Journal: Cell reports

Article Title: Control of Viral Infection by Natural Killer Cell Inhibitory Receptors

doi: 10.1016/j.celrep.2020.107969

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD122 Monoclonal Antibody (TM-b1 (TM-beta1)), eFluor 450 , eBioscience/Thermo Fisher Scientific , Cat# 48-1222-82; RRID: AB_2016697.

Techniques: Labeling, Purification, Blocking Assay, Protein Purification, Virus, Recombinant, Transgenic Assay, Expressing, Generated, Mutagenesis, Knock-In, Disruption, Synthesized, Sequencing, Plasmid Preparation, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr −/− mice

doi: 10.4049/jimmunol.1601429

Figure Lengend Snippet: 16 week DDC fed Stat4−/−Ldlr−/− and Ldlr−/− splenic cell suspensions were stained with anti-CD3, CD8, CXCR5, CD275, CD122 Abs and analyzed by flow cytometry. (A) Flow cytometry gating scheme for CD8+ Tregs. (B) Increased number of CD8+ Tregs despite decreased content of total splenic CD8+ T cells (C) in Stat4−/−Ldlr−/− (black bars) compared with Ldlr−/− (grey bars) mice (n=7–8 mice per genotype). Representative histograms are shown. (D) In vitro differentiation of Stat4-deficient and Stat4-sufficient CD8+ Tregs with plate bound ant-CD3 (1ug/mL) in complete RPMI media supplemented with soluble anti-CD28 (1ug/mL), rhIL-2 (100 U/mL), and rhTGFβ (2ng/mL) for 3 days (n=6, 3 independent experiments). Representative histograms in the CD8+CXCR5+CD275+ gates are shown. *p < 0.05, **p < 0.01

Article Snippet: The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA).

Techniques: Staining, Flow Cytometry, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr −/− mice

doi: 10.4049/jimmunol.1601429

Figure Lengend Snippet: (A) Elevated number of CD8+ Tregs within the aortas of Stat4−/−Ldlr−/− (black bars) compared with Ldlr−/− (grey bar) mice (3 pooled aortas per genotype, n=6 in 3 independent experiments). Representative histograms of aortic CD8+ Tregs (CD8+CXCR5+CD275+CD122+) from Stat4−/−Ldlr−/− and Ldlr−/− mice are shown. Gates are based on isotype control staining. (B) Reduced number of Th cells within the aortas of Stat4−/−Ldlr−/− (black bars) compared with Ldlr−/− (grey bar) mice (n=8–12 per genotype). Representative FACS plots and histograms for aortic Tfh cells from Stat4−/−Ldlr−/− and Ldlr−/− mice. (C) Content of CD11b+F4/80+ MΦs from the aortas of Stat4−/−Ldlr−/− (black bars) versus Ldlr−/− (grey bars) mice (n=8–9 per genotype). **p < 0.01, ****p<0.0001.

Article Snippet: The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA).

Techniques: Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr −/− mice

doi: 10.4049/jimmunol.1601429

Figure Lengend Snippet: (A–B) Splenic cell suspension from 16 week DDC fed Stat4−/−Ldlr−/− (black bars) and Ldlr−/− (grey bars) mice were stained with anti-CD45, CD11b, CD68, I-Ab, and Ly6C Abs and analyzed by flow cytometry (n=5–6 per genotype). (C) RT-PCR expression of Nos2 and Mrc1 in MΦs isolated from Stat4−/−Ldlr−/− (black bars) and Ldlr−/− (grey bars) mice (n=4 mice). Results show mean±SE as the ratio of Stat4−/−Ldlr−/− (black bar) to Ldlr−/− (grey bar) MΦs. (D) Peritoneal MΦs from 16 week DDC fed Stat4−/−Ldlr−/− (black bars) and Ldlr−/− (grey bars) were collected, stimulated with LPS, and analyzed by RT-PCR. The ratio from Stat4−/−Ldlr−/− and Ldlr−/− MΦs is shown (n=5–6 per genotype). (E–G) Stat4−/−Ldlr−/− and Ldlr−/− peritoneal MΦs were isolated and activated with LPS (500ng/mL) in culture for 48 hours. Isolated CD4+ cells were cultured with plate bound anti-CD3, soluble anti-CD28, and the conditioned media (CM) for 72 hrs. Differentiated cells were stained for Foxp3, Tbet, CXCR5, CD8, CD4, CD122, and analyzed by flow cytometry.

Article Snippet: The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA).

Techniques: Suspension, Staining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr −/− mice

doi: 10.4049/jimmunol.1601429

Figure Lengend Snippet: STAT4-sufficient or STAT4-deficient sorter CD8+CD122+ (CD45.2+) Tregs were adoptively transferred into C57BL/6 (CD45.1+) recipient mice (n=4/per group). A group of C57BL/6 mice that did not receive T cells was used as a control. All mice were immunized subcutaneously with 1000 μg KLH emulsified in CFA. Seven days after immunization, cell suspensions from draining lymph nodes of the recipients were stained with anti-Bcl6, CD4, CXCR5, GL-7, Fas, B220, and Tfh cells and germinal center B cells were analyzed. (A) Representative FACS plots from recipients that received WT CD8+ Tregs or STAT4-deficient CD8+ Tregs or no cells (ctrl). Numbers in the boxes represent the percentages. (B) The sera from the recipients were subject to a 3-fold serial dilution, and the concentrations of KLH-specific IgG, IgM, and IgA were analyzed by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA).

Techniques: Control, Staining, Serial Dilution, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: STAT4 regulates CD8+Treg/Tfh cell axis and promotes atherogenesis in insulin-resistant Ldlr −/− mice

doi: 10.4049/jimmunol.1601429

Figure Lengend Snippet: Stat4−/−Ldlr−/− and Ldlr−/− sorted CD8+CD122+ Tregs were adoptively transferred into Ldlr−/− recipient mice (n=5/per group). After 11 weeks of DDC feeding, aortas were analyzed for plaque burden and cell suspensions from para-aortic and peripheral LN were stained with anti-Bcl6, CD4, CXCR5, GL-7, FAS, B220, and Tfh cells and germinal center B cells were analyzed. (A) Representative en face Oil Red O staining of aortas from DDC fed Ldlr−/− recipients that received either Stat4−/−Ldlr−/− (KO) or Ldlr−/− (WT) sorted CD8+CD122+ Tregs. Lesion size (% of whole aorta) is shown. Each symbol represents 1 animal; horizontal bars represent means. (B) Representative FACS plots from recipients that received either Stat4−/−Ldlr−/− (KO) CD8+ Tregs or Ldlr−/− (KO) CD8+ Tregs. Numbers in the boxes represent the percentages. (C) Decreased percentage of Tfh cells and FAS+GL7+B-220+ cells in para-aortic LN and Tfh cells in the spleen of Ldlr−/− recipients that received Stat4−/−Ldlr−/− CD8+ Tregs (blue bars) vs Ldlr−/− mice that received Ldlr−/− CD8+ Tregs (red bars) (n=4–5 per genotype). (D) Reduced levels of IgG1, IgG3, and IgA in the plasma of Ldlr−/− mice received Stat4−/−Ldlr−/− CD8+ Tregs (blue bars) vs Ldlr−/− mice that received Ldlr−/− CD8+ Tregs (red bars). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were as follows: CD19-PECy7 (1D3), CD8-APC (53-6.7), Ly6C-FITC (AL-21), CD68-PE (FA11), CD44-FITc (IM7), CD45-PerCP (30-F11), CD69-PE (H1.2F3), CXCR5-ef450 (SPRCL5), CD138-APC (281-2), CD21-PE (7G6), CD275-PE (HK5.3), CD122-APC (TM-b1), CD11b-PB (M1/70.15), IFNγ-eFluor 450 (XMG1.2), CD4-PerCP (L3T4), F4/80-APC-eFluor780 (BM8), Foxp3-PE (MF23), CD3-APC-CY7/eFluor780 (17A2), Ly-6G-PE (1A8), from eBioscience (San Diego, CA) and anti-mouse CD16/CD32 (The Lymphocyte Culture Centre, UVA, Charlottesville, VA).

Techniques: Staining, Clinical Proteomics

Journal: eLife

Article Title: mTORC1 and mTORC2 differentially promote natural killer cell development

doi: 10.7554/eLife.35619

Figure Lengend Snippet: ( A ) BM and spleen lymphocytes count in WT and Rptor cKO mice. n = 4 pooled from two independent experiments. ( B ) The viability of freshly-isolated NK cells from BM and spleen of WT and Rptor cKO mice was evaluated by Annexin V (Ann V) and Propidium iodide (PI) staining (left). Percentage of live cells (Ann V − PI − ) in each population gated by CD27 and CD11b was quantified (right). n = 3 pooled from three independent experiments. ( C ) Number of iNKs (CD3 − CD122 + NK1.1 + DX5 − ), mNKs (CD3 − CD122 + NK1.1 + DX5 + ) in the spleen and NKPs (CD3 − Flt3 − CD27 + 2B4 + CD127 + CD122 + NK1.1 − ), iNKs, mNKs in the BM from Rptor cKO mice were quantified using flow cytometry. n = 3–5 pooled from two or three independent experiments. ( D ) Number of each NK cell population defined by CD27 and CD11b expression in BM and spleen was quantified as the number of cells per million lymphocytes. n = 7 pooled from four independent experiments. ( E ) CD27 and the CD11b expression on NK cells from liver, blood and lymph node (LN) were assessed by flow cytometry (left). Percentage of each NK subsets was quantified (right). n = 4 pooled from two to four independent experiments. ( F ) Expression of various maturation markers on NK cells was shown as fold change in MFI normalized to WT. n = 4 pooled from two independent experiments. All bar graphs present the mean ± SD. Statistical significance was calculated using two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CD3 (17A2), NK1.1 (PK136), CD49b (DX5), CD27 (LG.7F9), CD11b (M1/70), KLRG1 (2F1), NCR1 (29A1.4), NKG2D (CX5), Ly49H (3D10), NKG2A/C/E (20d5), CD122 (5H4 or TM-b1), Ki-67 (SolA15), Eomes (Dan11mag), T-bet (4B10), CD127 (A7R34), CD244.2 (eBio244F4), CD107a (eBio1D4B), IFN-γ (XMG1.2), Streptavidin-PE, Donkey anti-Rabbit second antibodies are from Thermo-Fisher Scientific (Waltham, MA); Ly49D (4E5), CD45.2 (104), CD132 (TUGm2), CD135 (A2F10), Biotin-Ccr7 (4B12) are from Biolegend (San Diego, CA); Ly49A (A1), Ly49G2 (4D11), Ly49C/I (5E6), p-STAT5 Y694 (47) are from BD Pharmingen (San Jose, CA); Raptor (24C12), Rictor (53A2), p-Akt S473 (D9E), p-rpS6 S240/244 (D68F8), p-4E-BP1 T37/46 (236B4), p-FoxO1 T24 /FoxO3a T32 are from Cell Signaling Technology (Danvers, MA); β-Actin (ACTBD11B7) is from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Staining, Flow Cytometry, Expressing

Journal: eLife

Article Title: mTORC1 and mTORC2 differentially promote natural killer cell development

doi: 10.7554/eLife.35619

Figure Lengend Snippet: ( A ) BM and spleen lymphocytes count in WT and Rictor cKO mice. n = 4 pooled from two independent experiments. ( B ) The viability of freshly isolated NK cells from BM and spleen of WT and Rictor cKO mice was evaluated by Annexin V (Ann V) and Propidium iodide (PI) staining (left). Percentage of live cells (Ann V − PI − ) in each population gated by CD27 and CD11b was quantified (right). n = 3 pooled from three independent experiments. ( C ) Number of iNKs (CD3 − CD122 + NK1.1 + DX5 − ), mNKs (CD3 − CD122 + NK1.1 + DX5 + ) in the spleen and NKPs (CD3 − Flt3 − CD27 + 2B4 + CD127 + CD122 + NK1.1 − ), iNKs, mNKs in the BM from Rictor cKO mice were quantified using flow cytometry. n = 3–4 pooled from two or four independent experiments. ( D ) Number of each NK cell population defined by CD27 and CD11b expression in BM and spleen of WT and Rictor cKO mice was quantified as the number of cells per million lymphocytes. n = 6 pooled from four independent experiments. ( E ) CD27 and the CD11b expression on NK cells from liver, blood and lymph node (LN) of WT and Rictor cKO mice were assessed by flow cytometry (left). Percentage of each NK subsets was quantified (right). n = 3–4 pooled from two or three independent experiments. ( F ) Expression of various maturation markers on NK cells was shown as fold change in MFI normalized to WT. n = 4 pooled from two independent experiments. All bar graphs present the mean ± SD. Statistical significance was calculated using two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CD3 (17A2), NK1.1 (PK136), CD49b (DX5), CD27 (LG.7F9), CD11b (M1/70), KLRG1 (2F1), NCR1 (29A1.4), NKG2D (CX5), Ly49H (3D10), NKG2A/C/E (20d5), CD122 (5H4 or TM-b1), Ki-67 (SolA15), Eomes (Dan11mag), T-bet (4B10), CD127 (A7R34), CD244.2 (eBio244F4), CD107a (eBio1D4B), IFN-γ (XMG1.2), Streptavidin-PE, Donkey anti-Rabbit second antibodies are from Thermo-Fisher Scientific (Waltham, MA); Ly49D (4E5), CD45.2 (104), CD132 (TUGm2), CD135 (A2F10), Biotin-Ccr7 (4B12) are from Biolegend (San Diego, CA); Ly49A (A1), Ly49G2 (4D11), Ly49C/I (5E6), p-STAT5 Y694 (47) are from BD Pharmingen (San Jose, CA); Raptor (24C12), Rictor (53A2), p-Akt S473 (D9E), p-rpS6 S240/244 (D68F8), p-4E-BP1 T37/46 (236B4), p-FoxO1 T24 /FoxO3a T32 are from Cell Signaling Technology (Danvers, MA); β-Actin (ACTBD11B7) is from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Staining, Flow Cytometry, Expressing

Journal: eLife

Article Title: mTORC1 and mTORC2 differentially promote natural killer cell development

doi: 10.7554/eLife.35619

Figure Lengend Snippet: ( A ) Histogram of CD122 expression on each NK population gated by CD27 and CD11b of WT and Rptor cKO mice (left). MFI of CD122 was normalized to WT CD27 SP population (right). n = 3 pooled from three independent experiments. ( B ) Splenocytes from WT or Rptor cKO mice were stimulated with either medium or 100 ng/mL IL-15 for 1 hr. Phosphorylation of STAT5 Y694 was detected by phosphor-flow and shown as the representative histogram of three independent experiments. ( C, D ) CD122 expression ( C ) and IL-15-mediated STAT5 activation ( D ) in WT or Rictor-deficient NK cells. Same experimental procedures as A and B. All bar graphs present the mean ± SD. Statistical significance was calculated using two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CD3 (17A2), NK1.1 (PK136), CD49b (DX5), CD27 (LG.7F9), CD11b (M1/70), KLRG1 (2F1), NCR1 (29A1.4), NKG2D (CX5), Ly49H (3D10), NKG2A/C/E (20d5), CD122 (5H4 or TM-b1), Ki-67 (SolA15), Eomes (Dan11mag), T-bet (4B10), CD127 (A7R34), CD244.2 (eBio244F4), CD107a (eBio1D4B), IFN-γ (XMG1.2), Streptavidin-PE, Donkey anti-Rabbit second antibodies are from Thermo-Fisher Scientific (Waltham, MA); Ly49D (4E5), CD45.2 (104), CD132 (TUGm2), CD135 (A2F10), Biotin-Ccr7 (4B12) are from Biolegend (San Diego, CA); Ly49A (A1), Ly49G2 (4D11), Ly49C/I (5E6), p-STAT5 Y694 (47) are from BD Pharmingen (San Jose, CA); Raptor (24C12), Rictor (53A2), p-Akt S473 (D9E), p-rpS6 S240/244 (D68F8), p-4E-BP1 T37/46 (236B4), p-FoxO1 T24 /FoxO3a T32 are from Cell Signaling Technology (Danvers, MA); β-Actin (ACTBD11B7) is from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Phospho-proteomics, Activation Assay

Journal: eLife

Article Title: mTORC1 and mTORC2 differentially promote natural killer cell development

doi: 10.7554/eLife.35619

Figure Lengend Snippet: ( A ) The MFI of CD132 in NK cells from BM and Spleen of Rptor cKO mice were normalized to WT control demonstrated as a bar graph. n = 4 pooled from two independent experiments. ( B ) Phosphorylation of rpS6 S240/244 in different NK cell subsets defined by CD27 and CD11b from BM and spleen of WT mice was evaluated by flow cytometry (left). MFI of rpS6 S240/244 was normalized to CD27 SP population and shown as percentages (right). n = 3 pooled from two independent experiments. ( C ) MFI of FSC of each NK subset was normalized to CD27 SP population from BM and spleen of WT mice and shown as percentages. n = 7 pooled from four independent experiments. ( D ) Eomes expression in three NK cell subsets is shown in fold change normalized to CD27 SP population. n = 8 pooled from four independent experiments. ( E ) CD122 expression in three NK cell subsets is shown in fold change normalized to CD27 SP population. n = 5 pooled from four independent experiments. ( F ) The MFI of CD132 in NK cells from BM and Spleen of Rictor cKO mice were normalized to WT control demonstrated as a bar graph. n = 3 pooled from three independent experiments. All bar graphs present the mean ± SD. Statistical significance was calculated using two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CD3 (17A2), NK1.1 (PK136), CD49b (DX5), CD27 (LG.7F9), CD11b (M1/70), KLRG1 (2F1), NCR1 (29A1.4), NKG2D (CX5), Ly49H (3D10), NKG2A/C/E (20d5), CD122 (5H4 or TM-b1), Ki-67 (SolA15), Eomes (Dan11mag), T-bet (4B10), CD127 (A7R34), CD244.2 (eBio244F4), CD107a (eBio1D4B), IFN-γ (XMG1.2), Streptavidin-PE, Donkey anti-Rabbit second antibodies are from Thermo-Fisher Scientific (Waltham, MA); Ly49D (4E5), CD45.2 (104), CD132 (TUGm2), CD135 (A2F10), Biotin-Ccr7 (4B12) are from Biolegend (San Diego, CA); Ly49A (A1), Ly49G2 (4D11), Ly49C/I (5E6), p-STAT5 Y694 (47) are from BD Pharmingen (San Jose, CA); Raptor (24C12), Rictor (53A2), p-Akt S473 (D9E), p-rpS6 S240/244 (D68F8), p-4E-BP1 T37/46 (236B4), p-FoxO1 T24 /FoxO3a T32 are from Cell Signaling Technology (Danvers, MA); β-Actin (ACTBD11B7) is from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control, Phospho-proteomics, Flow Cytometry, Expressing