apc cy7 cd62l tonbo Search Results


apc cy7 cd62l tonbo  (Cytek Biosciences)
93
Cytek Biosciences apc cy7 cd62l tonbo
Apc Cy7 Cd62l Tonbo, supplied by Cytek Biosciences, 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/apc+cy7+cd62l+tonbo/10__1113_slash_jp281698-79-44-45?v=Cytek+Biosciences
Average 93 stars, based on 1 article reviews
apc cy7 cd62l tonbo - by Bioz Stars, 2026-06
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cd62l apc  (Cytek Biosciences)
93
Cytek Biosciences cd62l apc
W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory <t>(CD62L</t> lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.
Cd62l Apc, supplied by Cytek Biosciences, 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/apc+cy7+cd62l+tonbo/pmc05339679-240-12-14?v=Cytek+Biosciences
Average 93 stars, based on 1 article reviews
cd62l apc - by Bioz Stars, 2026-06
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pe cy7 conjugated cd62l  (Cytek Biosciences)
93
Cytek Biosciences pe cy7 conjugated cd62l
W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory <t>(CD62L</t> lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.
Pe Cy7 Conjugated Cd62l, supplied by Cytek Biosciences, 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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pe cy7 conjugated cd62l - by Bioz Stars, 2026-06
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pe conjugated cd62l  (Cytek Biosciences)
93
Cytek Biosciences pe conjugated cd62l
W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory <t>(CD62L</t> lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.
Pe Conjugated Cd62l, supplied by Cytek Biosciences, 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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Average 93 stars, based on 1 article reviews
pe conjugated cd62l - by Bioz Stars, 2026-06
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cd4-pe-cy7  (Becton Dickinson)
90
Becton Dickinson cd4-pe-cy7
W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory <t>(CD62L</t> lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.
Cd4 Pe Cy7, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/apc+cy7+cd62l+tonbo/10__2147_slash_ijn__s234850-104-10-13?v=Becton+Dickinson
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cd4-pe-cy7 - by Bioz Stars, 2026-06
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pe cy7 conjugated cd4  (Cytek Biosciences)
93
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.
Pe Cy7 Conjugated Cd4, supplied by Cytek Biosciences, 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/apc+cy7+cd62l+tonbo/pmc06719416-162-7-11?v=Cytek+Biosciences
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pe cy7 conjugated cd4 - by Bioz Stars, 2026-06
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tcr-β-apc  (Becton Dickinson)
90
Becton Dickinson tcr-β-apc
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.
Tcr β Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/apc+cy7+cd62l+tonbo/10__2147_slash_ijn__s234850-104-7-9?v=Becton+Dickinson
Average 90 stars, based on 1 article reviews
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pe-conjugated rorγ (q31-378  (Becton Dickinson)
90
Becton Dickinson pe-conjugated rorγ (q31-378
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.
Pe Conjugated Rorγ (Q31 378, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/apc+cy7+cd62l+tonbo/ppr0288898-182-53-56?v=Becton+Dickinson
Average 90 stars, based on 1 article reviews
pe-conjugated rorγ (q31-378 - by Bioz Stars, 2026-06
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ghost uv 450  (Cytek Biosciences)
95
Cytek Biosciences ghost uv 450
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.
Ghost Uv 450, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/apc+cy7+cd62l+tonbo/pmc06719416-162-131-134?v=Cytek+Biosciences
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pe conjugated cd8  (Cytek Biosciences)
93
Cytek Biosciences pe conjugated cd8
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.
Pe Conjugated Cd8, supplied by Cytek Biosciences, 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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pe conjugated cd8 - by Bioz Stars, 2026-06
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violetfluor450 cd45  (Cytek Biosciences)
93
Cytek Biosciences violetfluor450 cd45
Figure 1. Anti-CD3 F(ab’)2 fragment treatment results in depletion of T cells in the blood and spleen Blood (100 μL) from young isotype (n = 10), young anti-CD3 (n = 10), old isotype (n = 9) and old anti-CD3 (n = 10) mice was directly stained. Spleens from young isotype (n = 11–14), young anti-CD3 (n = 8–11), old isotype (n = 15–17) and old anti-CD3 (n = 18–19) mice were enzymatically digested and passed through a cell strainer and then stained for <t>CD45</t> (total leukocytes), CD3 (pan T cells), CD4 and CD8. Percentages of CD3+ cells and the CD4-to-CD8 ratio in (A) blood and (B) spleen were assessed by flow cytometry. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]
Violetfluor450 Cd45, supplied by Cytek Biosciences, 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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violetfluor450 cd45 - by Bioz Stars, 2026-06
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percp 448 cy5 5 conjugated cd4  (Revvity)
98
Revvity percp 448 cy5 5 conjugated cd4
Figure 1. Anti-CD3 F(ab’)2 fragment treatment results in depletion of T cells in the blood and spleen Blood (100 μL) from young isotype (n = 10), young anti-CD3 (n = 10), old isotype (n = 9) and old anti-CD3 (n = 10) mice was directly stained. Spleens from young isotype (n = 11–14), young anti-CD3 (n = 8–11), old isotype (n = 15–17) and old anti-CD3 (n = 18–19) mice were enzymatically digested and passed through a cell strainer and then stained for <t>CD45</t> (total leukocytes), CD3 (pan T cells), CD4 and CD8. Percentages of CD3+ cells and the CD4-to-CD8 ratio in (A) blood and (B) spleen were assessed by flow cytometry. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]
Percp 448 Cy5 5 Conjugated Cd4, supplied by Revvity, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory (CD62L lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.

Journal: The Journal of Experimental Medicine

Article Title: Destabilizing the autoinhibitory conformation of Zap70 induces up-regulation of inhibitory receptors and T cell unresponsiveness

doi: 10.1084/jem.20161575

Figure Lengend Snippet: W131A mice with a polyclonal TCR repertoire have grossly normal T cell development and TCR signaling. (A) Targeting strategy used to produce W131A knock-in mice. Residue W131A present in exon 2 was mutated to alanine. (B) CD4 versus CD8 profiles of thymocytes (top) and LN cells (bottom) from WT and W131A mice. Numbers in quadrants are the percentages of each T cell subset. Results are representative of seven experiments. (C) Total cell numbers in thymi (top) and LN (bottom) in age-matched 2-mo-old WT and W131A mice. (D) Increased percentages of memory (CD62L lo CD44 hi ) CD4 + T cells in W131A mice ( n = 5 per group). All error bars represent SEM. *, P < 0.05; **, P < 0.01. (E) Comparison of CD4 + Foxp3 + T cells from LNs of WT and W131A mice of the indicated ages ( n = 4 per group). (F) Anti–CD3-induced calcium changes in DP thymocytes (top), CD4SP (middle), and CD8SP thymocytes (bottom) from WT and W131A mice. (G) DP, CD4SP, and CD8SP thymocytes from WT or W131A mice were analyzed for levels of phospho-ERK by flow cytometry. Unstimulated T cells (filled gray) and anti-CD3–stimulated T cells (2.5 min) from WT mice (red line), and W131A (blue line) are shown. Data are representative of three experiments.

Article Snippet: The following antibodies were used for staining: CD4 PerCp-Cy5.5, TCRβ PerCp-Cy5.5, and CD62L APC (TONBO); CD44 FITC, PD-1 (29F.1A12) PE-Cy7, CD69 PE-Cy7, CD5 PerCp-Cy5.5, CD8α Pacific Blue, and TCRβ Pacific Blue (BioLegend); CD44 PE-Cy7, CD8α (APC-eFluor 780), CD4 Qdot606, and Zap70 FITC (Life Technology).

Techniques: Knock-In, Residue, Comparison, Flow Cytometry

Restricting the TCR repertoire in W131A mice leads to a profound impairment in T cell development. (A) Representative plots showing expression of CD4/CD8 (top) and OTII TCR tg (bottom) in total thymocytes from OTII and W131AOTII mice. Numbers in each histogram of the bottom panels indicate the frequency of OTII (Vα2 + ) tg-positive cells in CD4SP thymocytes. (B) Absolute numbers of CD4 + T cells from thymi and LNs of either OTII or W131AOTII are shown ( n = 10 per genotype). All error bars represent SEM. ***, P < 0.001; ****, P < 0.0001. (C) Representative plots showing expression of CD4/CD8 (top) and CD62L/CD44 (bottom) in LN cells from OTII and W131AOTII mice. (D) Representative flow cytometric profiles of CD4 and Foxp3 expression in CD4 SP thymocytes (top) and peripheral CD4 + T cells (bottom) from OTII and W131AOTII mice. Data are representative of seven experiments.

Journal: The Journal of Experimental Medicine

Article Title: Destabilizing the autoinhibitory conformation of Zap70 induces up-regulation of inhibitory receptors and T cell unresponsiveness

doi: 10.1084/jem.20161575

Figure Lengend Snippet: Restricting the TCR repertoire in W131A mice leads to a profound impairment in T cell development. (A) Representative plots showing expression of CD4/CD8 (top) and OTII TCR tg (bottom) in total thymocytes from OTII and W131AOTII mice. Numbers in each histogram of the bottom panels indicate the frequency of OTII (Vα2 + ) tg-positive cells in CD4SP thymocytes. (B) Absolute numbers of CD4 + T cells from thymi and LNs of either OTII or W131AOTII are shown ( n = 10 per genotype). All error bars represent SEM. ***, P < 0.001; ****, P < 0.0001. (C) Representative plots showing expression of CD4/CD8 (top) and CD62L/CD44 (bottom) in LN cells from OTII and W131AOTII mice. (D) Representative flow cytometric profiles of CD4 and Foxp3 expression in CD4 SP thymocytes (top) and peripheral CD4 + T cells (bottom) from OTII and W131AOTII mice. Data are representative of seven experiments.

Article Snippet: The following antibodies were used for staining: CD4 PerCp-Cy5.5, TCRβ PerCp-Cy5.5, and CD62L APC (TONBO); CD44 FITC, PD-1 (29F.1A12) PE-Cy7, CD69 PE-Cy7, CD5 PerCp-Cy5.5, CD8α Pacific Blue, and TCRβ Pacific Blue (BioLegend); CD44 PE-Cy7, CD8α (APC-eFluor 780), CD4 Qdot606, and Zap70 FITC (Life Technology).

Techniques: 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

Figure 1. Anti-CD3 F(ab’)2 fragment treatment results in depletion of T cells in the blood and spleen Blood (100 μL) from young isotype (n = 10), young anti-CD3 (n = 10), old isotype (n = 9) and old anti-CD3 (n = 10) mice was directly stained. Spleens from young isotype (n = 11–14), young anti-CD3 (n = 8–11), old isotype (n = 15–17) and old anti-CD3 (n = 18–19) mice were enzymatically digested and passed through a cell strainer and then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4 and CD8. Percentages of CD3+ cells and the CD4-to-CD8 ratio in (A) blood and (B) spleen were assessed by flow cytometry. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 1. Anti-CD3 F(ab’)2 fragment treatment results in depletion of T cells in the blood and spleen Blood (100 μL) from young isotype (n = 10), young anti-CD3 (n = 10), old isotype (n = 9) and old anti-CD3 (n = 10) mice was directly stained. Spleens from young isotype (n = 11–14), young anti-CD3 (n = 8–11), old isotype (n = 15–17) and old anti-CD3 (n = 18–19) mice were enzymatically digested and passed through a cell strainer and then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4 and CD8. Percentages of CD3+ cells and the CD4-to-CD8 ratio in (A) blood and (B) spleen were assessed by flow cytometry. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Single-cell suspensions were labelled with the following anti-mouse antibodies at a 1:100 concentration: violetfluor450-CD45, Tonbo #75-0454 (total leukocytes), APC-CD3, Tonbo #20-0032 (pan T cells), FITC-CD4, Tonbo #30-0041 (T helper cells), PE-Cy7-CD8, Tonbo #60-0081 (cytotoxic T cells), PerCP Cy5.5-CD44, Tonbo #65-00441 (naïve vs. memory) and APC-Cy7-CD62L Tonbo #25-0621 (central vs. effector).

Techniques: Staining, Flow Cytometry, Standard Deviation

Figure 2. Aortic T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of thoracic aortas from young isotype (n = 7–12), young anti-CD3 (n = 9–11), old isotype (n = 8–14) and old anti-CD3 (n = 10–19) mice were stained with antibodies against CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per aorta. B, sample aortic CD4 and CD8 flow cytometry plot. C, aortic pan, CD4 and CD8 T cell counts. D, percentage of CD3 cells out of total aortic immune cells. E, aortic CD4:CD8 ratio. F, sample aortic naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. G, aortic CD8 effector memory T cell proportion and counts. To assess aortic macrophage and B cell accumulation, thoracic aorta single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype. H, aortic B cell counts. I, aortic macrophage counts. J, macrophage M1:M2 ratio. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 2. Aortic T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of thoracic aortas from young isotype (n = 7–12), young anti-CD3 (n = 9–11), old isotype (n = 8–14) and old anti-CD3 (n = 10–19) mice were stained with antibodies against CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per aorta. B, sample aortic CD4 and CD8 flow cytometry plot. C, aortic pan, CD4 and CD8 T cell counts. D, percentage of CD3 cells out of total aortic immune cells. E, aortic CD4:CD8 ratio. F, sample aortic naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. G, aortic CD8 effector memory T cell proportion and counts. To assess aortic macrophage and B cell accumulation, thoracic aorta single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype. H, aortic B cell counts. I, aortic macrophage counts. J, macrophage M1:M2 ratio. A two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment, P values for age, treatment and the age × treatment interaction are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Single-cell suspensions were labelled with the following anti-mouse antibodies at a 1:100 concentration: violetfluor450-CD45, Tonbo #75-0454 (total leukocytes), APC-CD3, Tonbo #20-0032 (pan T cells), FITC-CD4, Tonbo #30-0041 (T helper cells), PE-Cy7-CD8, Tonbo #60-0081 (cytotoxic T cells), PerCP Cy5.5-CD44, Tonbo #65-00441 (naïve vs. memory) and APC-Cy7-CD62L Tonbo #25-0621 (central vs. effector).

Techniques: Staining, Flow Cytometry, Comparison, Standard Deviation

Figure 3. Ageing results in an enhanced proinflammatory phenotype of aortic accumulating T cells Single-cell suspensions of thoracic aortas from young (n = 6) and old (n = 8) mice were activated in vitro and stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and (C) number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test, P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 3. Ageing results in an enhanced proinflammatory phenotype of aortic accumulating T cells Single-cell suspensions of thoracic aortas from young (n = 6) and old (n = 8) mice were activated in vitro and stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and (C) number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test, P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Single-cell suspensions were labelled with the following anti-mouse antibodies at a 1:100 concentration: violetfluor450-CD45, Tonbo #75-0454 (total leukocytes), APC-CD3, Tonbo #20-0032 (pan T cells), FITC-CD4, Tonbo #30-0041 (T helper cells), PE-Cy7-CD8, Tonbo #60-0081 (cytotoxic T cells), PerCP Cy5.5-CD44, Tonbo #65-00441 (naïve vs. memory) and APC-Cy7-CD62L Tonbo #25-0621 (central vs. effector).

Techniques: In Vitro, Staining, Flow Cytometry, Comparison, Standard Deviation

Figure 5. Mesenteric T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of the mesenteric vascular arcade from young isotype (n = 8–14), young anti-CD3 (n = 10–12), old isotype (n = 9–21) and old anti-CD3 (n = 7–11) mice (excluding lymph nodes) were stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per mesentery. B, proportion of CD3 cells out of all mesenteric leukocytes. C, mesenteric CD3 cell counts. D, mesenteric arcade mass. E, mesenteric CD3 cell counts normalized to tissue mass. F, sample CD4 and CD8 flow cytometry plot. G, mesenteric CD4 (left) and CD8 (right) T cell counts. H, mesenteric CD4:CD8 ratio. I, sample mesenteric naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. J, proportion of mesenteric (left) and counts (right) of CD8 CD44hi/CD62Llo effector memory T cell counts. To assess mesenteric macrophage and B cell accumulation, mesenteric single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype). K, mesenteric B cell counts. L, mesenteric macrophage counts. M, macrophage M1:M2 ratio. Two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment. P values for age, treatment and the age × treatment inter- action are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 5. Mesenteric T cell accumulation with age and anti-CD3 F(ab’)2 Fragment treatment Single-cell suspensions of the mesenteric vascular arcade from young isotype (n = 8–14), young anti-CD3 (n = 10–12), old isotype (n = 9–21) and old anti-CD3 (n = 7–11) mice (excluding lymph nodes) were stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, CD44 (memory) and CD62L (central vs. effector). A, number of total leukocytes per mesentery. B, proportion of CD3 cells out of all mesenteric leukocytes. C, mesenteric CD3 cell counts. D, mesenteric arcade mass. E, mesenteric CD3 cell counts normalized to tissue mass. F, sample CD4 and CD8 flow cytometry plot. G, mesenteric CD4 (left) and CD8 (right) T cell counts. H, mesenteric CD4:CD8 ratio. I, sample mesenteric naïve (CD44lo) vs. memory (CD44hi) flow cytometry plots. J, proportion of mesenteric (left) and counts (right) of CD8 CD44hi/CD62Llo effector memory T cell counts. To assess mesenteric macrophage and B cell accumulation, mesenteric single-cell suspensions were stained for CD45 (total leukocytes), CD19 (B cells), CD64 (macrophages), CD11c (exclusion of dendritic cells) and CD206 (M1/M2 macrophage phenotype). K, mesenteric B cell counts. L, mesenteric macrophage counts. M, macrophage M1:M2 ratio. Two-way ANOVA was employed to assess the effects of age and anti-CD3 treatment. P values for age, treatment and the age × treatment inter- action are inset on each panel. When a significant age × treatment interaction occurred, Tukey’s post hoc test was employed to determine group differences. Significant post hoc test P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Single-cell suspensions were labelled with the following anti-mouse antibodies at a 1:100 concentration: violetfluor450-CD45, Tonbo #75-0454 (total leukocytes), APC-CD3, Tonbo #20-0032 (pan T cells), FITC-CD4, Tonbo #30-0041 (T helper cells), PE-Cy7-CD8, Tonbo #60-0081 (cytotoxic T cells), PerCP Cy5.5-CD44, Tonbo #65-00441 (naïve vs. memory) and APC-Cy7-CD62L Tonbo #25-0621 (central vs. effector).

Techniques: Staining, Flow Cytometry, Comparison, Standard Deviation

Figure 6. Ageing results in an enhanced proinflammatory phenotype of mesenteric accumulating T cells To assess cytokine production, mesenteric single-cell suspensions from young (n = 6) and old (n = 8) mice were activated in vitro. Cells were then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and C, number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test. P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Journal: The Journal of Physiology

Article Title: T cells mediate cell non‐autonomous arterial ageing in mice

doi: 10.1113/jp281698

Figure Lengend Snippet: Figure 6. Ageing results in an enhanced proinflammatory phenotype of mesenteric accumulating T cells To assess cytokine production, mesenteric single-cell suspensions from young (n = 6) and old (n = 8) mice were activated in vitro. Cells were then stained for CD45 (total leukocytes), CD3 (pan T cells), CD4, CD8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. A, sample IFN-γ flow cytometry plots. B, proportion and C, number of IFN-γ -producing T cells. D, sample TNF-α flow cytometry plots. E, proportion and (F) number of TNF-α-producing T cells. Group differences were assessed with an independent samples t test. P values are included on the panel with a horizontal line indicating the group comparison. Data are shown as means ± standard deviation, n represents the number of independent animals in each group. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Single-cell suspensions were labelled with the following anti-mouse antibodies at a 1:100 concentration: violetfluor450-CD45, Tonbo #75-0454 (total leukocytes), APC-CD3, Tonbo #20-0032 (pan T cells), FITC-CD4, Tonbo #30-0041 (T helper cells), PE-Cy7-CD8, Tonbo #60-0081 (cytotoxic T cells), PerCP Cy5.5-CD44, Tonbo #65-00441 (naïve vs. memory) and APC-Cy7-CD62L Tonbo #25-0621 (central vs. effector).

Techniques: In Vitro, Staining, Flow Cytometry, Comparison, Standard Deviation