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cd3e, mouse(145-2c11)  (Biotium)
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cd4 antibody  (NSJ Bioreagents)
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cd4 pe cy7  (Cytek Biosciences)
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pe cy7 anti cd8  (Cytek Biosciences)
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cd8 pe cy7 rpa t8 tonbo biosciences  (Cytek Biosciences)
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pe cy7 conjugated anti cd8 antibody  (Cytek Biosciences)
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biolegend cd8 pe cy7  (Cytek Biosciences)
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cd8 apc cy7  (Cytek Biosciences)
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apc cy7 cd62l tonbo  (Cytek Biosciences)
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cd8 fitc  (Cytek Biosciences)
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FIGURE 3 | Comparison of intracellular cytotoxic molecules in resting CD103+ and <t>CD103−CD8+</t> T cells. (A) Representative dot plots and (B) graphs of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells. (C) Comparison of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells in the endometrium (EM), endocervix and ectocervix (CX/ECX). (D) Comparison of pre- vs. postmenopausal (white) women. Each dot represents a different patient. *p < 0.05, **p < 0.01, ***p < 0.001.
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pe conjugated cd8  (Cytek Biosciences)
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FIGURE 3 | Comparison of intracellular cytotoxic molecules in resting CD103+ and <t>CD103−CD8+</t> T cells. (A) Representative dot plots and (B) graphs of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells. (C) Comparison of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells in the endometrium (EM), endocervix and ectocervix (CX/ECX). (D) Comparison of pre- vs. postmenopausal (white) women. Each dot represents a different patient. *p < 0.05, **p < 0.01, ***p < 0.001.
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pe cy7 conjugated cd4  (Cytek Biosciences)
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Cytek Biosciences pe cy7 conjugated cd4
ATF7ip is required for in vitro Th17 differentiation . (A) Relative expression of Atf7ip mRNA in tissues and immune cells downloaded from the BioGPS expression database. Red bars highlight hematopoietic cells or T cells. (B–D) Relative expression of Atf7ip mRNA in mouse tissues (B), <t>CD4</t> + T cell subsets (C), or naive CD4 + T cells (D) stimulated with either anti-CD3 (2 µg) or both anti-CD3 (2 µg) and anti-CD28 (2 µg) for the indicated times. (E–H) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated under Th17 (E), Th1 (F), iT reg cell (G), or Th2 (H) conditions and analyzed for intracellular cytokine or Foxp3 expression. (I) Rorγt protein expression under Th17 conditions. A histogram of Rorγt expression under Th0 conditions is included for reference. Each data point in D–H represents an individual mouse with three mice per genotype. Data are representative of five (E) or two (B–D and F–I) independent experiments. Error bars in B and C show mean with SEM of technical replicates. Error bars (D–H) show mean with SD. **, P < 0.01 by Student’s t test.
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Image Search Results


FIGURE 3 | Comparison of intracellular cytotoxic molecules in resting CD103+ and CD103−CD8+ T cells. (A) Representative dot plots and (B) graphs of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells. (C) Comparison of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells in the endometrium (EM), endocervix and ectocervix (CX/ECX). (D) Comparison of pre- vs. postmenopausal (white) women. Each dot represents a different patient. *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: Frontiers in immunology

Article Title: Differential Cytotoxic Function of Resident and Non-resident CD8+ T Cells in the Human Female Reproductive Tract Before and After Menopause.

doi: 10.3389/fimmu.2020.01096

Figure Lengend Snippet: FIGURE 3 | Comparison of intracellular cytotoxic molecules in resting CD103+ and CD103−CD8+ T cells. (A) Representative dot plots and (B) graphs of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells. (C) Comparison of intracellular levels of perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells in the endometrium (EM), endocervix and ectocervix (CX/ECX). (D) Comparison of pre- vs. postmenopausal (white) women. Each dot represents a different patient. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103–BV711 (Biolegend), CD4-PE-Cy5.5, CD103–PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience).

Techniques: Comparison

FIGURE 4 | Comparison of degranulation and release of cytotoxic molecules in CD103+ and CD103−CD8+ T cells after activation. (A) Representative dot plot and (B) graph of CD107a expression in CD103+ and CD103−CD8+ T cells after activation. EM, CX, and ECX are shown combined. (C) Comparison of pre and postmenopausal women for CD107a expression. (D) Comparison of the percent of cells expressing perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX). Each dot represents a different patient. **p < 0.01, ***p < 0.001.

Journal: Frontiers in immunology

Article Title: Differential Cytotoxic Function of Resident and Non-resident CD8+ T Cells in the Human Female Reproductive Tract Before and After Menopause.

doi: 10.3389/fimmu.2020.01096

Figure Lengend Snippet: FIGURE 4 | Comparison of degranulation and release of cytotoxic molecules in CD103+ and CD103−CD8+ T cells after activation. (A) Representative dot plot and (B) graph of CD107a expression in CD103+ and CD103−CD8+ T cells after activation. EM, CX, and ECX are shown combined. (C) Comparison of pre and postmenopausal women for CD107a expression. (D) Comparison of the percent of cells expressing perforin, granzyme B (GZB) and granzyme A (GZA) in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX). Each dot represents a different patient. **p < 0.01, ***p < 0.001.

Article Snippet: Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103–BV711 (Biolegend), CD4-PE-Cy5.5, CD103–PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience).

Techniques: Comparison, Activation Assay, Expressing

FIGURE 5 | Effect of menopausal status on release of cytotoxic molecules in CD103+ and CD103−CD8+ T cells after activation. (A) Comparison of the percent of cells expressing granzyme B (GZB), granzyme A (GZA), and perforin in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX) in premenopausal and postmenopausal women. Each dot represents a different patient. (B) Pie chart representing the mean value of the percentage for all the patients shown in (A). (C) Changes in the ratio of granzyme A: granzyme B (GZA/GZB) in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX) in premenopausal compared to postmenopausal women. *p < 0.05, **p < 0.01.

Journal: Frontiers in immunology

Article Title: Differential Cytotoxic Function of Resident and Non-resident CD8+ T Cells in the Human Female Reproductive Tract Before and After Menopause.

doi: 10.3389/fimmu.2020.01096

Figure Lengend Snippet: FIGURE 5 | Effect of menopausal status on release of cytotoxic molecules in CD103+ and CD103−CD8+ T cells after activation. (A) Comparison of the percent of cells expressing granzyme B (GZB), granzyme A (GZA), and perforin in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX) in premenopausal and postmenopausal women. Each dot represents a different patient. (B) Pie chart representing the mean value of the percentage for all the patients shown in (A). (C) Changes in the ratio of granzyme A: granzyme B (GZA/GZB) in CD103+ and CD103−CD8+ T cells from the endometrium (EM), endocervix and ectocervix (CX/ECX) in premenopausal compared to postmenopausal women. *p < 0.05, **p < 0.01.

Article Snippet: Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103–BV711 (Biolegend), CD4-PE-Cy5.5, CD103–PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience).

Techniques: Activation Assay, Comparison, Expressing

ATF7ip is required for in vitro Th17 differentiation . (A) Relative expression of Atf7ip mRNA in tissues and immune cells downloaded from the BioGPS expression database. Red bars highlight hematopoietic cells or T cells. (B–D) Relative expression of Atf7ip mRNA in mouse tissues (B), CD4 + T cell subsets (C), or naive CD4 + T cells (D) stimulated with either anti-CD3 (2 µg) or both anti-CD3 (2 µg) and anti-CD28 (2 µg) for the indicated times. (E–H) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated under Th17 (E), Th1 (F), iT reg cell (G), or Th2 (H) conditions and analyzed for intracellular cytokine or Foxp3 expression. (I) Rorγt protein expression under Th17 conditions. A histogram of Rorγt expression under Th0 conditions is included for reference. Each data point in D–H represents an individual mouse with three mice per genotype. Data are representative of five (E) or two (B–D and F–I) independent experiments. Error bars in B and C show mean with SEM of technical replicates. Error bars (D–H) show mean with SD. **, P < 0.01 by Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses

doi: 10.1084/jem.20182316

Figure Lengend Snippet: ATF7ip is required for in vitro Th17 differentiation . (A) Relative expression of Atf7ip mRNA in tissues and immune cells downloaded from the BioGPS expression database. Red bars highlight hematopoietic cells or T cells. (B–D) Relative expression of Atf7ip mRNA in mouse tissues (B), CD4 + T cell subsets (C), or naive CD4 + T cells (D) stimulated with either anti-CD3 (2 µg) or both anti-CD3 (2 µg) and anti-CD28 (2 µg) for the indicated times. (E–H) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated under Th17 (E), Th1 (F), iT reg cell (G), or Th2 (H) conditions and analyzed for intracellular cytokine or Foxp3 expression. (I) Rorγt protein expression under Th17 conditions. A histogram of Rorγt expression under Th0 conditions is included for reference. Each data point in D–H represents an individual mouse with three mice per genotype. Data are representative of five (E) or two (B–D and F–I) independent experiments. Error bars in B and C show mean with SEM of technical replicates. Error bars (D–H) show mean with SD. **, P < 0.01 by Student’s t test.

Article Snippet: Antibodies used for flow-cytometry were as follows: PE-Cy7-conjugated CD4 (clone RM4-5; Tonbo), PE-Cy7-conjugated B220 (clone RA3-6B2; BioLegend), PE-Cy7-conjugated CD62L (clone MEL-14; Tonbo), PE-Cy7-conjugated CD45 (clone 30-F11; Invitrogen), PE-conjugated IL-17A (clone eBio17B7; Invitrogen), PE-conjugated CD62L (clone MEL-14; Tonbo), PE-conjugated CD44 (clone IM7; BioLegend), PE-conjugated CD8 (clone 53-6.7; Tonbo), PE-conjugated RORγ (Q31-378; BD Biosciences), PE-conjugated IL-13 (clone eBio13A; Invitrogen), FITC-conjugated Thy1.2 (clone 53-2.1; BD Biosciences), FITC-conjugated IFNγ (clone xMG1.2; Invitrogen), FITC-conjugated Foxp3 (clone FJK-16s; Invitrogen), FITC-conjugated CD44 (clone IM7; BioLegend), FITC-conjugated CD8 (clone 53-6.7; Tonbo), allophycocyanin (APC)-conjugated Thy1.1 (clone OX-7; BioLegend), APC-conjugated CD25 (clone PC61.5; Tonbo), APC-conjugated CD4 (clone GK1.5; BioLegend), APC-conjugated IL-2 (clone JES6-5H4; Invitrogen), APC-conjugated CD62L (clone MEL-14; Tonbo), PerCP-conjugated CD45 (clone 30-F11; Invitrogen), eFluor 450-conjugated TCRβ (clone H57-597; Tonbo), eFluor 450-conjugated CD8 (clone 53-6.7; Tonbo), PerCP-Cy5.5-conjugated CD4 (clone GK1.5; BioLegend), Ghost UV 450 (Tonbo), and Ghost 510 (Tonbo).

Techniques: In Vitro, Expressing

Atf7ip deletion attenuates colitis in vivo . (A–E) CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were injected intraperitoneal with anti-CD3 antibodies and analyzed 48 h after injection. (A) Flow cytometric analysis of CD4 + IL17A + INFγ + small intestine IELs. (B) Percentage and absolute numbers intraepithelial CD4 + T cells in the small intestine expressing IL-17A. (C) Serum IL-17A levels measured by ELISA. (D) Histological score of the small intestine. (E) H&E staining of the small intestine. Bars, 100 µm. (F) Weight change in Rag1 −/− recipients of RB hi naive T cells from either CD4 -Cre/ Atf7ip +/fl or CD4 -Cre/ Atf7ip fl/fl mice measured on day 0, 14, 21, 28, 35, and 42. Results are the combination of two experiments with 13 mice per genotype. (G) Absolute number of CD4 + IL17A + T cells in the colonic lamina propria of Rag1 −/− mice. Each data point in B–D and G represents an individual mouse. Data are the combination of three (B) or two (C, D, F, and G) independent experiments with three to four mice per group in each experiment. Error bars in B–D and G are mean with SD. Error bars in F are SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; significance by Student’s t test (B, C, and G); Mann–Whitney nonparametric test (D); and two-way ANOVA followed by multiple t tests using the Holm–Sidak method (F).

Journal: The Journal of Experimental Medicine

Article Title: The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses

doi: 10.1084/jem.20182316

Figure Lengend Snippet: Atf7ip deletion attenuates colitis in vivo . (A–E) CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were injected intraperitoneal with anti-CD3 antibodies and analyzed 48 h after injection. (A) Flow cytometric analysis of CD4 + IL17A + INFγ + small intestine IELs. (B) Percentage and absolute numbers intraepithelial CD4 + T cells in the small intestine expressing IL-17A. (C) Serum IL-17A levels measured by ELISA. (D) Histological score of the small intestine. (E) H&E staining of the small intestine. Bars, 100 µm. (F) Weight change in Rag1 −/− recipients of RB hi naive T cells from either CD4 -Cre/ Atf7ip +/fl or CD4 -Cre/ Atf7ip fl/fl mice measured on day 0, 14, 21, 28, 35, and 42. Results are the combination of two experiments with 13 mice per genotype. (G) Absolute number of CD4 + IL17A + T cells in the colonic lamina propria of Rag1 −/− mice. Each data point in B–D and G represents an individual mouse. Data are the combination of three (B) or two (C, D, F, and G) independent experiments with three to four mice per group in each experiment. Error bars in B–D and G are mean with SD. Error bars in F are SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; significance by Student’s t test (B, C, and G); Mann–Whitney nonparametric test (D); and two-way ANOVA followed by multiple t tests using the Holm–Sidak method (F).

Article Snippet: Antibodies used for flow-cytometry were as follows: PE-Cy7-conjugated CD4 (clone RM4-5; Tonbo), PE-Cy7-conjugated B220 (clone RA3-6B2; BioLegend), PE-Cy7-conjugated CD62L (clone MEL-14; Tonbo), PE-Cy7-conjugated CD45 (clone 30-F11; Invitrogen), PE-conjugated IL-17A (clone eBio17B7; Invitrogen), PE-conjugated CD62L (clone MEL-14; Tonbo), PE-conjugated CD44 (clone IM7; BioLegend), PE-conjugated CD8 (clone 53-6.7; Tonbo), PE-conjugated RORγ (Q31-378; BD Biosciences), PE-conjugated IL-13 (clone eBio13A; Invitrogen), FITC-conjugated Thy1.2 (clone 53-2.1; BD Biosciences), FITC-conjugated IFNγ (clone xMG1.2; Invitrogen), FITC-conjugated Foxp3 (clone FJK-16s; Invitrogen), FITC-conjugated CD44 (clone IM7; BioLegend), FITC-conjugated CD8 (clone 53-6.7; Tonbo), allophycocyanin (APC)-conjugated Thy1.1 (clone OX-7; BioLegend), APC-conjugated CD25 (clone PC61.5; Tonbo), APC-conjugated CD4 (clone GK1.5; BioLegend), APC-conjugated IL-2 (clone JES6-5H4; Invitrogen), APC-conjugated CD62L (clone MEL-14; Tonbo), PerCP-conjugated CD45 (clone 30-F11; Invitrogen), eFluor 450-conjugated TCRβ (clone H57-597; Tonbo), eFluor 450-conjugated CD8 (clone 53-6.7; Tonbo), PerCP-Cy5.5-conjugated CD4 (clone GK1.5; BioLegend), Ghost UV 450 (Tonbo), and Ghost 510 (Tonbo).

Techniques: In Vivo, Injection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

CD4- Cre /Atf7ip fl/fl T cells have increased Il2 and a decreased Th17 gene signature secondary to less H3K9me3 at the Il2-Il21 intergenic region. (A) Volcano plots from RNA-seq data comparing global gene expression analysis of T cells from CD4- Cre /Atf7ip fl/fl mice and CD4- Cre/ Atf7ip +/fl mice. Left plot represents naive T cells, middle plot is T cells differentiated for 24 h under Th17-inducing conditions, and right plot is 72 h of Th17-inducing conditions. Red numbers indicate the number of genes that are significantly increased (FDR <0.01) in CD4- Cre /Atf7ip fl/fl T cells, and blue numbers indicate the number of genes that are significantly increased in CD4- Cre /Atf7ip +/fl T cells. RNA-seq was performed in triplicate for each condition. (B) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated for 4 d under Th17-inducing conditions (IL-6 + TGFβ) and analyzed by flow cytometric analysis for intracellular cytokines (IL-17A, IL-2) or Foxp3. (C) Summary of flow cytometric data in B. (D) ELISA of secreted cytokines (IL-17A, IL-2, IL-17F, and IL-21) from Th17 culture supernatant. (E–G) ChIP-seq data for H3K9me3 from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) naive T cells performed in duplicate. (E) Scatterplot comparing log2 fold change of H3K9me3 in CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) naive T cells. Blue numbers represent the number of loci with twofold increased H3K9me3 in CD4 -Cre/ Atf7ip +/fl naive T cells compared with CD4 -Cre/ Atf7ip fl/fl naive T cells. Red numbers indicate the number of loci with twofold increased H3K9me3 in CD4 -Cre/ Atf7ip fl/fl naive T cells. (F) Integrated genome viewer H3K9me3 ChIP-seq tracings for the Il2-Il21 intergenic region. (G) Integrated genome viewer H3K9me3 ChIP-seq tracings for the indicated zinc finger proteins (Zfp). Green boxes show sites of twofold decreased H3K9me3 deposition. (H) H3K9me3 ChIP qPCR targeting the site of H3K9me3 deposition within the Il2-Il21 intergenic region and within the Zfp780b gene. Each data point represents an individual mouse. Data in B and C are one representative experiment of three experiments with three mice per group. Data in D are the combination of three experiments with two to three mice per group. Error bars are mean with SD (C and D) and mean and SD of technical replicates (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses

doi: 10.1084/jem.20182316

Figure Lengend Snippet: CD4- Cre /Atf7ip fl/fl T cells have increased Il2 and a decreased Th17 gene signature secondary to less H3K9me3 at the Il2-Il21 intergenic region. (A) Volcano plots from RNA-seq data comparing global gene expression analysis of T cells from CD4- Cre /Atf7ip fl/fl mice and CD4- Cre/ Atf7ip +/fl mice. Left plot represents naive T cells, middle plot is T cells differentiated for 24 h under Th17-inducing conditions, and right plot is 72 h of Th17-inducing conditions. Red numbers indicate the number of genes that are significantly increased (FDR <0.01) in CD4- Cre /Atf7ip fl/fl T cells, and blue numbers indicate the number of genes that are significantly increased in CD4- Cre /Atf7ip +/fl T cells. RNA-seq was performed in triplicate for each condition. (B) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated for 4 d under Th17-inducing conditions (IL-6 + TGFβ) and analyzed by flow cytometric analysis for intracellular cytokines (IL-17A, IL-2) or Foxp3. (C) Summary of flow cytometric data in B. (D) ELISA of secreted cytokines (IL-17A, IL-2, IL-17F, and IL-21) from Th17 culture supernatant. (E–G) ChIP-seq data for H3K9me3 from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) naive T cells performed in duplicate. (E) Scatterplot comparing log2 fold change of H3K9me3 in CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) naive T cells. Blue numbers represent the number of loci with twofold increased H3K9me3 in CD4 -Cre/ Atf7ip +/fl naive T cells compared with CD4 -Cre/ Atf7ip fl/fl naive T cells. Red numbers indicate the number of loci with twofold increased H3K9me3 in CD4 -Cre/ Atf7ip fl/fl naive T cells. (F) Integrated genome viewer H3K9me3 ChIP-seq tracings for the Il2-Il21 intergenic region. (G) Integrated genome viewer H3K9me3 ChIP-seq tracings for the indicated zinc finger proteins (Zfp). Green boxes show sites of twofold decreased H3K9me3 deposition. (H) H3K9me3 ChIP qPCR targeting the site of H3K9me3 deposition within the Il2-Il21 intergenic region and within the Zfp780b gene. Each data point represents an individual mouse. Data in B and C are one representative experiment of three experiments with three mice per group. Data in D are the combination of three experiments with two to three mice per group. Error bars are mean with SD (C and D) and mean and SD of technical replicates (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

Article Snippet: Antibodies used for flow-cytometry were as follows: PE-Cy7-conjugated CD4 (clone RM4-5; Tonbo), PE-Cy7-conjugated B220 (clone RA3-6B2; BioLegend), PE-Cy7-conjugated CD62L (clone MEL-14; Tonbo), PE-Cy7-conjugated CD45 (clone 30-F11; Invitrogen), PE-conjugated IL-17A (clone eBio17B7; Invitrogen), PE-conjugated CD62L (clone MEL-14; Tonbo), PE-conjugated CD44 (clone IM7; BioLegend), PE-conjugated CD8 (clone 53-6.7; Tonbo), PE-conjugated RORγ (Q31-378; BD Biosciences), PE-conjugated IL-13 (clone eBio13A; Invitrogen), FITC-conjugated Thy1.2 (clone 53-2.1; BD Biosciences), FITC-conjugated IFNγ (clone xMG1.2; Invitrogen), FITC-conjugated Foxp3 (clone FJK-16s; Invitrogen), FITC-conjugated CD44 (clone IM7; BioLegend), FITC-conjugated CD8 (clone 53-6.7; Tonbo), allophycocyanin (APC)-conjugated Thy1.1 (clone OX-7; BioLegend), APC-conjugated CD25 (clone PC61.5; Tonbo), APC-conjugated CD4 (clone GK1.5; BioLegend), APC-conjugated IL-2 (clone JES6-5H4; Invitrogen), APC-conjugated CD62L (clone MEL-14; Tonbo), PerCP-conjugated CD45 (clone 30-F11; Invitrogen), eFluor 450-conjugated TCRβ (clone H57-597; Tonbo), eFluor 450-conjugated CD8 (clone 53-6.7; Tonbo), PerCP-Cy5.5-conjugated CD4 (clone GK1.5; BioLegend), Ghost UV 450 (Tonbo), and Ghost 510 (Tonbo).

Techniques: RNA Sequencing, Gene Expression, In Vitro, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, ChIP-qPCR

CD4- Cre /Atf7ip fl/fl T cells produce increased IL-2 with TCR stimulation. Naive T cells were stimulated for 12–24 h in the presence of TCR stimulation (2 µg anti-CD3) or with both TCR stimulation (2 µg anti-CD3) and costimulation (2 µg anti-CD28). (A) qPCR for Il2 mRNA. (B) Flow cytometric data for IL-2 with ± SD shown. (C and D) IL-2 ELISA from culture supernatant. (E and G) Representative flow cytometry of IL-2 expression in the mesenteric LN (E) or small intestine IELs (G) 48 h after in vivo anti-CD3 treatment. (F and H) Summary of flow cytometry data from the mesenteric LN (E) and small intestine (SI) IELs (G). Each data point represents an individual mouse. Data in A and B are representative of two experiments with three mice per genotype. Data in C, D, F, and H are the combination of three experiments with three to four mice per genotype. Error bars are SEM (A) and mean with SD (B–D, F, and H). *, P < 0.05; ***, P < 0.001 by Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses

doi: 10.1084/jem.20182316

Figure Lengend Snippet: CD4- Cre /Atf7ip fl/fl T cells produce increased IL-2 with TCR stimulation. Naive T cells were stimulated for 12–24 h in the presence of TCR stimulation (2 µg anti-CD3) or with both TCR stimulation (2 µg anti-CD3) and costimulation (2 µg anti-CD28). (A) qPCR for Il2 mRNA. (B) Flow cytometric data for IL-2 with ± SD shown. (C and D) IL-2 ELISA from culture supernatant. (E and G) Representative flow cytometry of IL-2 expression in the mesenteric LN (E) or small intestine IELs (G) 48 h after in vivo anti-CD3 treatment. (F and H) Summary of flow cytometry data from the mesenteric LN (E) and small intestine (SI) IELs (G). Each data point represents an individual mouse. Data in A and B are representative of two experiments with three mice per genotype. Data in C, D, F, and H are the combination of three experiments with three to four mice per genotype. Error bars are SEM (A) and mean with SD (B–D, F, and H). *, P < 0.05; ***, P < 0.001 by Student’s t test.

Article Snippet: Antibodies used for flow-cytometry were as follows: PE-Cy7-conjugated CD4 (clone RM4-5; Tonbo), PE-Cy7-conjugated B220 (clone RA3-6B2; BioLegend), PE-Cy7-conjugated CD62L (clone MEL-14; Tonbo), PE-Cy7-conjugated CD45 (clone 30-F11; Invitrogen), PE-conjugated IL-17A (clone eBio17B7; Invitrogen), PE-conjugated CD62L (clone MEL-14; Tonbo), PE-conjugated CD44 (clone IM7; BioLegend), PE-conjugated CD8 (clone 53-6.7; Tonbo), PE-conjugated RORγ (Q31-378; BD Biosciences), PE-conjugated IL-13 (clone eBio13A; Invitrogen), FITC-conjugated Thy1.2 (clone 53-2.1; BD Biosciences), FITC-conjugated IFNγ (clone xMG1.2; Invitrogen), FITC-conjugated Foxp3 (clone FJK-16s; Invitrogen), FITC-conjugated CD44 (clone IM7; BioLegend), FITC-conjugated CD8 (clone 53-6.7; Tonbo), allophycocyanin (APC)-conjugated Thy1.1 (clone OX-7; BioLegend), APC-conjugated CD25 (clone PC61.5; Tonbo), APC-conjugated CD4 (clone GK1.5; BioLegend), APC-conjugated IL-2 (clone JES6-5H4; Invitrogen), APC-conjugated CD62L (clone MEL-14; Tonbo), PerCP-conjugated CD45 (clone 30-F11; Invitrogen), eFluor 450-conjugated TCRβ (clone H57-597; Tonbo), eFluor 450-conjugated CD8 (clone 53-6.7; Tonbo), PerCP-Cy5.5-conjugated CD4 (clone GK1.5; BioLegend), Ghost UV 450 (Tonbo), and Ghost 510 (Tonbo).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, In Vivo

iT reg cell induction is augmented in Atf7ip fl/fl T cells and Atf7ip fl/fl T cells suppress IL-17A production in trans. (A–C) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (A), Th2 (B), or iT reg cell (C) conditions and analyzed for intracellular cytokine or Foxp3 expression. (D–F) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (D), Th2 (E), or iT reg cell (F) conditions and analyzed for IL-2 expression. (G and H) WT (Thy1.1) naive T cells were mixed at a 50:50 ratio with either CD4 -Cre/ Atf7ip +/fl (Thy1.2) or CD4 -Cre/ Atf7ip fl/fl (Thy1.2) naive T cells and cultured for 96 h under Th17-inducing conditions with the addition of no IL-2 blocking antibody (No Antibody), 5 µg isotype control antibody (Isotype), or 5 µg S4B6. (G) Flow cytometric analysis of T cells expressing Thy1.1, Thy1.2, and IL-17A. Green box shows that S4B6 is able to rescue the trans defect in IL-17A production. (H) Summary of flow cytometric data. Each data point represents an individual mouse. Each data point in A–F and H represents an individual mouse. Data are representative of two independent experiments (A and D) or one of two experiments (B, C, E, and F) with three mice per group; data in G and H are representative of two experiments with two to four mice per genotype. Error bars (A–F and H) show mean with SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses

doi: 10.1084/jem.20182316

Figure Lengend Snippet: iT reg cell induction is augmented in Atf7ip fl/fl T cells and Atf7ip fl/fl T cells suppress IL-17A production in trans. (A–C) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (A), Th2 (B), or iT reg cell (C) conditions and analyzed for intracellular cytokine or Foxp3 expression. (D–F) Naive T cells from CD4 -Cre/ Atf7ip +/fl ( Atf7ip +/fl ) and CD4 -Cre/ Atf7ip fl/fl ( Atf7ip fl/fl ) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (D), Th2 (E), or iT reg cell (F) conditions and analyzed for IL-2 expression. (G and H) WT (Thy1.1) naive T cells were mixed at a 50:50 ratio with either CD4 -Cre/ Atf7ip +/fl (Thy1.2) or CD4 -Cre/ Atf7ip fl/fl (Thy1.2) naive T cells and cultured for 96 h under Th17-inducing conditions with the addition of no IL-2 blocking antibody (No Antibody), 5 µg isotype control antibody (Isotype), or 5 µg S4B6. (G) Flow cytometric analysis of T cells expressing Thy1.1, Thy1.2, and IL-17A. Green box shows that S4B6 is able to rescue the trans defect in IL-17A production. (H) Summary of flow cytometric data. Each data point represents an individual mouse. Each data point in A–F and H represents an individual mouse. Data are representative of two independent experiments (A and D) or one of two experiments (B, C, E, and F) with three mice per group; data in G and H are representative of two experiments with two to four mice per genotype. Error bars (A–F and H) show mean with SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.

Article Snippet: Antibodies used for flow-cytometry were as follows: PE-Cy7-conjugated CD4 (clone RM4-5; Tonbo), PE-Cy7-conjugated B220 (clone RA3-6B2; BioLegend), PE-Cy7-conjugated CD62L (clone MEL-14; Tonbo), PE-Cy7-conjugated CD45 (clone 30-F11; Invitrogen), PE-conjugated IL-17A (clone eBio17B7; Invitrogen), PE-conjugated CD62L (clone MEL-14; Tonbo), PE-conjugated CD44 (clone IM7; BioLegend), PE-conjugated CD8 (clone 53-6.7; Tonbo), PE-conjugated RORγ (Q31-378; BD Biosciences), PE-conjugated IL-13 (clone eBio13A; Invitrogen), FITC-conjugated Thy1.2 (clone 53-2.1; BD Biosciences), FITC-conjugated IFNγ (clone xMG1.2; Invitrogen), FITC-conjugated Foxp3 (clone FJK-16s; Invitrogen), FITC-conjugated CD44 (clone IM7; BioLegend), FITC-conjugated CD8 (clone 53-6.7; Tonbo), allophycocyanin (APC)-conjugated Thy1.1 (clone OX-7; BioLegend), APC-conjugated CD25 (clone PC61.5; Tonbo), APC-conjugated CD4 (clone GK1.5; BioLegend), APC-conjugated IL-2 (clone JES6-5H4; Invitrogen), APC-conjugated CD62L (clone MEL-14; Tonbo), PerCP-conjugated CD45 (clone 30-F11; Invitrogen), eFluor 450-conjugated TCRβ (clone H57-597; Tonbo), eFluor 450-conjugated CD8 (clone 53-6.7; Tonbo), PerCP-Cy5.5-conjugated CD4 (clone GK1.5; BioLegend), Ghost UV 450 (Tonbo), and Ghost 510 (Tonbo).

Techniques: In Vitro, Expressing, Cell Culture, Blocking Assay, Control