (orange) CD45RO+CD4+ T cells. Cells were stimulated as indicated for 16 hours, and Brefeldin A was added in the last 4 hours. Flow plots indicate

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Staining

Figure 3. IL-7 and TGF-β reciprocally regulate the expansion of prenatal PLZF+CD4+ T cells. (A) Whole prenatal tissue concentrations of IL-7 and IL-15 measured by cytokine bead array identify higher cytokine levels in the SI compared with the MLNs (n = 6). (B–E) Specific accumulation of PLZF+CD4+ T cells in response to IL-7. (B) Representative flow plots and (C) frequencies of PLZF+CD4+ T cells derived from naive CD4+ T cells before initiation of cul- ture (d0) and after 6 days of culture in the presence of indicated cytokines (10 ng/mL). (D) Representative histograms of Cell Trace Violet (CTV) dilution and (E) expansion indices after 6 days of culture depict the enhanced IL-7–driven proliferation of PLZF+CD4+ T cells. (F) Representative flow plots and (G) proportions of PLZF+CD4+ T cells indicate that addition of TGF-β (10 ng/mL) inhibits their accumulation after 6 days of culture in the presence of IL-7. (H) Representative histograms of CTV dilution and (I) expansion indices indicate the specific TGF-β–mediated inhibition of IL-7–driven proliferation within the PLZF+ subset of CD4+ T cells. Circles represent individual donors. Wilcoxon’s signed rank test (A, G, and I) and paired ANOVA with Tukey’s multiple-comparison test (C and E). *P < 0.05, **P < 0.01, ***P < 0.001. Numbers in flow cytometry plots represent the mean frequency of gated populations ± SD.

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 3. IL-7 and TGF-β reciprocally regulate the expansion of prenatal PLZF+CD4+ T cells. (A) Whole prenatal tissue concentrations of IL-7 and IL-15 measured by cytokine bead array identify higher cytokine levels in the SI compared with the MLNs (n = 6). (B–E) Specific accumulation of PLZF+CD4+ T cells in response to IL-7. (B) Representative flow plots and (C) frequencies of PLZF+CD4+ T cells derived from naive CD4+ T cells before initiation of cul- ture (d0) and after 6 days of culture in the presence of indicated cytokines (10 ng/mL). (D) Representative histograms of Cell Trace Violet (CTV) dilution and (E) expansion indices after 6 days of culture depict the enhanced IL-7–driven proliferation of PLZF+CD4+ T cells. (F) Representative flow plots and (G) proportions of PLZF+CD4+ T cells indicate that addition of TGF-β (10 ng/mL) inhibits their accumulation after 6 days of culture in the presence of IL-7. (H) Representative histograms of CTV dilution and (I) expansion indices indicate the specific TGF-β–mediated inhibition of IL-7–driven proliferation within the PLZF+ subset of CD4+ T cells. Circles represent individual donors. Wilcoxon’s signed rank test (A, G, and I) and paired ANOVA with Tukey’s multiple-comparison test (C and E). *P < 0.05, **P < 0.01, ***P < 0.001. Numbers in flow cytometry plots represent the mean frequency of gated populations ± SD.

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Derivative Assay, Inhibition, Comparison, Flow Cytometry

Figure 4. Differences in IL-7 signaling contribute to the preferential expansion of PLZF+CD4+ T cells. (A) Representative histograms, (B) frequencies, and (C) mean fluorescence intensity (MFI) of IL-7R expression within subsets of naive CD4+ T cells (n = 6). (D) Representative histograms and (E) normalized MFI of phosphorylated STAT5B (pSTAT5B) within subsets of naive CD4+ T cells after 30 minutes of stimulation with IL-7 (n = 6). (F) Normalized propor- tions of PLZF+CD4+ T cells relative to DMSO control after 6 days of stimulation with IL-7 in the presence of indicated signaling inhibitors. Inhibitors used: SIS (SIS3, SMAD3), PD0 (PD0325901, MEK), PIC (pictilisib, PI3Kα/δ), SH (SH-4-54, STAT3 and STAT5), S3 (STAT3-IN-1, STAT3), J1 (JANEX-1, JAK3), and Sol (solcitinib, JAK1). Gray lines connect values derived from the same donor. (G) Normalized expansion indices relative to DMSO control within subsets of CD4+ T cells after 6 days of stimulation with IL-7 in the presence of the indicated signaling inhibitors and (H) representative histograms of CTV dilution within indicated populations of naive CD4+ T cells after treatment with IL-7 and PD0 depict the selective sensitivity of PLZF+CD4+ T cells to MEK/ERK signaling. Circles represent individual donors. Wilcoxon’s signed rank test (B, C, and E) and paired ANOVA with Tukey’s multiple-comparison test (F and G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 4. Differences in IL-7 signaling contribute to the preferential expansion of PLZF+CD4+ T cells. (A) Representative histograms, (B) frequencies, and (C) mean fluorescence intensity (MFI) of IL-7R expression within subsets of naive CD4+ T cells (n = 6). (D) Representative histograms and (E) normalized MFI of phosphorylated STAT5B (pSTAT5B) within subsets of naive CD4+ T cells after 30 minutes of stimulation with IL-7 (n = 6). (F) Normalized propor- tions of PLZF+CD4+ T cells relative to DMSO control after 6 days of stimulation with IL-7 in the presence of indicated signaling inhibitors. Inhibitors used: SIS (SIS3, SMAD3), PD0 (PD0325901, MEK), PIC (pictilisib, PI3Kα/δ), SH (SH-4-54, STAT3 and STAT5), S3 (STAT3-IN-1, STAT3), J1 (JANEX-1, JAK3), and Sol (solcitinib, JAK1). Gray lines connect values derived from the same donor. (G) Normalized expansion indices relative to DMSO control within subsets of CD4+ T cells after 6 days of stimulation with IL-7 in the presence of the indicated signaling inhibitors and (H) representative histograms of CTV dilution within indicated populations of naive CD4+ T cells after treatment with IL-7 and PD0 depict the selective sensitivity of PLZF+CD4+ T cells to MEK/ERK signaling. Circles represent individual donors. Wilcoxon’s signed rank test (B, C, and E) and paired ANOVA with Tukey’s multiple-comparison test (F and G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Fluorescence, Expressing, Control, Derivative Assay, Comparison

Figure 5. IL-7 and TGF-β sustain reciprocal regulation of MHC class II–restricted PLZF+CD4+ naive T cells in the presence of TCR signaling. (A and B) Blocking of MHC class II–TCR interactions inhibits the activation of PLZF+CD4+ T cells. (A) Representative flow plots of intracellular CD40L/CD154 expres- sion within SI PLZF+CD4+ T cells (top) and IFN-γ production within CD154+PLZF+CD4+ T cells (bottom) cocultured with allogeneic adult CD14+ APCs in the presence of a pan–MHC-II blocking antibody (αHLA DR-DP-DQ) or isotype control. (B) Stimulation index, calculated as the percentage of CD154+IFN-γ+ within PLZF+CD4+ T cells after 16 hours of coculture with allogeneic adult CD14+ APCs in the presence of a pan MHC II blocking antibody (αHLA DR-DP-DQ) compared with isotype control (IgG2). (C–E) TCR signaling does not interfere with the IL-7–driven accumulation of PLZF+CD4+ T cells. (C) Representative flow plots and (D) frequencies of PLZF+CD4+ T cells derived from naive CD4+ T cells stimulated with αCD3/CD28 in the presence of IL-2 or IL-7 for 6 days. Gray lines connect values derived from the same donor. (E) Proportions of PLZF+CD4+ T cells stimulated with indicated concentrations of αCD3/CD28 in the presence of IL-7 for 12 days. (F) Representative flow plots and (G) proportions of PLZF+CD4+ T cells indicate that stimulation with αCD3/CD28 does not interfere with the TGF-β–mediated inhibition of their IL-7–driven accumulation. (H) Representative histograms of CTV dilution and (I) expansion indices

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 5. IL-7 and TGF-β sustain reciprocal regulation of MHC class II–restricted PLZF+CD4+ naive T cells in the presence of TCR signaling. (A and B) Blocking of MHC class II–TCR interactions inhibits the activation of PLZF+CD4+ T cells. (A) Representative flow plots of intracellular CD40L/CD154 expres- sion within SI PLZF+CD4+ T cells (top) and IFN-γ production within CD154+PLZF+CD4+ T cells (bottom) cocultured with allogeneic adult CD14+ APCs in the presence of a pan–MHC-II blocking antibody (αHLA DR-DP-DQ) or isotype control. (B) Stimulation index, calculated as the percentage of CD154+IFN-γ+ within PLZF+CD4+ T cells after 16 hours of coculture with allogeneic adult CD14+ APCs in the presence of a pan MHC II blocking antibody (αHLA DR-DP-DQ) compared with isotype control (IgG2). (C–E) TCR signaling does not interfere with the IL-7–driven accumulation of PLZF+CD4+ T cells. (C) Representative flow plots and (D) frequencies of PLZF+CD4+ T cells derived from naive CD4+ T cells stimulated with αCD3/CD28 in the presence of IL-2 or IL-7 for 6 days. Gray lines connect values derived from the same donor. (E) Proportions of PLZF+CD4+ T cells stimulated with indicated concentrations of αCD3/CD28 in the presence of IL-7 for 12 days. (F) Representative flow plots and (G) proportions of PLZF+CD4+ T cells indicate that stimulation with αCD3/CD28 does not interfere with the TGF-β–mediated inhibition of their IL-7–driven accumulation. (H) Representative histograms of CTV dilution and (I) expansion indices

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Blocking Assay, Activation Assay, Control, Derivative Assay, Inhibition

Figure 6. IL-7 is sufficient to promote the emergence of memory phenotype PLZF+CD4+ T cells. (A) Representative flow plots and (B) frequencies of CD45RO+ cells within PLZF+ and PLZF– CD4+ T cells after 12 days of culture with IL-7 in the presence of indicated concentrations of αCD3/CD28 stimulation. (C) Representative flow plots of intracellular cytokine staining and (D and E) frequencies of (D) TNF-α+ and (E) IFN-γ+ cells within subsets of CD4+ T cells following restimulation with PMA/ionomycin after 12 days of culture with IL-7 and indicated concentrations of αCD3/CD28 stim- ulation. Circles represent individual donors. Paired ANOVA with Tukey’s multiple-comparison test (B, D, and E). **P < 0.01, ***P < 0.001. Numbers in flow cytometry plots represent the mean frequency ± SD of gated populations.

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 6. IL-7 is sufficient to promote the emergence of memory phenotype PLZF+CD4+ T cells. (A) Representative flow plots and (B) frequencies of CD45RO+ cells within PLZF+ and PLZF– CD4+ T cells after 12 days of culture with IL-7 in the presence of indicated concentrations of αCD3/CD28 stimulation. (C) Representative flow plots of intracellular cytokine staining and (D and E) frequencies of (D) TNF-α+ and (E) IFN-γ+ cells within subsets of CD4+ T cells following restimulation with PMA/ionomycin after 12 days of culture with IL-7 and indicated concentrations of αCD3/CD28 stim- ulation. Circles represent individual donors. Paired ANOVA with Tukey’s multiple-comparison test (B, D, and E). **P < 0.01, ***P < 0.001. Numbers in flow cytometry plots represent the mean frequency ± SD of gated populations.

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Staining, Comparison, Flow Cytometry

Figure 7. IL-7 modulates the effector maturation of prenatal PLZF+CD4+ T cells. Functional studies following a 2-step in vitro culture in which naive CD4+ T cells were first expanded in the presence of IL-7 alone, followed by 7 days of matura- tion in the indicated conditions: IL-7 alone, IL-7 + TCR (stimulated with αCD3/CD28 mAbs), Th0 (TCR + IL-2), Th1 (TCR + IL-12), Th17 (TCR + IL-23 + IL-1β). (A–D) IL-7 enhances the emergence of IFN-γ+PLZF+CD4+ T cells. (A, B, and D) Proportion of IFN-γ+ cells within CD4+ T cell subsets exposed to the indicated differentiation conditions and following 24-hour restimulation with αCD3/CD28 (TCR) or IL-12/IL-18 (Bystander). (C) Representative flow plots of intracellular cytokine staining within subsets of CD4+ T cells differentiated under indicated conditions and following 24-hour restimulation with αCD3/CD28. (E and F) IL-7 dampens the emergence of IL-17+PLZF+CD4+ T cells. (E) Representative flow plots of intracellular cytokine staining and (F) proportions of IL-17+ cells within indicated CD4+ T cell subsets matured under Th17-skewing conditions and following PMA/ionomycin stimulation. Circles represent individual donors. Wilcoxon’s signed rank test to compare PLZF+ to PLZF– fractions and paired stimulations across maturation conditions (A, B, D, and F). *P < 0.05, **P < 0.01. Numbers in flow cytometry plots represent the mean frequency of gated populations ± SD.

Journal: JCI insight

Article Title: Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

doi: 10.1172/jci.insight.164672

Figure Lengend Snippet: Figure 7. IL-7 modulates the effector maturation of prenatal PLZF+CD4+ T cells. Functional studies following a 2-step in vitro culture in which naive CD4+ T cells were first expanded in the presence of IL-7 alone, followed by 7 days of matura- tion in the indicated conditions: IL-7 alone, IL-7 + TCR (stimulated with αCD3/CD28 mAbs), Th0 (TCR + IL-2), Th1 (TCR + IL-12), Th17 (TCR + IL-23 + IL-1β). (A–D) IL-7 enhances the emergence of IFN-γ+PLZF+CD4+ T cells. (A, B, and D) Proportion of IFN-γ+ cells within CD4+ T cell subsets exposed to the indicated differentiation conditions and following 24-hour restimulation with αCD3/CD28 (TCR) or IL-12/IL-18 (Bystander). (C) Representative flow plots of intracellular cytokine staining within subsets of CD4+ T cells differentiated under indicated conditions and following 24-hour restimulation with αCD3/CD28. (E and F) IL-7 dampens the emergence of IL-17+PLZF+CD4+ T cells. (E) Representative flow plots of intracellular cytokine staining and (F) proportions of IL-17+ cells within indicated CD4+ T cell subsets matured under Th17-skewing conditions and following PMA/ionomycin stimulation. Circles represent individual donors. Wilcoxon’s signed rank test to compare PLZF+ to PLZF– fractions and paired stimulations across maturation conditions (A, B, D, and F). *P < 0.05, **P < 0.01. Numbers in flow cytometry plots represent the mean frequency of gated populations ± SD.

Article Snippet: Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

Techniques: Functional Assay, In Vitro, Staining, Flow Cytometry

Primer sequence for real-time quantitative PCR

Journal: The World Journal of Men's Health

Article Title: Bisphenol Analogs Downregulate the Self-Renewal Potential of Spermatogonial Stem Cells

doi: 10.5534/wjmh.230166

Figure Lengend Snippet: Primer sequence for real-time quantitative PCR

Article Snippet: The primary antibodies used were as follows: anti-GFRα1 (1:200, ab8026; Abcam), anti-promyelocytic leukemia zinc finger (PLZF) (1:200, NBP1-80894; Novus Biologicals), anti-DEAD-box polypeptide 4 (DDX4) (1:200, ab13840; Abcam), and anti-KIT, a receptor tyrosine kinase (c-Kit) (1:200, sc-365504; Santa Cruz Biotechnology).

Techniques: Sequencing, Virus

BPA, BPF, and BPS do not alter specific biomarker expression. Fluorescence microscopic images of immunocytochemistry analysis of BPA-, BPF-, and BPS-treated (0, 25, and 100 µM) spermatogonial stem cells (SSCs) (A), scale bar=50 µm, 40X magnification. Localization of specific proteins GFRα1 and PLZF (undifferentiated spermatogonia, red), VASA (germ cells, red), and c-Kit (differentiated spermatogonia, red), and DAPI (nuclei, blue), GFP (cultured germ cells enriched for SSCs, green). The ratio of marker-expressing cells per GFP + germ cells is presented by bar graph as mean±standard error of the mean (SEM) from three independent experiments. (B) Graphical representation of A is shown as the ratio of marker-expressing cells to GFP + germ cells presented as mean±SEM from three independent experiments. (C) Relative gene expression of undifferentiation biomarker genes ( Id4 , Bcl6b , Lhx1 , and Bmi1 ) are presented by bar graph as mean fold change±SEM from four independent experiments. Statistical analysis with one-way ANOVA and multiple comparisons by Dunnett’s test was conducted. There were no significant differences between treatments. BPA: bisphenol-A, BPF: bisphenol-F, BPS: bisphenol-S.

Journal: The World Journal of Men's Health

Article Title: Bisphenol Analogs Downregulate the Self-Renewal Potential of Spermatogonial Stem Cells

doi: 10.5534/wjmh.230166

Figure Lengend Snippet: BPA, BPF, and BPS do not alter specific biomarker expression. Fluorescence microscopic images of immunocytochemistry analysis of BPA-, BPF-, and BPS-treated (0, 25, and 100 µM) spermatogonial stem cells (SSCs) (A), scale bar=50 µm, 40X magnification. Localization of specific proteins GFRα1 and PLZF (undifferentiated spermatogonia, red), VASA (germ cells, red), and c-Kit (differentiated spermatogonia, red), and DAPI (nuclei, blue), GFP (cultured germ cells enriched for SSCs, green). The ratio of marker-expressing cells per GFP + germ cells is presented by bar graph as mean±standard error of the mean (SEM) from three independent experiments. (B) Graphical representation of A is shown as the ratio of marker-expressing cells to GFP + germ cells presented as mean±SEM from three independent experiments. (C) Relative gene expression of undifferentiation biomarker genes ( Id4 , Bcl6b , Lhx1 , and Bmi1 ) are presented by bar graph as mean fold change±SEM from four independent experiments. Statistical analysis with one-way ANOVA and multiple comparisons by Dunnett’s test was conducted. There were no significant differences between treatments. BPA: bisphenol-A, BPF: bisphenol-F, BPS: bisphenol-S.

Article Snippet: The primary antibodies used were as follows: anti-GFRα1 (1:200, ab8026; Abcam), anti-promyelocytic leukemia zinc finger (PLZF) (1:200, NBP1-80894; Novus Biologicals), anti-DEAD-box polypeptide 4 (DDX4) (1:200, ab13840; Abcam), and anti-KIT, a receptor tyrosine kinase (c-Kit) (1:200, sc-365504; Santa Cruz Biotechnology).

Techniques: Biomarker Discovery, Expressing, Fluorescence, Immunocytochemistry, Cell Culture, Marker, Gene Expression

Figure 3 DBCP and 2-BP reduce PLZF+ spermatogonia and HILI+ spermatocytes, respectively, in in vitro spermatogenic cultures. (A) Graphical representation showing that 100 μM DBCP and 2-BP reduce overall colony size (left graph) and PLZF expression intensity (right graph) compared to controls. DBCP significantly reduces mean colony size area, whereas 2-BP and DBCP significantly reduce PLZF staining intensity. p b 0.01. (B) Representative 5X images obtained by the Cellomics ArrayScan VT1 of PLZF+ colonies used for analyses in (A). All images are taken under the same imaging conditions and parameters. Scale bar, 5000 μm. Second row depicts zoomed up images of representative individual colonies. Scale bar, 100 μm. (C) 2-BP reduces the number of HILI+ cells in culture. Representative images of HILI staining (red). Scale bar, 5000 μm. Second row shows zoomed representative images of individual colonies. Scale bar, 100 μm. Secondary-only control image is shown in Supplemental Fig. 2.

Journal: Stem cell research

Article Title: Assessing reproductive toxicity of two environmental toxicants with a novel in vitro human spermatogenic model.

doi: 10.1016/j.scr.2015.03.002

Figure Lengend Snippet: Figure 3 DBCP and 2-BP reduce PLZF+ spermatogonia and HILI+ spermatocytes, respectively, in in vitro spermatogenic cultures. (A) Graphical representation showing that 100 μM DBCP and 2-BP reduce overall colony size (left graph) and PLZF expression intensity (right graph) compared to controls. DBCP significantly reduces mean colony size area, whereas 2-BP and DBCP significantly reduce PLZF staining intensity. p b 0.01. (B) Representative 5X images obtained by the Cellomics ArrayScan VT1 of PLZF+ colonies used for analyses in (A). All images are taken under the same imaging conditions and parameters. Scale bar, 5000 μm. Second row depicts zoomed up images of representative individual colonies. Scale bar, 100 μm. (C) 2-BP reduces the number of HILI+ cells in culture. Representative images of HILI staining (red). Scale bar, 5000 μm. Second row shows zoomed representative images of individual colonies. Scale bar, 100 μm. Secondary-only control image is shown in Supplemental Fig. 2.

Article Snippet: Other reagents used that are not listed above include the following: 2-bromopropane (2-BP), 1,2-dibromo-3-chloropropane (DBCP), and l-sulforaphane (all from Sigma-Aldrich, St. Louis, MO); PLZF antibody (R&D Systems, Minneapolis, MN); and HILI antibody (Bioss, Inc., Woburn, MA).

Techniques: In Vitro, Expressing, Staining, Imaging, Control