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Lactate Induces IL17 via Nuclear PKM2- and FAS-Mediated STAT3 Phosphorylation <t>CD4</t> + T cells were isolated from HC <t>PBMCs</t> and activated with anti-CD3 and anti-CD28 mAb. (A) ROS levels in CD4 + T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. PBS and H 2 O 2 were used as negative and positive control, respectively. (B) Representative western blots showing nuclear PKM1/2, P-STAT3, STAT3, and cytosolic PKM1/2 in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Histone H3 and β-actin were used as controls for nuclear and cytosolic fraction, respectively. Data representative of n = 3 independent experiments. (C) Representative western blots (left) and densitometric quantification (right; n = 3) of P-STAT3, STAT3, P-STAT1, and STAT1 expression by activated CD4 + T cells treated with sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Untreated CD4 + T cells (CN, dotted line) set to 1. (D) Representative western blots (left) and densitometric quantification (right; n = 3) of P-ACC, ACC, P-AMPK, and AMPK expression by activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (E) Representative western blots showing cytosolic and mitochondrial P-ACC and ACC in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). β-actin and VDAC were used as controls for cytosolic and mitochondrial fraction, respectively. Data representative of n = 2 independent experiments. (F) Mass spectrometry carbon tracer analysis of palmitate in 48-h [U 13 C]-lactate-fed activated CD4 + T cells treated as in Figure 2 D (n = 4, time points 0, 24, and 48 h; n = 2, time points 72 and 96 h). (G) Representative western blots (left) and densitometric quantification (right; n = 2) of P-STAT3 and STAT3 expression by activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), and DHEA (20 μM) or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (H) IL-17A and IFNγ ELISAs from supernatants of activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), DHEA (20 μM), DASA (20 μM), AICAR (1 mM), or left untreated (n = 5, each in duplicate; for lactate + DASA + C75 or lactate + DASA + TOFA, n = 2, each in duplicate). (I) Representative western blots (left) and densitometric quantifications (right; n = 3) of P-ACC, ACC, P-STAT3, and STAT3 expression by activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (CN). Also, IL-17A ELISA from supernatants of activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (n = 3, each in duplicate). Two-tailed Student’s t test (A), (C), (F), (H), and (I) or one-way ANOVA (G). Data expressed as mean ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ###p ≤ 0.001 versus lactate (H). (J) Schematic depicting the described findings: lactate modulates IL17 expression by activating two pathways, PKM2 translocation into the nucleus and FAS induction, converging on STAT3-induced transcription of IL17. See also Figure S4 .
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1) Product Images from "Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring"

Article Title: Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring

Journal: Cell Metabolism

doi: 10.1016/j.cmet.2019.10.004

Lactate Induces IL17 via Nuclear PKM2- and FAS-Mediated STAT3 Phosphorylation CD4 + T cells were isolated from HC PBMCs and activated with anti-CD3 and anti-CD28 mAb. (A) ROS levels in CD4 + T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. PBS and H 2 O 2 were used as negative and positive control, respectively. (B) Representative western blots showing nuclear PKM1/2, P-STAT3, STAT3, and cytosolic PKM1/2 in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Histone H3 and β-actin were used as controls for nuclear and cytosolic fraction, respectively. Data representative of n = 3 independent experiments. (C) Representative western blots (left) and densitometric quantification (right; n = 3) of P-STAT3, STAT3, P-STAT1, and STAT1 expression by activated CD4 + T cells treated with sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Untreated CD4 + T cells (CN, dotted line) set to 1. (D) Representative western blots (left) and densitometric quantification (right; n = 3) of P-ACC, ACC, P-AMPK, and AMPK expression by activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (E) Representative western blots showing cytosolic and mitochondrial P-ACC and ACC in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). β-actin and VDAC were used as controls for cytosolic and mitochondrial fraction, respectively. Data representative of n = 2 independent experiments. (F) Mass spectrometry carbon tracer analysis of palmitate in 48-h [U 13 C]-lactate-fed activated CD4 + T cells treated as in Figure 2 D (n = 4, time points 0, 24, and 48 h; n = 2, time points 72 and 96 h). (G) Representative western blots (left) and densitometric quantification (right; n = 2) of P-STAT3 and STAT3 expression by activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), and DHEA (20 μM) or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (H) IL-17A and IFNγ ELISAs from supernatants of activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), DHEA (20 μM), DASA (20 μM), AICAR (1 mM), or left untreated (n = 5, each in duplicate; for lactate + DASA + C75 or lactate + DASA + TOFA, n = 2, each in duplicate). (I) Representative western blots (left) and densitometric quantifications (right; n = 3) of P-ACC, ACC, P-STAT3, and STAT3 expression by activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (CN). Also, IL-17A ELISA from supernatants of activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (n = 3, each in duplicate). Two-tailed Student’s t test (A), (C), (F), (H), and (I) or one-way ANOVA (G). Data expressed as mean ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ###p ≤ 0.001 versus lactate (H). (J) Schematic depicting the described findings: lactate modulates IL17 expression by activating two pathways, PKM2 translocation into the nucleus and FAS induction, converging on STAT3-induced transcription of IL17. See also Figure S4 .
Figure Legend Snippet: Lactate Induces IL17 via Nuclear PKM2- and FAS-Mediated STAT3 Phosphorylation CD4 + T cells were isolated from HC PBMCs and activated with anti-CD3 and anti-CD28 mAb. (A) ROS levels in CD4 + T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. PBS and H 2 O 2 were used as negative and positive control, respectively. (B) Representative western blots showing nuclear PKM1/2, P-STAT3, STAT3, and cytosolic PKM1/2 in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Histone H3 and β-actin were used as controls for nuclear and cytosolic fraction, respectively. Data representative of n = 3 independent experiments. (C) Representative western blots (left) and densitometric quantification (right; n = 3) of P-STAT3, STAT3, P-STAT1, and STAT1 expression by activated CD4 + T cells treated with sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Untreated CD4 + T cells (CN, dotted line) set to 1. (D) Representative western blots (left) and densitometric quantification (right; n = 3) of P-ACC, ACC, P-AMPK, and AMPK expression by activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (E) Representative western blots showing cytosolic and mitochondrial P-ACC and ACC in activated CD4 + T cells treated with sodium lactate (10 mM) for the indicated time points or left untreated (CN). β-actin and VDAC were used as controls for cytosolic and mitochondrial fraction, respectively. Data representative of n = 2 independent experiments. (F) Mass spectrometry carbon tracer analysis of palmitate in 48-h [U 13 C]-lactate-fed activated CD4 + T cells treated as in Figure 2 D (n = 4, time points 0, 24, and 48 h; n = 2, time points 72 and 96 h). (G) Representative western blots (left) and densitometric quantification (right; n = 2) of P-STAT3 and STAT3 expression by activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), and DHEA (20 μM) or left untreated (CN). Untreated CD4 + T cells (CN, dotted line) set to 1. (H) IL-17A and IFNγ ELISAs from supernatants of activated CD4 + T cells treated with sodium lactate (10 mM) alone or in combination with C75 (10 μM), TOFA (20 μM), DHEA (20 μM), DASA (20 μM), AICAR (1 mM), or left untreated (n = 5, each in duplicate; for lactate + DASA + C75 or lactate + DASA + TOFA, n = 2, each in duplicate). (I) Representative western blots (left) and densitometric quantifications (right; n = 3) of P-ACC, ACC, P-STAT3, and STAT3 expression by activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (CN). Also, IL-17A ELISA from supernatants of activated CD4 + T cells from Slc5a12 WT or KO mice, treated with sodium lactate (10 mM) or left untreated (n = 3, each in duplicate). Two-tailed Student’s t test (A), (C), (F), (H), and (I) or one-way ANOVA (G). Data expressed as mean ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ###p ≤ 0.001 versus lactate (H). (J) Schematic depicting the described findings: lactate modulates IL17 expression by activating two pathways, PKM2 translocation into the nucleus and FAS induction, converging on STAT3-induced transcription of IL17. See also Figure S4 .

Techniques Used: Isolation, Activation Assay, Positive Control, Western Blot, Expressing, Mass Spectrometry, Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Translocation Assay

Lactate Uptake by CD4 + T Cells Impacts Intracellular Utilization of Central Carbon Metabolic Pathways (A) Glucose and glutamine uptake rates for CD4 + T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAbs for 24 h followed by further 48-h culture with lactate alone or in the presence of SLC5A12 Ab, or left untreated, in medium containing low glucose (5 mM) and 5% FBS (n = 3, each in duplicate). (B) NAD + and NADH intracellular levels in CD4 + T cells (n = 2) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation and shown as NAD + /NADH ratio. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. (C) Seahorse measurements of extracellular acidification (left) and oxygen consumption (right) rates (ECAR and OCR, respectively) by 12-h-activated CD4 + T cells (n = 3, technical replicates). 1 h prior to the experiment, cells were seeded in a 96-well microplate in XF Assay medium in the presence of 10 mM of glucose. Sodium lactate (10 mM) or PBS was injected during measurement. Data representative of n = 2 independent experiments. (D and E) 13 C tracing of [U 13 C]-lactate into pyruvate and citrate. Activated CD4 + T cells were incubated for 48 h with [U 13 C]-lactate in the presence or absence of SLC5A12 Ab in medium containing low glucose (5 mM) and 5% FBS (n = 2, each in duplicate). Polar metabolites were extracted, analyzed by LC-MS and peak areas of mass isotopologues normalized to cell number are represented. (F and G) Acetyl-CoA (F) and citrate (G) intracellular levels in CD4 + T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. Two tailed Student’s t test. Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.
Figure Legend Snippet: Lactate Uptake by CD4 + T Cells Impacts Intracellular Utilization of Central Carbon Metabolic Pathways (A) Glucose and glutamine uptake rates for CD4 + T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAbs for 24 h followed by further 48-h culture with lactate alone or in the presence of SLC5A12 Ab, or left untreated, in medium containing low glucose (5 mM) and 5% FBS (n = 3, each in duplicate). (B) NAD + and NADH intracellular levels in CD4 + T cells (n = 2) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation and shown as NAD + /NADH ratio. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. (C) Seahorse measurements of extracellular acidification (left) and oxygen consumption (right) rates (ECAR and OCR, respectively) by 12-h-activated CD4 + T cells (n = 3, technical replicates). 1 h prior to the experiment, cells were seeded in a 96-well microplate in XF Assay medium in the presence of 10 mM of glucose. Sodium lactate (10 mM) or PBS was injected during measurement. Data representative of n = 2 independent experiments. (D and E) 13 C tracing of [U 13 C]-lactate into pyruvate and citrate. Activated CD4 + T cells were incubated for 48 h with [U 13 C]-lactate in the presence or absence of SLC5A12 Ab in medium containing low glucose (5 mM) and 5% FBS (n = 2, each in duplicate). Polar metabolites were extracted, analyzed by LC-MS and peak areas of mass isotopologues normalized to cell number are represented. (F and G) Acetyl-CoA (F) and citrate (G) intracellular levels in CD4 + T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. Two tailed Student’s t test. Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Techniques Used: Isolation, Activation Assay, XF Assay, Injection, Incubation, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Lactate Shapes the Effector Phenotype of CD4 + T Cells at the Site of Inflammation via SLC5A12 (A) Relative mRNA expression levels of IL17A , IL22 , IFNγ , IL6 , IL10 , and TGFβ as assessed by qRT-PCR in tonsil CD4 + T cells treated with sodium lactate (10 mM) and/or SLC5A12 Ab or left untreated (n = 5). Levels of mRNA of each cytokine expressed by lactate-untreated CD4 + T cells were set to 1 (CN, dotted line). (B) IL-17A and IFNγ ELISAs from supernatants of tonsil CD4 + T cells treated as in (A), (n = 5, each in duplicate). (C) Relative mRNA expression levels of RORγT , FOXO1 , FOXP3 , PD1 , CXCR5 , and BCL6 as assessed by qRT-PCR in tonsil CD4 + T cells treated as in (A), (n = 5). Levels of mRNA of each cytokine expressed by lactate-untreated CD4 + T cells set to 1 (CN, dotted line). (D) Representative flow cytometry plots of CD4 + IL17 + , CD4 + FOXP3 + , CD4 + PD1 + CXCR5 + , CD4 + IFNγ + , and CD4 + IL10 + tonsil CD4 + T cells incubated in the presence or absence of SLC5A12 Ab (left; n = 3). Quantification bar charts (right). (E) Percentage of IFNγ + , IL17A + , IL21 + , Treg (CD25 + Foxp3 + ), and cytokine-negative (Neg CKS; left) or RORγt + , Treg (CD25 + Foxp3 + ), Tfh (CXCR5 + PD-1 + ICOS + ), and Tbet + (right) CD4 + SLC5A12 + T cell subsets in 48-h activated human HC PBMCs (n = 5). Two-tailed Student’s t test. Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.
Figure Legend Snippet: Lactate Shapes the Effector Phenotype of CD4 + T Cells at the Site of Inflammation via SLC5A12 (A) Relative mRNA expression levels of IL17A , IL22 , IFNγ , IL6 , IL10 , and TGFβ as assessed by qRT-PCR in tonsil CD4 + T cells treated with sodium lactate (10 mM) and/or SLC5A12 Ab or left untreated (n = 5). Levels of mRNA of each cytokine expressed by lactate-untreated CD4 + T cells were set to 1 (CN, dotted line). (B) IL-17A and IFNγ ELISAs from supernatants of tonsil CD4 + T cells treated as in (A), (n = 5, each in duplicate). (C) Relative mRNA expression levels of RORγT , FOXO1 , FOXP3 , PD1 , CXCR5 , and BCL6 as assessed by qRT-PCR in tonsil CD4 + T cells treated as in (A), (n = 5). Levels of mRNA of each cytokine expressed by lactate-untreated CD4 + T cells set to 1 (CN, dotted line). (D) Representative flow cytometry plots of CD4 + IL17 + , CD4 + FOXP3 + , CD4 + PD1 + CXCR5 + , CD4 + IFNγ + , and CD4 + IL10 + tonsil CD4 + T cells incubated in the presence or absence of SLC5A12 Ab (left; n = 3). Quantification bar charts (right). (E) Percentage of IFNγ + , IL17A + , IL21 + , Treg (CD25 + Foxp3 + ), and cytokine-negative (Neg CKS; left) or RORγt + , Treg (CD25 + Foxp3 + ), Tfh (CXCR5 + PD-1 + ICOS + ), and Tbet + (right) CD4 + SLC5A12 + T cell subsets in 48-h activated human HC PBMCs (n = 5). Two-tailed Student’s t test. Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Incubation, Two Tailed Test

SLC5A12 Expression by CD4 + T Cells Is Regulated by Activating and Inflammatory Stimuli (A–C) Representative flow cytometry plots of SLC5A12 expression by CD4 + or CD8 + T cells from non-activated (n = 3; A) or anti-CD3 mAb-activated (n = 6; B) HC PBMCs. Quantification shown in (C). (D–F) Representative flow cytometry histograms (D and E) and quantification (F) of SLC5A12 expression by CD4 + T cells from non-activated HC (n = 4) and RA (n = 4; D and F), or anti-CD3 mAb-activated HC (n = 4) and RA (n = 5; E and F) PBMCs. CD4 + T cells from non-activated RA SFMCs (n = 8; E and F) were also analyzed. Briefly, PBMCs were cultured in RPMI medium supplemented with 5% RA or HC autologous blood serum (BS), or 5% RA synovial fluid (SF); SFMCs were cultured in RPMI medium supplemented with 5% autologous SF. (G) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4 + T cells from non-activated or anti-CD3 mAb-activated RA SFMCs. Briefly, cells were cultured in RPMI medium supplemented with 5% FBS (n = 3), 5% autologous BS (n = 8) or 5% autologous RA SF (n = 8). Activated RA PBMCs cultured in 5% BS RPMI (n = 5) were used as controls (H). MFI, mean fluorescent intensity. (H) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4 + T cells from RA SFMCs (n = 5) incubated with 3C7 mAb or control rat sera. (I and J) SLC5A12 mRNA (n = 5; I) and protein (representative western blots [left] and densitometric quantification [right; n = 3]; J) expression by CD4 + T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAb in the presence of sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. One-way ANOVA (C and F) or two-tailed Student’s t test (G–J). Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. See also Figures S1 , S2 , and S3 .
Figure Legend Snippet: SLC5A12 Expression by CD4 + T Cells Is Regulated by Activating and Inflammatory Stimuli (A–C) Representative flow cytometry plots of SLC5A12 expression by CD4 + or CD8 + T cells from non-activated (n = 3; A) or anti-CD3 mAb-activated (n = 6; B) HC PBMCs. Quantification shown in (C). (D–F) Representative flow cytometry histograms (D and E) and quantification (F) of SLC5A12 expression by CD4 + T cells from non-activated HC (n = 4) and RA (n = 4; D and F), or anti-CD3 mAb-activated HC (n = 4) and RA (n = 5; E and F) PBMCs. CD4 + T cells from non-activated RA SFMCs (n = 8; E and F) were also analyzed. Briefly, PBMCs were cultured in RPMI medium supplemented with 5% RA or HC autologous blood serum (BS), or 5% RA synovial fluid (SF); SFMCs were cultured in RPMI medium supplemented with 5% autologous SF. (G) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4 + T cells from non-activated or anti-CD3 mAb-activated RA SFMCs. Briefly, cells were cultured in RPMI medium supplemented with 5% FBS (n = 3), 5% autologous BS (n = 8) or 5% autologous RA SF (n = 8). Activated RA PBMCs cultured in 5% BS RPMI (n = 5) were used as controls (H). MFI, mean fluorescent intensity. (H) Representative flow cytometry histograms (left) and quantification (right) of SLC5A12 expression by CD4 + T cells from RA SFMCs (n = 5) incubated with 3C7 mAb or control rat sera. (I and J) SLC5A12 mRNA (n = 5; I) and protein (representative western blots [left] and densitometric quantification [right; n = 3]; J) expression by CD4 + T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAb in the presence of sodium lactate (10 mM) and/or SLC5A12 Ab, or left untreated. Lactate-untreated CD4 + T cells (CN, dotted line) set to 1. One-way ANOVA (C and F) or two-tailed Student’s t test (G–J). Data expressed as mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. See also Figures S1 , S2 , and S3 .

Techniques Used: Expressing, Flow Cytometry, Cytometry, Cell Culture, Incubation, Western Blot, Isolation, Two Tailed Test

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Mouse Assay:

Article Title: Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring
Article Snippet: .. CD4+ T cells were purified from human PBMCs or MCs, or from spleen and lymph nodes of Slc5a12 WT or KO female mice by negative selection using a magnetic cell separation (EasySep, Stem Cell Technology), cultured in RPMI 1640 plus 10% FBS (37°C, 5% CO2 ) and then stimulated for 3 days in the presence of anti-CD3 and anti-CD28 mAbs coated Dynabeads (0.1 beads per cell; Thermo Fisher Scientific). .. Mouse Models We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders as well as INFRAFRONTIER/EMMA ( www.infrafrontier.eu ), for accepting our gene nomination for Slc5a12, and for generating and providing the Crispr/Cas9 mutant mouse line (Allele: Slc5a12em1(IMPC)Wtsi ) in the C57BL/6N background ( , , , ).

Plasmid Preparation:

Article Title: Engineering Antigen-Specific T Cells from Genetically Modified Human Hematopoietic Stem Cells in Immunodeficient Mice
Article Snippet: .. TCR-Containing Vector Transduction of CD8+ PBMC CD8+ T cells were purified from fresh human PBMC using the EasySep CD8+ T cell enrichment Kit (StemCell Technologies) and were stimulated with anti-CD3 and irradiated allogeneic PBMCs for 4 days. .. Cells were then transduced with the lentiviral vector overnight and incubated for 2 more days.

Purification:

Article Title: Engineering Antigen-Specific T Cells from Genetically Modified Human Hematopoietic Stem Cells in Immunodeficient Mice
Article Snippet: .. TCR-Containing Vector Transduction of CD8+ PBMC CD8+ T cells were purified from fresh human PBMC using the EasySep CD8+ T cell enrichment Kit (StemCell Technologies) and were stimulated with anti-CD3 and irradiated allogeneic PBMCs for 4 days. .. Cells were then transduced with the lentiviral vector overnight and incubated for 2 more days.

Article Title: Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells
Article Snippet: .. In another experiment, T cells were first enriched from human PBMC or BLT splenocytes using the EasySep Human T cell purification kit (Stem Cell Technologies, Vancouver, BC, Canada). .. Cells were cultured in medium alone or with human T-cell cytokines (R & D Systems, Minneapolis, MN), e.g. IL-2 at 20 U/ml and IL-7 (50 ng/ml).

Article Title: Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring
Article Snippet: .. CD4+ T cells were purified from human PBMCs or MCs, or from spleen and lymph nodes of Slc5a12 WT or KO female mice by negative selection using a magnetic cell separation (EasySep, Stem Cell Technology), cultured in RPMI 1640 plus 10% FBS (37°C, 5% CO2 ) and then stimulated for 3 days in the presence of anti-CD3 and anti-CD28 mAbs coated Dynabeads (0.1 beads per cell; Thermo Fisher Scientific). .. Mouse Models We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders as well as INFRAFRONTIER/EMMA ( www.infrafrontier.eu ), for accepting our gene nomination for Slc5a12, and for generating and providing the Crispr/Cas9 mutant mouse line (Allele: Slc5a12em1(IMPC)Wtsi ) in the C57BL/6N background ( , , , ).

Magnetic Cell Separation:

Article Title: Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring
Article Snippet: .. CD4+ T cells were purified from human PBMCs or MCs, or from spleen and lymph nodes of Slc5a12 WT or KO female mice by negative selection using a magnetic cell separation (EasySep, Stem Cell Technology), cultured in RPMI 1640 plus 10% FBS (37°C, 5% CO2 ) and then stimulated for 3 days in the presence of anti-CD3 and anti-CD28 mAbs coated Dynabeads (0.1 beads per cell; Thermo Fisher Scientific). .. Mouse Models We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders as well as INFRAFRONTIER/EMMA ( www.infrafrontier.eu ), for accepting our gene nomination for Slc5a12, and for generating and providing the Crispr/Cas9 mutant mouse line (Allele: Slc5a12em1(IMPC)Wtsi ) in the C57BL/6N background ( , , , ).

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    STEMCELL Technologies Inc human b lymphocytes peripheral blood mononuclear cells pbmcs
    Endogenous expression of C3 is very low in <t>human</t> B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, <t>PBMCs,</t> and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.
    Human B Lymphocytes Peripheral Blood Mononuclear Cells Pbmcs, supplied by STEMCELL Technologies Inc, 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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    STEMCELL Technologies Inc human treg isolation human pbmcs
    <t>Xeno-Treg</t> suppress rejection of islet xenografts in humanized mice. (A) Percentage of graft survival in mice administered 1×10 7 CD25 + cell-depleted human <t>PBMCs</t> with or without 1×10 6 Poly-Treg or Xeno-Treg. Graft survival was monitored 18, 21, 28, 48, 56, 63 and 84 days post cell transfer. (B) Flow cytometric analysis of the percentage of human leukocyte engraftment in the spleen and peripheral blood of NOD-SCID interleukin-2 receptor γ −/− mice after PBMC plus Treg adoptive transfer. Data were acquired on day 63 for Poly-Treg or day 84 for Xeno-Treg. Data are presented as the means ± standard deviation of three independent experiments. CD, cluster of differentiation; PBMC, peripheral blood mononuclear cell; Poly-Treg, polyclonal Treg; Tregs, regulatory T cells; Xeno-Treg, Treg with xenoantigen specificity.
    Human Treg Isolation Human Pbmcs, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    STEMCELL Technologies Inc human cd163 m2 macrophages in vitro pbmcs
    Expression of interleukin‐33 ( IL ‐33) and candidate TLR s in SG s from patients with IgG4‐ RD . A , Expression levels of mRNA for IL ‐33 in SG s from healthy controls (n = 10), patients with chronic sialadenitis (n = 10), patients with SS (n = 15), and patients with IgG4‐ RD (n = 15). B , Distribution of IL ‐33 in SG s from a representative healthy control, patient with chronic sialadenitis, patient with SS , and patient with IgG4‐ RD . Mayer's hematoxylin (blue) counterstained; bars = 100 μm. C , Correlation between expression levels of IL ‐33 mRNA and candidate TLR s in SG s from patients with IgG4‐ RD (n = 15), as determined by Spearman's rank correlation test. D , Schematic illustration of the extraction of CD 163+ <t>M2</t> macrophages stimulated with TLR ‐7 agonist R848. Cells were cultivated as described in Patients and Methods. <t>PBMC</t> = peripheral blood mononuclear cell. E , Production of IL ‐33 by CD 163+ M2 macrophages stimulated with R848, as determined by enzyme‐linked immunosorbent assay. IL ‐33 levels increased in a concentration‐dependent manner. In A and E , bars show the mean ± SD . * = P
    Human Cd163 M2 Macrophages In Vitro Pbmcs, supplied by STEMCELL Technologies Inc, 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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    STEMCELL Technologies Inc human pbmcs
    The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and <t>SEB</t> super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 low (PC3) and PD-L1 high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ TNFα). d Workflow of the SEB super-antigen assay. Total primary human <t>PBMCs</t> were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA
    Human Pbmcs, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

    doi: 10.3389/fimmu.2019.00493

    Figure Lengend Snippet: Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

    Article Snippet: Isolation of Human B Lymphocytes Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell Technologies) density gradient centrifugation from superfluous buffy coat obtained from the Medical Service (Clinical Immunology and Transfusion Medicine, Lund) according to standard procedures ( ) and permit granted by the local ethics committee of Lund.

    Techniques: Expressing, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Positive Control, Sequencing, Western Blot, Incubation

    Xeno-Treg suppress rejection of islet xenografts in humanized mice. (A) Percentage of graft survival in mice administered 1×10 7 CD25 + cell-depleted human PBMCs with or without 1×10 6 Poly-Treg or Xeno-Treg. Graft survival was monitored 18, 21, 28, 48, 56, 63 and 84 days post cell transfer. (B) Flow cytometric analysis of the percentage of human leukocyte engraftment in the spleen and peripheral blood of NOD-SCID interleukin-2 receptor γ −/− mice after PBMC plus Treg adoptive transfer. Data were acquired on day 63 for Poly-Treg or day 84 for Xeno-Treg. Data are presented as the means ± standard deviation of three independent experiments. CD, cluster of differentiation; PBMC, peripheral blood mononuclear cell; Poly-Treg, polyclonal Treg; Tregs, regulatory T cells; Xeno-Treg, Treg with xenoantigen specificity.

    Journal: Molecular Medicine Reports

    Article Title: Adoptive transfer of xenoantigen-stimulated T cell receptor Vβ-restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice

    doi: 10.3892/mmr.2018.9471

    Figure Lengend Snippet: Xeno-Treg suppress rejection of islet xenografts in humanized mice. (A) Percentage of graft survival in mice administered 1×10 7 CD25 + cell-depleted human PBMCs with or without 1×10 6 Poly-Treg or Xeno-Treg. Graft survival was monitored 18, 21, 28, 48, 56, 63 and 84 days post cell transfer. (B) Flow cytometric analysis of the percentage of human leukocyte engraftment in the spleen and peripheral blood of NOD-SCID interleukin-2 receptor γ −/− mice after PBMC plus Treg adoptive transfer. Data were acquired on day 63 for Poly-Treg or day 84 for Xeno-Treg. Data are presented as the means ± standard deviation of three independent experiments. CD, cluster of differentiation; PBMC, peripheral blood mononuclear cell; Poly-Treg, polyclonal Treg; Tregs, regulatory T cells; Xeno-Treg, Treg with xenoantigen specificity.

    Article Snippet: Peripheral blood mononuclear cell (PBMC) isolation and human Treg isolation Human PBMCs were isolated from the blood of 4 healthy donors (age, 28–58; gender, 2 male and 2 female) by density gradient centrifugation using Lymphoprep™ (STEMCELL Technologies China Co., Ltd, Shanghai, China).

    Techniques: Mouse Assay, Flow Cytometry, Adoptive Transfer Assay, Standard Deviation

    Histology and immunohistochemical analysis of NICC xenografts. Representative hematoxylin and eosin staining images; and immunohistochemical staining images of porcine insulin and human CD45 in NICC xenograft samples from mice receiving (A-C) no human cells (NICC alone 84 days post-transplantation), (D-F) only human PBMCs (NICC + PBMC 28 days post-PBMC transfer), (G-I) human PBMCs and Xeno-Treg (NICC + PBMC + Xeno-Treg 84 days post-cell transfer) or (J-L) human PBMCs and Poly-Treg (NICC + PBMC + Poly-Treg 63 days post-cell transfer). (A, B, E-I, K and L) Magnification, ×200; (C, D and J) magnification, ×100. CD, cluster of differentiation; NICC, neonatal porcine islet cell clusters; PBMC, peripheral blood mononuclear cell; Poly-Treg, polyclonal Treg; Tregs, regulatory T cells; Xeno-Treg, Treg with xenoantigen specificity.

    Journal: Molecular Medicine Reports

    Article Title: Adoptive transfer of xenoantigen-stimulated T cell receptor Vβ-restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice

    doi: 10.3892/mmr.2018.9471

    Figure Lengend Snippet: Histology and immunohistochemical analysis of NICC xenografts. Representative hematoxylin and eosin staining images; and immunohistochemical staining images of porcine insulin and human CD45 in NICC xenograft samples from mice receiving (A-C) no human cells (NICC alone 84 days post-transplantation), (D-F) only human PBMCs (NICC + PBMC 28 days post-PBMC transfer), (G-I) human PBMCs and Xeno-Treg (NICC + PBMC + Xeno-Treg 84 days post-cell transfer) or (J-L) human PBMCs and Poly-Treg (NICC + PBMC + Poly-Treg 63 days post-cell transfer). (A, B, E-I, K and L) Magnification, ×200; (C, D and J) magnification, ×100. CD, cluster of differentiation; NICC, neonatal porcine islet cell clusters; PBMC, peripheral blood mononuclear cell; Poly-Treg, polyclonal Treg; Tregs, regulatory T cells; Xeno-Treg, Treg with xenoantigen specificity.

    Article Snippet: Peripheral blood mononuclear cell (PBMC) isolation and human Treg isolation Human PBMCs were isolated from the blood of 4 healthy donors (age, 28–58; gender, 2 male and 2 female) by density gradient centrifugation using Lymphoprep™ (STEMCELL Technologies China Co., Ltd, Shanghai, China).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Transplantation Assay

    Expression of interleukin‐33 ( IL ‐33) and candidate TLR s in SG s from patients with IgG4‐ RD . A , Expression levels of mRNA for IL ‐33 in SG s from healthy controls (n = 10), patients with chronic sialadenitis (n = 10), patients with SS (n = 15), and patients with IgG4‐ RD (n = 15). B , Distribution of IL ‐33 in SG s from a representative healthy control, patient with chronic sialadenitis, patient with SS , and patient with IgG4‐ RD . Mayer's hematoxylin (blue) counterstained; bars = 100 μm. C , Correlation between expression levels of IL ‐33 mRNA and candidate TLR s in SG s from patients with IgG4‐ RD (n = 15), as determined by Spearman's rank correlation test. D , Schematic illustration of the extraction of CD 163+ M2 macrophages stimulated with TLR ‐7 agonist R848. Cells were cultivated as described in Patients and Methods. PBMC = peripheral blood mononuclear cell. E , Production of IL ‐33 by CD 163+ M2 macrophages stimulated with R848, as determined by enzyme‐linked immunosorbent assay. IL ‐33 levels increased in a concentration‐dependent manner. In A and E , bars show the mean ± SD . * = P

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    Article Title: Activated M2 Macrophages Contribute to the Pathogenesis of IgG4‐Related Disease via Toll‐like Receptor 7/Interleukin‐33 Signaling

    doi: 10.1002/art.41052

    Figure Lengend Snippet: Expression of interleukin‐33 ( IL ‐33) and candidate TLR s in SG s from patients with IgG4‐ RD . A , Expression levels of mRNA for IL ‐33 in SG s from healthy controls (n = 10), patients with chronic sialadenitis (n = 10), patients with SS (n = 15), and patients with IgG4‐ RD (n = 15). B , Distribution of IL ‐33 in SG s from a representative healthy control, patient with chronic sialadenitis, patient with SS , and patient with IgG4‐ RD . Mayer's hematoxylin (blue) counterstained; bars = 100 μm. C , Correlation between expression levels of IL ‐33 mRNA and candidate TLR s in SG s from patients with IgG4‐ RD (n = 15), as determined by Spearman's rank correlation test. D , Schematic illustration of the extraction of CD 163+ M2 macrophages stimulated with TLR ‐7 agonist R848. Cells were cultivated as described in Patients and Methods. PBMC = peripheral blood mononuclear cell. E , Production of IL ‐33 by CD 163+ M2 macrophages stimulated with R848, as determined by enzyme‐linked immunosorbent assay. IL ‐33 levels increased in a concentration‐dependent manner. In A and E , bars show the mean ± SD . * = P

    Article Snippet: Cell culture and stimulation of human CD163+ M2 macrophages in vitro PBMCs were obtained from a healthy donor, and CD14+ monocytes were isolated using an EasySep Human Monocyte Isolation Kit (StemCell Technologies).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

    The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and SEB super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 low (PC3) and PD-L1 high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ TNFα). d Workflow of the SEB super-antigen assay. Total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

    doi: 10.1186/s40425-018-0454-3

    Figure Lengend Snippet: The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and SEB super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 low (PC3) and PD-L1 high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ TNFα). d Workflow of the SEB super-antigen assay. Total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA

    Article Snippet: Additionally, the SEB assay was performed in human PBMCs depleted of CD4, CD8, or both CD4/CD8 cells, using magnetic positive selection kits (StemCell Technologies).

    Techniques: Functional Assay, Activity Assay, In Vitro, Ex Vivo, Co-Culture Assay, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Activation Assay, Antigen Assay