Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p < 0.001. One representative out of three independent experiments is shown (B) .

Article Snippet: One day prior to challenge, the neutralizing Abs against CXCL9 (2 μg, R&D Systems, MAB554, USA), CXCL10 (2 μg, R&D Systems, MAB554, USA), and CXCL11 (2 μg, R&D Systems, MAB554, USA) were delivered to the vagina of mice, respectively, or in combination.

Techniques: Infection, Injection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: One day prior to challenge, the neutralizing Abs against CXCL9 (2 μg, R&D Systems, MAB554, USA), CXCL10 (2 μg, R&D Systems, MAB554, USA), and CXCL11 (2 μg, R&D Systems, MAB554, USA) were delivered to the vagina of mice, respectively, or in combination.

Techniques: Infection, Transfection, Control, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: One day prior to challenge, the neutralizing Abs against CXCL9 (2 μg, R&D Systems, MAB554, USA), CXCL10 (2 μg, R&D Systems, MAB554, USA), and CXCL11 (2 μg, R&D Systems, MAB554, USA) were delivered to the vagina of mice, respectively, or in combination.

Techniques: Infection, Migration, Control, Neutralization, Incubation, Recombinant, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: One day prior to challenge, the neutralizing Abs against CXCL9 (2 μg, R&D Systems, MAB554, USA), CXCL10 (2 μg, R&D Systems, MAB554, USA), and CXCL11 (2 μg, R&D Systems, MAB554, USA) were delivered to the vagina of mice, respectively, or in combination.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Control, Migration, Neutralization, Infection, Incubation

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p < 0.001. One representative out of three independent experiments is shown (G) .

Article Snippet: One day prior to challenge, the neutralizing Abs against CXCL9 (2 μg, R&D Systems, MAB554, USA), CXCL10 (2 μg, R&D Systems, MAB554, USA), and CXCL11 (2 μg, R&D Systems, MAB554, USA) were delivered to the vagina of mice, respectively, or in combination.

Techniques: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: a Naïve WT C57BL/6 mice were infected intranasally with 30 PFU influenza (A/PR8/34; “PR8”) for 8 days. Single-cell suspensions were generated from the lung-draining lymph nodes (DLN), and analysis of Aiolos protein expression in the indicated populations was performed via flow cytometry. Data are compiled from 2 independent experiments ( n = 6 ± s.e.m; **** P < 0.0001; one-way ANOVA with Tukey’s multiple comparison test). b – d Analysis of the percentage of influenza nucleoprotein (NP)-specific PD-1 HI Cxcr5 HI (T FH ) populations generated in response to influenza infection. Following single-cell suspension, cells were stained with fluorochrome-labeled tetramers to identify NP-specific populations. b , c Analysis of the percentage of influenza nucleoprotein (NP)-specific Bcl-6 HI Cxcr5 HI (T FH ) populations generated in response to influenza infection (For ‘ b ’, n = 14 for WT and 13 for Aiolos KO. For ‘ c ’, n = 14. Data are presented as mean ± s.e.m; data are compiled from four independent experiments; **** P < 0.0001; two-sided, unpaired Student’s t test). d Total NP-specific CD4 + T cells generated in WT versus Aiolos-deficient animals following influenza infection were enumerated. ( n = 14 for WT and 13 for Aiolos KO. Data are presented as mean ± s.e.m; data are compiled from 4 independent experiments; **** P < 0.0001; two-sided, unpaired Student’s t test. e ELISA analysis of indicated serum antibody levels in ng/mL at 8 d.p.i. Data are compiled from three independent experiments ( n = 11 ± s.e.m, ** P < 0.01, *** P < 0.001; two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Infection, Generated, Expressing, Flow Cytometry, Comparison, Suspension, Staining, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve WT or Aiolos-deficient ( Ikzf3 −/− ) CD4 + T cells were cultured under T FH -polarizing conditions for 3 days. RNA-seq analysis was performed to assess differentially expressed genes (DEGs) between WT and Aiolos-deficient cells. a Volcano plot displaying gene expression changes in Aiolos-deficient vs WT cells; genes of particular interest are labeled. Genes were color-coded as follows: no significant changes in expression (gray), upregulated genes with >2-fold change in expression with a P < 0.05 (red), downregulated genes with >2-fold change in expression with a P < 0.05 (blue) ( n = 3; two-sided DESeq2 analysis). b Representative heat-maps of DEGs positively and negatively associated with the T FH gene program in WT vs. Aiolos-deficient cells; changes in gene expression are presented as row (gene) Z-score. c Pre-ranked (sign of fold change × −log 10 ( p -value)) genes were analyzed using the Broad Institute Gene Set Enrichment Analysis (GSEA) software for comparison against ‘hallmark’, ‘gene ontology’, and ‘immunological signature’ gene sets. Enrichment plots for indicated gene sets are shown. Data are compiled from three biological replicates from three independent experiments ( n = 3; pre-ranked GSEA analysis). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Cell Culture, RNA Sequencing, Gene Expression, Labeling, Expressing, Software, Comparison

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve CD4 + T cells were cultured under T FH -like polarizing conditions for 3 days. a Representative heatmap showing normalized counts from Assay for Transposase-Accessible Chromatin (ATAC)-seq of WT and Aiolos-deficient CD4 + cells. Loci shown exhibit statistically significant differences in accessibility between WT and Aiolos-deficient samples. Data are compiled from 4 independent experiments. b CPM-normalized representative IGV tracks for the Zfp831 locus from four independent experiments are shown. Approximate location of primers used for chromatin immunoprecipitation (ChIP) is indicated with gray arrows. c Transcript analysis for Zfp831 was performed via qRT-PCR. Data were normalized to Rps18 control and are presented relative to the WT sample ( n = 7 ± s.e.m; ** P < 0.01, two-sided, unpaired Student’s t test). d ChIP analysis of Aiolos enrichment at the indicated Zfp831 regulatory region or negative control region in T FH -like cells are shown. Data were normalized to total input and are presented as percent enrichment of total minus IgG. ( n = 5 ± s.e.m; * P < 0.05, ** P < 0.01; two-sided, paired Student’s t test). e ChIP analysis of H3K27Ac at the site of Aiolos enrichment in WT versus Ikzf3 −/− cells. Data were normalized to total input, and IgG background was subtracted from each sample. Data are presented relative to the WT sample ( n = 5 ± s.e.m; * P < 0.05, ** P < 0.01; two-sided, unpaired Student’s t test). f , g HOMER motif enrichment analysis of regions of increased ( f ) and decreased ( g ) accessibility in Aiolos-deficient samples. Data are compiled from analysis of four independent ATAC-seq experiments ( n = 4, HOMER hypergeometric analysis). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Cell Culture, Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Negative Control

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naive WT or Aiolos-deficient ( Ikzf3 −/− ) CD4 + T cells were cultured under T H 1-polarizing conditions for 3 days. a – c Transcript analysis for the indicated genes was performed via qRT-PCR. Data are normalized to Rps18 control and presented relative to the WT sample for each gene. Data are compiled from 6 independent experiments. ( n = 9 ± s.e.m; ** P < 0.01, *** P < 0.00; two-sided, unpaired Student’s t test). d – f Analysis of cytotoxic effector molecule production by flow cytometry. Cells were cultured on stimulation with protein transport inhibitors for 3 h prior to analysis. ( n = 10 ± s.e.m; ** P < 0.01, *** P < 0.001, **** P < 0.0001; two-sided, unpaired Student’s t test). g – i RNA-seq analysis was performed to assess differentially expressed genes (DEGs) between WT and Aiolos-deficient cells. g Representative heatmap of DEGs positively and negatively associated with the T H 1, CD4-CTL, and T FH gene programs in WT vs. Aiolos-deficient cells is shown ; changes in gene expression are presented as row (gene) Z-score. Data are compiled from three independent experiments. h – i Pre-ranked (sign of fold change × −log 10 ( p -value)) genes were analyzed using the Broad Institute Gene Set Enrichment Analysis (GSEA) software for comparison against ‘hallmark’ and ‘immunological signature’ gene sets. Enrichment plots for indicated gene sets are shown. Data are compiled from three biological replicates from three independent experiments ( n = 3; pre-ranked GSEA analysis). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Flow Cytometry, RNA Sequencing, Gene Expression, Software, Comparison

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve WT or Aiolos-deficient ( Ikzf3 −/− ) mice were infected intranasally with 30 PFU influenza (A/PR8/34; “PR8”). After 8 days, draining lymph nodes (DLN) were harvested and viable CD4 + effector populations were analyzed via flow cytometry. a – c Analysis of of T-bet and Eomes expression by influenza nucleoprotein (NP)-specific CD4 + . Data are compiled from 7 independent experiments ( n = 22 ± s.e.m; ** P < 0.01; two-sided, unpaired Student’s t test). d Single-cell suspensions from the DLN were incubated in culture medium in the presence of protein transport inhibitors for 3 h. Perforin production by Eomes + populations was analyzed via flow cytometry. Data are compiled from four independent experiments ( n = 11 ± s.e.m; * P < 0.05, ** P < 0.01; two-sided, unpaired Student’s t test). e CD4 + NP-specific cells were enumerated. Data are compiled from 7 independent experiments ( n = 22 ± s.e.m; * P < 0.05; two-sided, unpaired Student’s t test). f CD25 (IL-2Rα) surface expression was evaluated on viable antigen-specific CD4 + CD44 + CD62L - Foxp3 - (effector) T cells from the lung-draining lymph nodes (DLN) via flow cytometry. Percentage of CD25 + cells are shown. Data are compiled from three independent experiments ( n = 9 ± s.e.m; ** P < 0.01, **** P < 0.0001; two-sided, unpaired Student’s t test). g Production of IL-2 by CD4 + T cell populations was analyzed in WT vs. Aiolos-deficient cells from the DLN by flow cytometry. Cells were cultured in the presence of PMA and Ionomycin and protein transport inhibitors for 3 h prior to analysis. Data are compiled from two independent experiments ( n = 7 ± s.e.m; two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Infection, Flow Cytometry, Expressing, Incubation, Cell Culture

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve CD4 + T cells were isolated from OT-II-WT and OT-II- Ikzf3 −/−/ mice. 5 × 10 5 cells were adoptively transferred into naïve CD45.1 mice which were then infected with 40 PFU OVA 323–339 -expressing A/PR8/34 (“PR8-OT-II”). After 8 days, lungs were harvested and viable CD45.2 + CD4 + populations were analyzed via flow cytometry. a , b Analysis of Eomes expression in antigen-specific (CD45.2 + CD4 + ) cells. Data are representative of 6 independent experiments ( n = 20 ± s.e.m; * P < 0.05, two-sided, unpaired Student’s t test). c , d Analysis of T-bet expression in antigen-specific (CD45.2 + CD4 + ) cells . Data are representative of five independent experiments ( n = 17 ± s.e.m; two-sided, unpaired Student’s t test). e – i Whole-tissue homogenates from the lung were stimulated ex vivo in culture media with OVA 323-339 peptide for 48 h. Suspensions were then incubated in the presence of protein transport inhibitors for 3 h. e , f Flow cytometry analyses of antigen-specific (CD45.2 + CD4 + ) cells for the expression of NKG2A/C/E and perforin. Data are representative of 4 independent experiments ( n = 13 ± s.e.m; ** P < 0.01, **** P < 0.0001, two-sided, unpaired Student’s t test). g Analysis of the number of anti g en - specific (CD45.2 + CD4 + ) cells from whole lung tissue. Data are representative of 5 independent experiments ( n = 17 ± s.e.m; two-sided, unpaired Student’s t test). h , i Flow cytometry analyses of ex vivo stimulated antigen-specific (CD45.2 + CD4 + ) cells for the expression of CD25 (IL-2Rα) and CD122 (IL-2Rβ). Data are representative of three independent experiments ( n = 10 ± s.e.m; *** P < 0.001, two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Isolation, Infection, Expressing, Flow Cytometry, Ex Vivo, Incubation

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve WT and Aiolos-deficient CD4 + T cells were cultured in the presence of T H 1-polarizing conditions for 3 days. Assay for Transposase-Accessible Chromatin (ATAC)-seq was performed to assess changes in accessibility at differentially expressed gene loci. a Representative heatmap of significantly differentially accessible regions associated with CD4-CTL and T FH gene programs in WT vs Aiolos-deficient cells, presented as row (gene) Z-score with distance from TSS indicated. Regions shown each exhibit statistically significant differences in accessibility. Data are compiled from three independent experiments. b – f ATAC-seq analyses of T H 1 samples overlaid with published STAT5 ChIP-seq ( GSM1865310 ; dark purple) data. WT (light blue, top track) and Aiolos-deficient (red, middle track) samples are displayed as CPM-normalized Integrative Genomics Viewer (IGV) tracks (representative from three independent experiments). Regulatory regions of significant differential accessibility are indicated by gray boxes and asterisks. g , h HOMER motif analysis of sites of significantly increased ( g ) and decreased ( h ) accessibility in the absence of Aiolos. Data are compiled from 3 independent experiments ( n = 3, HOMER hypergeometric analysis). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Cell Culture, ChIP-sequencing

Journal: Nature Communications

Article Title: Aiolos represses CD4 + T cell cytotoxic programming via reciprocal regulation of T FH transcription factors and IL-2 sensitivity

doi: 10.1038/s41467-023-37420-0

Figure Lengend Snippet: Naïve WT and Aiolos-deficient CD4 + T cells were cultured in the presence of T H 1-polarizing conditions for 3 days. a Immunoblot analysis of the indicated proteins. β-actin serves as a loading control. A representative image from 4 independent experiments is shown. b – e Naïve WT and Aiolos-deficient CD4 + T cells were cultured in the presence of T H 1-polarizing conditions for 4 days. ChIP analysis was performed using anti-STAT5, anti-H3K27Ac, and IgG control antibodies for the indicated regions. Data are normalized to total input sample. IgG values were subtracted from percent enrichment and data are displayed relative to the WT sample. Distance from TSS are indicated. Data are compiled from three independent experiments ( n = 4 ± s.e.m; * P < 0.05; ** P < 0.01; two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: For all flow cytometry antibodies, example catalog numbers provided for each antibody, but catalog numbers will vary based on fluorochrome and volume of product: CD4 (1:300; clone GK1.5; R&D Systems; catalog # for anti-CD4:AF488: FAB554G); CD44 (1:300; clone IM7; BD Biosciences; catalog # for anti-CD44:V450: 560451); CD62L (1:300; clone MEL-14; ThermoFisher; catalog # for anti-CD62L-APC-eFluor780: 47-0621-82); PD-1 (1:50; clone 29 F.1A12; BioLegend; catalog # for anti-PE-Cy7: 135216); Cxcr5 (1:50; clone SPRCL5; ThermoFisher; catalog # for anti-Cxcr5:PE:12-7185-82); CD25 (1:25; clone PC61.5; ThermoFisher; catalog # for anti-CD25:PE: 12-0251-82), CD45.2 (1:100; clone 104; Biolegend; catalog # for anti-CD45.2:AF488: 109816), CD122 (1:50, clone 5H4; Biolegend; catalog # for anti-CD122 BUV711: B741537), NKG2A/C/E (1:100; clone 20D5; Biolegend, catalog # for anti-NKG2A/C/E:BUV737: B741808).

Techniques: Cell Culture, Western Blot, Control