Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) can inhibit the stimulation of cluster of differentiation 4 positive T (CD4 + T) cells on microglia activation in collagen-induced arthritis (CIA). (A) The expression level of CD4 was conducted by Western blot (left) and blot quantification and statistics (right) ( n = 3 per group). (B) Transwell assay showed the cell number across the upper chamber in CD4 + T cells from different groups ( n = 15 per group). (C) The double-immunofluorescence staining of ionized calcium-binding adapter molecule 1 (IBA1) and CD4 ( n = 3 per group). (D) A schematic illustration of the microglia intervened with supernatants of CD4 + T cells derived from different groups of mice. (E) Representative pictures of immunofluorescence staining and the relative fluorescence for IBA1, CD68, and CD40 of the microglia intervened with supernatants of CD4 + T cells in different groups of mice ( n = 3 per group). (F) The level of interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) conducted by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control or control CD4 + T cell group; # P < 0.05, compared with CIA or CIA/rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Activation Assay, Expressing, Western Blot, Transwell Assay, Double Immunofluorescence Staining, Binding Assay, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibits T helper 17 (Th17) differentiation and spinal infiltration. (A) Volcano plot of differentially expressed genes (DEGs) between cluster of differentiation 4 positive T (CD4 + T) cells of control and collagen-induced arthritis (CIA) group. (B) Volcano plot of DEGs between CD4 + T cells of CIA mice with or without LUT treatment. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated genes in CIA CD4 + T cells. (D) KEGG pathway enrichment analysis of downregulated genes in CIA + LUT CD4 + T cell. (E) Representative flow cytometry plots of spleen CD4 + interleukin-17 (IL-17) + T cells measured as CD4 + IL-17A + cell percentages (left), and the percentage of spleen CD4 + IL-17A + T cell was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (F) Enzyme-linked immunosorbent assay (ELISA) was employed to quantify IL-17A and IL-22 concentrations in spinal dorsal horn (SDH) samples collected from mice in each experimental group ( n = 6 per group). (G) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to quantify IL-17 a and IL-22 messenger ribonucleic acid (mRNA) in SDH samples collected from mice in each experimental group ( n = 6 per group). (H) The Venn diagram showed the intersection of the genes upregulated in CIA mice, genes downregulated after LUT treatment, and genes enriched in the Th17 differentiation pathway. (I) The heat map showed the mRNA expression profile of the overlapping genes ( n = 3 per group). (J) qRT-PCR validation of mRNA expression of the overlapping genes (nuclear factor of activated T cells 2 ( Nfatc2 ) , IL23α , IL1 receptor accessory protein ( IL1rap ), signal transducer and activator of transcription 5A ( Stat5a ), and zeta chain of T cell receptor associated protein kinase 70 ( Zap70 )) ( n = 4 per group). (K) The immunofluorescence intensity of NFATC2 in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (L) The double-immunofluorescence staining was used to verify the level of co-localization of NFATC2 and CD4 in SDH ( n = 4 per group). The insets and white arrows indicate representative images of NFATC2-CD4 co-localization and cells exhibiting NFATC2-CD4 co-localization, respectively. ∗ P < 0.05 and ∗∗ P < 0.01, compared with control group; # P < 0.05 vs. CIA or rheumatoid arthritis (RA) group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; HIF-1: hypoxia inducible factor-1; NF-κB: nuclear factor kappa B; FoxO: forkhead box O; VEGF: vascular endothelial growth factor; PE-A: phycoerythrin-area; APC-A: allophycocyanin-area; UL: upper left; UR: upper right; LL: lower left; LR: lower right; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Biomarker Discovery, Immunofluorescence, Double Immunofluorescence Staining

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibited nuclear factor of activated T cells 2 (NFATC2)-drived T helper 17 (Th17) differentiation and spinal infiltration in collagen-induced arthritis (CIA) model. (A) Representative flow cytometry plots of cluster of differentiation 4 (CD4) + interleukin-17 (IL-17) + T cells measured as IL-17A + CD4 + cell percentages (left), and the percentage of spleen Th17 cells was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (B) Quantitative live cell tracking of motility reveals significant increases in migration speed and distance of Th17 polarization cytokine cocktail-stimulated CD4 + T cells by transfected with NFATC2 short hairpin RNA (shRNA) plasmid in vitro (top). Visual narrative of cell morphology through time-lapse imaging and tracking (middle). Distinct cellular structures such as lamellipodia and uropodia are annotated with red arrow ( n = 20 per group) (bottom). (C) The double-immunofluorescence staining was used to verify the level of co-localization of IL-17A and CD4 in spinal dorsal horn (SDH) ( n = 4 per group). The arrows indicate IL-17A + CD4 + cells. The inset provides a detailed view of cells where IL-17A and CD4 are co-localized. (D) The Spearman correlation analysis of the percentage of IL-17A + CD4 + T cells and the expression level of NFATC2 ( n = 6). (E–G) The immunofluorescence staining and Western blot were used to verify the level of IL-17A (E), phosphorylated signal transducer and activator of transcription 3 (pSTAT3) (F), and ionized calcium-binding adapter molecule 1 (IBA1) (G) in SDH ( n = 3–4 per group). (H) Mechanical pain threshold and thermal withdrawal latency in different groups ( n = 8 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA, control, or CD4 c yclization recombination enzyme (Cre) + scramble shRNA groups; # P < 0.05 and ## P < 0.01, compared with CIA, or CD4 Cre + scramble shRNA + CIA group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Dunnett's multiple comparisons test. SSC-A: side scatter-area; PE-A: phycoerythrin-area; DAPI: 4′,6-diamidino-2-phenylindole; CIA + LUT-H: CIA + LUT high dose; mRNA: messenger RNA; AAV: adeno-associated virus.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Flow Cytometry, Cell Tracking Assay, Migration, Transfection, shRNA, Plasmid Preparation, In Vitro, Imaging, Double Immunofluorescence Staining, Expressing, Immunofluorescence, Staining, Western Blot, Binding Assay, Control, Two Tailed Test, Virus

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Nuclear factor of activated T cells 2 (NFATC2) orchestrates T helper 17 (Th17) pathogenicity via a protein kinase C epsilon (PRKCE)-signal transducer and activator of transcription 3 (STAT3) cascade. (A) The binding density of NFATC2 was visualized by deepTools: the heatmap presents the cleavage under targets and tagmentation (CUT&Tag) counts on the different NFATC2 binding peaks in cluster of differentiation 4 positive T (CD4 + T) cells derived from control and collagen-induced arthritis (CIA) mice ( n = 3 per group). (B) Genome-wide distribution of upregulated and downregulated NFATC2-binding peaks in CD4 + T cells derived from control and CIA mice. (C) Gene Ontology biological process (GO-BP) analysis of the NFATC2 binding peaks at promoter genes. (D) Genome browser tracks of CUT&Tag signal at the representative target gene loci ( n = 3 per group). (E) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) assay detected NFATC2 levels at the PRKCE promoter regions in CD4 + T cells derived from control and CIA mice ( n = 3 per group). (F) The co-localization of PRKCE and pSTAT3 was detected by laser confocal microscopy ( n = 3 per group). (G) The levels of interleukin-17A (IL-17A) and retinoic acid-related orphan receptor gamma t (RORγt) were detected by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). (H, I) Transwell assay (H) and quantification (I) showed the cell number across the upper chamber in cells treated with scramble short hairpin RNA (shRNA) or NFATC2 shRNA ( n = 5 per group). (J) The co-localization of C–C chemokine receptor type 6 (CCR6) and C–C motif chemokine ligand 20 (CCL20) was detected by laser confocal microscopy ( n = 3 per group). (K) The levels of CCR6 and CCL20 were detected by ELISA ( n = 6 per group). (L) The levels of IL-17A and RORγt were detected by ELISA under different interventions ( n = 6 per group). (M, N) Transwell assay (M) and quantification (N) showed the cell number across the upper chamber in cells under different interventions ( n = 5 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA or control group; # P < 0.05, compared with NFATC2 shRNA group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. TSS: transcription start site; TTS: transcription termination site; bp: base pairs; Ifna11 : interferon alpha 11; Msh2 : mutator S (MutS) homolog 2; Psen2 : presenilin 2; Sanbr : switch 3 (SWI3), alteration/deficiency in activation 2 (ADA2), nuclear receptor co-repressor (N-CoR), transcription factor III B (TFIIIB) and bric-a-brac, tramtrack, broad complex domain regulator of class switch recombination (CSR); Tmem98 : transmembrane protein 98; DAPI: 4′,6-diamidino-2-phenylindole; OE: overexpression.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Binding Assay, Derivative Assay, Control, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Transwell Assay, shRNA, Two Tailed Test, Activation Assay, Over Expression

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Effects of luteolin (LUT) treatment on nuclear factor of activated T cells 2 (NFATC2) transcription and lactic acid levels. (A, B) The total ion chromatogram of metabolites between cluster of differentiation 4 positive T (CD4 + T) cells derived from collagen-induced arthritis (CIA) mice with or without LUT (50 mg/kg) treatment: positive (A) and negative (B) ion models. (C, D) The principal component analysis (PCA) score plot of CD4 + T cells. It represents samples in the groups were closely cluster to one another: positive (C) and negative (D) ion models ( n = 3 per group). (E) Heatmap of differential metabolites derived from CIA mice with or without LUT treatment. The upregulated metabolites were marked in red, and the downregulated metabolites levels were presented in blue ( n = 3 per group). (F) Bubble map of the impact values of metabolic pathways, where the horizontal coordinate is the impact values enriched into different metabolic pathways, and the vertical coordinate is the enrichment pathway. The size of the dots indicates the corresponding number of metabolites on the pathway. The color of the dot reflects the P value, where the redder the color, the smaller the P value, and the bluer the color, the larger the P value. (G) The lactate level of CD4 + T cells derived from CIA mice treated with LUT or not based on global untargeted metabolomics ( n = 3 per group). (H) The lactate level in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (I) Lactate content assay kit and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the lactate level and Nfatc2 expression in T helper 17 (Th17) polarization cytokine cocktail-induced CD4 + T cells at different LUT concentrations (10, 20, and 40 μM) ( n = 6 per group). (J) The correlation of the lactate and the expression level of Nfatc2 in Th17 polarization cytokine cocktail-induced CD4 + T cells at 40 μM LUT concentrations ( n = 6). ∗ P < 0.05, compared with control CD4 + T cell group; # P < 0.05, compared with rheumatoid arthritis (RA) CD4 + T cell group; && P < 0.01, compared with CIA CD4 + T cell group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. PC: principal component; HIF-1: hypoxia-inducible factor-1; mTOR: mammalian target of rapamycin.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) could suppress nuclear factor of activated T cells 2 (NFATC2) transcription by decreasing histone H3 lysine 9 lactylation (H3K9la) in cluster of differentiation 4 positive T (CD4 + T) cells. (A) Western blot analysis showed the level of global lactylation in spleen CD4 + T cells of collagen-induced arthritis (CIA) mice with or without LUT treatment ( n = 5 per group). (B) The immunofluorescence staining was used to verify the localization of global lactylation and 4′,6-diamidino-2-phenylindole (DAPI) in CD4 + T cells. The white arrow shows the nucleus. (C–E) The Western blot was used to verify the expression of H3K9la (C), histone h3 lysine 18 lactylation (H3K18la) (D), and histone h3 lysine 23 lactylation (H3K23la) (E) in spleen CD4 + T cells of CIA mice with or without LUT treatment ( n = 4 per group). (F) The immunofluorescence intensity of H3K9la in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (G) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) detected the H3K9la level at the NFATC2 promoter regions in CD4 + T cells with or without sodium lactate (NaLa) treatment ( n = 3 per group). (H) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays monitoring expression of the NFATC2 in CD4 + T cells treated with different concentrations of NaLa (0, 5, 10, and 25 mM) for 24 h ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05, compared with CIA or rheumatoid arthritis (RA) CD4 + T cell group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's or Tukey multiple comparisons test. Pan Kla: pan-lysine lactylation.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) could inhibit the expression and activity of lactate dehydrogenase A (LDHA). (A) Venn gram showed the intersection of upregulated genes in spleen cluster of differentiation 4 positive T (CD4 + T) cells of collagen-induced arthritis (CIA) mice and downregulated genes in spleen CD4 + T cells of CIA mice with LUT treatment and the genes enriched in the glycolytic pathway according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. (B) Heat map of differential shows the messenger ribonucleic acid (mRNA) level of hexokinase domain containing 1 ( Hkdc1 ) , phosphoglycerate kinase 1 ( Pgk1 ), Ldha , pyruvate kinase m1/2 ( Pkm ), bisphosphoglycerate mutase ( Bpgm ), phosphofructokinase platelet ( Pfkp ), phosphoglucomutase 1 ( Pgm1 ), and glucose-6-phosphate isomerase 1 ( Gpi1 ) in CD4 + T cells of CIA mice with or without LUT administration ( n = 3 per group). (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) monitors the mRNA expression of Hkdc1 , Pgk1 , Ldha , Bpgm , Pkm , Pfkp , Pgm1 , and Gpi1 in CD4 + T cells of CIA mice with or without LUT administration ( n = 4 per group). The dashed lines serves to highlight the genes that were significantly upregulated in CD4 + T cells of the CIA group and significantly downregulated upon LUT treatment, thereby drawing clearer attention to the key findings. (D) The immunofluorescence intensity of LDHA in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (E–H) A model of LUT binding with LDHA protein generated by molecular docking: surface diagram of LUT and LDHA binding (E), enlarged view of the surface diagram of LUT and LDHA binding (F), three-dimensional (3D) diagram of the interaction between LUT and LDHA (G), and 2D diagram of the interaction between LUT and LDHA (H). (I–K) The surface plasmon resonance (SPR) analysis of LUT-LDHA interaction (1.9–62.5 μmol/L): steady-state affinity fitting curve, showing the binding signal as a function of analyte concentration (I), kinetic fitting using a 1:1 binding model, where the affinity is calculated from the ratio of association to dissociation rates (J), and sensogram depicting real-time binding responses, with data points recorded every 0.1 s (K). (L) The root mean square deviation (RMSD) analysis of 100 ns-length molecular dynamics (MD) simulation results for LDHA and LUT. (M) Radius of gyration (Rg) graph of LDHA docked with LUT from 0 to 100 ns time scale. (N) The 3D free energy landscape of LDHA docked with LUT. (O) The 2D free energy landscape of LDHA docked with LUT. (P) The average number of hydrogen bonds between each ligand throughout 100 ns simulations. (Q) LDHA activity was measured in CD4 + T cells derived from control, CIA, and CIA + LUT mice ( n = 6 per group). (R) LDHA activity was measured in CD4 + T cells derived from different group ( n = 15 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05 and ## P < 0.05, compared with CIA or rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; DAPI: 4′,6-diamidino-2-phenylindole; K D : dissociation constant.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Expressing, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Binding Assay, Generated, SPR Assay, Concentration Assay, Derivative Assay, Control

Journal: Materials Today Bio

Article Title: GTKO rabbit: A novel animal model for preclinical assessment of decellularized xenogeneic grafts via in situ implantation

doi: 10.1016/j.mtbio.2022.100505

Figure Lengend Snippet: Analysis of lymphocyte subsets in rabbit PBMCs. A) Proportions of B cells; B) CD4 + cells; C) CD8 + cells; and D) CD4+/CD8+. Experiments were performed in triplicate. WT, wild type; GTKO, α1,3-galactosyltransferase gene-knockout; Con, control; NBM, natural bone material; PCB, porcine cancellous bone.

Article Snippet: CD4 (Anti-Rabbit CD4 Purified, MRB4020; Antingenix America Inc., Melville, NY, USA), CD8 (Anti-Rabbit CD8 Purified, MRB8020, Antigenix America Inc.) purified antibodies coupled with goat anti-mouse fluorescent secondary antibody (Alexa Fluor®647, ab150115, Abcam), and B-PE (Anti-Rabbit B Cells-PE, MRB9997; Antigenix America Inc.) were used to identify lymphoid subsets in PBMCs.

Techniques: Gene Knockout, Control

Journal: Nature Communications

Article Title: Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects

doi: 10.1038/s41467-023-41066-3

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4, VioBright FITC (REA604) , Miltenyi Biotec , 130-118-692.

Techniques: In Vivo

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Hepatitis B (Engerix-B) vaccination and experimental design. (Top) Timeline of vaccination and blood collection. (Bottom) Memory CD4 T cells were magnetically enriched and FACS-sorted from two time points (day 0 and day 60) for TCRβ repertoire sequencing. Peptide matrix pools were used to map CD4 T cell epitopes of the vaccine from peripheral blood mononuclear cells (PBMCs) collected at day 60 and to select single peptides. After 7 days of in vitro expansion, single peptide-specific and master peptide pool-specific CFSE low CD4 T cells from PBMCs collected at day 60 were FACS-sorted in two technical replicates for TCRβ repertoire sequencing. PBMCs collected at days 0, 60, 180, and 365 were stimulated with the master peptide pool (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry. ( B ) Vaccinee cohort can be classified into three groups as determined by anti-hepatitis B surface (anti-HBs) titer over four times points. Early-converters seroconverted at day 60, late-converters seroconverted at day 180 or day 365, and non–converters did not have an anti-HBs titer higher than 10 IU/ml at any of the time points.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Sequencing, In Vitro, Expressing, Flow Cytometry

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Scatter plot of the DNA-based TCRβ reads for each vaccinee at each time point. ( B ) Scatter plot of number of unique TCRβ amino acid sequences for each vaccinee at each time point, where the shape denotes the response as based on antibody titer. ( C ) Overview of unique TCRβ amino acid sequences in the memory CD4 T cell repertoire of each vaccinee. The bottom blue bar denotes those TCR sequences that were found at both time points. The green and red bars denote the number of unique TCR sequences at each time point. The total bar height thus represents the total number of unique memory CD4 T cell clonotypes sequences for a specific vaccinee. ( D ) Frequency of unique HBsAg-specific TCRβ sequences out of total sequenced TCRβ sequences between two time points for all vaccinees colored and faceted by group. ( E ) Change in frequency (clone read count/total counts) of those HBsAg-specific CD4 T cells present at both time points. The (ns) mark denotes a non-significant paired Wilcoxon signed-rank test (p-value = 0.7577). ( F ) Receiver operating characteristic (ROC) curve using R hbs to differentiate between age-matched early-converters and late-converters in a leave-one-out cross-validation at day 0. Age-matching was accomplished retaining only samples in the age range 40–55. A Wilcoxon test was used to confirm that there was no difference in age distributions between early- and late-converters (p-value = 0.60, mean EC = 44.5 years, mean LC 45.1 years). Diagonal line denotes a random classifier. Reported is the area under the curve (AUC) and its 95% confidence interval.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Biomarker Discovery

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Comparison of the memory CD4 TCRβ repertoire diversity, as shown by breadth (number of unique TCRs) and entropy (Shannon equitability index) between day 0 and day 60. Indices are available in . ( B ) Frequency of unique vaccine-specific TCRβ sequences out of total sequenced TCRβ sequences between two time points for all vaccinees colored by group. Frequencies are available in . ( C ) Sequenced CD4 + TCR memory repertoire of vaccinee H35 at day 60. Each TCR clonotype is represented by a node. TCRs are connected by an edge if their Hamming distance is one. Only clusters with at least three TCRs are shown. TCR clonotypes in red are the vaccine-specific TCRβ sequences that were not present prior to vaccination. ( D ) Frequency of vaccine-specific TCRβ sequences within memory CD4 T cell repertoire normalized by number of HBsAg-specific TCRβ sequences found for each vaccinee at time point 60. Frequencies are available in . Figure 2—source data 1. Breadth and entropy of T cell receptor β (TCRβ) repertoire. Breadth (number of unique TCRs) and entropy (Shannon equitability index) of the memory CD4 TCRβ repertoire at two time points, day 0 and day 60. Figure 2—source data 2. Frequency of unique hepatitis B surface antigen (HBsAg)-specific T cell receptor β (TCRβ) sequences. Frequency of unique HBsAg-specific TCRβ sequences out of unique total TCRβ sequences in the memory CD4 T cell repertoire at two time points, day 0 and day 60. Figure 2—source data 3. Frequency of normalized hepatitis B surface antigen (HBsAg)-specific T cell receptor β (TCRβ) sequences. Frequency of vaccine-specific TCRβ sequences within memory CD4 T cell repertoire normalized by number of HBsAg-specific TCRβ sequences found for each vaccinee at day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Comparison

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Shown is the frequency of CFSE low CD4 T cells out of total CD4 T cells for all vaccinees, vaccinees per group and for each vaccinee. Peripheral blood mononuclear cells from day 60 were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with a pool of peptides spanning hepatitis B (HB) surface antigen (HBsAg) (peptide pool) and single peptides selected based on epitope mapping of the entire antigen (single peptide). After day 7 of in vitro expansion, cells were stained with antibodies to surface markers (CD3, CD4, and CD8) that enable gating on viable CD4 T cells. CFSE intensity was used to identify and sort CFSE low cells for T cell receptor (TCR) repertoire analysis of antigen-specific CD4 T cells.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Labeling, In Vitro, Staining

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Overview of the detected HBsAg epitope-specific TCRβ sequences. Each bar corresponds to unique TCRβ sequences found against a single 15mer HBsAg peptide, with 11 amino acid overlap to each subsequent peptide. Bars in blue denote those epitopes for which 10 or more volunteers had a strong T cell reaction. Motif logos on top of bars denote a sampling of the most common TCRβ amino acid sequence motifs for those epitopes. ( B ) Scatter plot with the frequency of predicted HBsAg epitope-specific and bystander TCRβ sequences at day 60. These make up respectively the numerator and denominator of the HBsAg-predictive ratio, R hbs . Predictions done as a leave-one-out cross-validation. Each circle represents a vaccinee with the color denoting the response group (blue: early-converter, yellow: late-converter, red: non-converter). ( C ) HBsAg-predictive ratio, R hbs , when calculated on the memory CD4 TCRβ repertoires at day 60. ( D ) HBsAg-predictive ratio, R hbs , when calculated on the memory CD4 TCRβ repertoires at day 0. ( E ) Receiver operating characteristic (ROC) curve using R hbs to differentiate between early-converters and late-converters in a leave-one-out cross-validation at day 0. Reported is the area under the curve (AUC) and its 95% confidence interval. Data for B, C, D, and E are available in . ( F ) ROC curve using R hbs to differentiate between age-matched early-converters and late-converters in a leave-one-out cross-validation at day 0. Age-matching was accomplished retaining only samples in the age range 40–55. A Wilcoxon test was used to confirm that there was no difference in age distributions between early- and late-converters (p-value = 0.60, mean EC = 44.5 years, mean LC 45.1 years). Diagonal line denotes a random classifier. Reported is the area under the curve (AUC) and its 95% confidence interval. Figure 3—source data 1. Hepatitis B surface antigen (HBsAg)-predictive ratio ( R hb ) data. Frequency of predicted HBsAg epitope-specific and bystander T cell receptor β (TCRβ) sequences; and HBsAg-predictive ratio, R hbs , calculated on the memory CD4 TCRβ repertoires at two time points, day 0 and day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Sampling, Sequencing, Biomarker Discovery

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Gating strategy started by a lymphocyte gate, followed by gating on viable CD3 + CD8 − T cells. Doublets were excluded using doublet discrimination (area against the height of forward scatter pulse) before gating on CD4 + T cells. Next, CD45RA, CXCR5, CD25, and CD127 were used to identify main subsets of CD4 T cells using Boolean gates as specified in the accompanying table. ( B ) Shown an example of gating for CD154 (CD40L) and CD137 (4-1BB) for cells left unstimulated (left) and cells stimulated with a master peptide pool (right) for an early-converter vaccinee at day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques:

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of a pool of peptides of hepatitis B surface antigen (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry. ( A ) CD40L + 4-1BB − and CD40L − 4-1BB + CD4 T cells from day 60 were gated on and then overlaid in a contour plots of CD25 versus CD127 to assess T COV and T REG phenotype. ( B ) Summary plot of median fluorescence intensity (MFI) of CD25 and CD127 for all vaccinees. Wilcoxon signed-rank with paired analysis; statistical significance was indicated with ****p ≤ 0.0001.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of the master peptide pool (hepatitis B surface antigen [HBsAg]) and assessed for converse expression of 4-1BB and CD40L by flow cytometry on days 0, 60, 180, and 365. Shown is number of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells after subtraction of responses in negative control. ( A ) Aggregate analysis from vaccinees (including early-, late-, and non-converters) showing a peak of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60 (day 60 after first dose of the vaccine and day 30 after second dose), declining thereafter. Shown are numbers of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells. ( B ) Correlation between the difference in antibody titer between day 365 and day 0 and vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60. ( C ) Aggregate analysis from early- and late-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell in early-converters and lack thereof in late-converters. ( D ) Aggregate analysis from early- and late-converter vaccinees showing no significant differences in vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 0. Data for A, B, C, and D are available in . ( E ) Receiver operating characteristic (ROC) curves for R hbs from day 0 data in a leave-one-out cross-validation compared to the frequency of vaccine-specific CD40L + 4-1BB − memory CD4 T cell out of 10 6 memory CD4 T cells for each vaccinee at time points 60 (area under the curve [AUC] = 0.84), 180 (AUC = 0.56), and 365 (AUC = 0.57). ( F ) Receiver operating characteristic (ROC) curves for R hbs from day 0 data in a leave-one-out cross-validation compared to the frequency of vaccine-specific CD40L − 4-1BB + memory CD4 T cell out of 10 6 memory CD4 T cells for each vaccinee at time points 60 (AUC = 0.62), 180 (AUC = 0.56), and 365 (AUC = 0.52). Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 rs , Spearman’s correlation coefficient, −1≤ rs ≤ 1; rs and p-value by Spearman’s correlation test. Figure 4—source data 1. Ex vivo T cell assay and serological data. Numbers of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell out of 10 6 memory CD4 T cells and antibody titers at the four time points, days 0, 60, 180, and 365.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Negative Control, Biomarker Discovery, Ex Vivo

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Correlation between the difference in antibody titer between day 365 and day 0 and vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60 colored by vaccinee group and labeled with vaccinee ID. rs , Spearman’s correlation coefficient, −1≤ rs ≤ 1; rs and p-value by Spearman’s correlation test.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Labeling

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of a pool of peptides of hepatitis B surface antigen (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry on days 0, 60, 180, and 365. ( A ) Aggregate analysis from early-, late-, and non-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell in early-converters and lack thereof in late and non-converters. Shown are numbers of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells after subtraction of responses in negative control (see Materials and methods for details). ( B ) Aggregate analysis from early-, late-, and non-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + CD4 T cell in early-converters and lack thereof in late and non-converters. Shown are numbers of vaccine-specific CD4 T cells out of 10 6 CD4 T cells after subtraction of responses in negative control (see Materials and methods for details).

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Negative Control

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees at day 0 (prior to vaccination) were phenotyped for expression of markers of T REG . ( A ) Aggregate analysis of 4-1BB + CD45RA − T REG within CD45RA − T REG CD4 T cells in early- and late- and non-converter vaccinees before vaccination. ( B ) Aggregate analysis of the median fluorescence intensity of 4-1BB in T H , cT FH , T REG , and cT FR cells before vaccination. ( C ) Aggregate analysis of the median fluorescence intensity of 4-1BB (left panel) and CD25 (right panel) in CD45RA − T REG and CD45RA + T REG cells before vaccination. Data for A, B, and C are available in . ( D ) Frequency of T REG , CD45RA − T REG , and CD45RA + T REG cells within total CD4 T cells in early-, late-, and non-converter vaccinees before vaccination. ( E ) Composition of T REG compartment as determined by expression of 4-1BB and CD45RA in early-, late-, and non-converter vaccinees before vaccination. Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Figure 5—source data 1. Frequency of 4-1BB + CD45RA − T REG cells and median fluorescence intensity data. Frequency of 4-1BB + CD45RA − T REG within CD45RA− T REG CD4 T cells, and median fluorescence intensity of 4-1BB in T H , cT FH , T REG , and cT FR cells, and of 4-1BB and CD25 in CD45RA − T REG and CD45RA + T REG cells before vaccination.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Fluorescence

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Aggregate analysis of the frequency of 4-1BB + CD45RA − T REG within CD45RA − T REG CD4 T cells in early-, late-, and non-converter vaccinees at days 0, 60, 180, and 365. Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques:

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet:

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Recombinant, Sequencing, Software, Staining, Virus

Journal: STAR Protocols

Article Title: CyTOF mass cytometry analysis of human memory CD4 + T cells and memory B cells

doi: 10.1016/j.xpro.2022.101269

Figure Lengend Snippet: Surface marker antibody cocktail

Article Snippet: 174 Yb-conjugated anti-human CD4 (clone SK3) (1:50 dilution) , Fluidigm , Cat#3174004B.

Techniques: Marker

Journal: STAR Protocols

Article Title: CyTOF mass cytometry analysis of human memory CD4 + T cells and memory B cells

doi: 10.1016/j.xpro.2022.101269

Figure Lengend Snippet: CyTOF data analysis (A) PBMCs from four healthy donors were stained with a panel of metal-labeled antibodies and analyzed by CyTOF. After data pre-processing, manual gating was used to identify classical MBCs (CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ), atypical MBCs (CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) and activated MBCs (CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ). tSNE analysis was then performed in Cytobank ( Kotecha et al., 2010 ) before self-organizing maps (SOMs) were generated using hierarchical consensus clustering on the tSNE axes. (B) tSNE and FlowSOM analysis of classical MBCs. (C) tSNE and FlowSOM analysis of atypical MBCs. (D) tSNE and FlowSOM analysis of activated MBCs. (E) A similar approach was used to analyze the memory CD4 + T cell compartment. Manual gating was used to identify T H1 memory CD4 + T cells (CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ), T H2 memory CD4 + T cells (CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ), T H17 memory CD4 + T cells (CD19 - CD3 + CD4 + CD45RA - CCR6 + CXCR3 - ) and circulating memory T FH cells (CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) before tSNE analysis and FlowSOM clustering was performed for each memory CD4 + T cell sub-set. (F) tSNE and FlowSOM analysis of T H1 memory CD4 + T cells. (G) tSNE and FlowSOM analysis of T H2 memory CD4 + T cells. (H) tSNE and FlowSOM analysis of T H17 memory CD4 + T cells. (I) tSNE and FlowSOM analysis of circulating memory T FH cells. The tSNE plots in the upper left panel display cell density and represent the pooled data from four healthy donors, while the upper right panel shows a projection of the FlowSOM clusters on the tSNE plot. Heatmaps shows the mean marker expression for each FlowSOM cluster.

Article Snippet: 174 Yb-conjugated anti-human CD4 (clone SK3) (1:50 dilution) , Fluidigm , Cat#3174004B.

Techniques: Staining, Labeling, Generated, Marker, Expressing

Journal: STAR Protocols

Article Title: CyTOF mass cytometry analysis of human memory CD4 + T cells and memory B cells

doi: 10.1016/j.xpro.2022.101269

Figure Lengend Snippet: Parameters and setting used for tSNE analysis

Article Snippet: 174 Yb-conjugated anti-human CD4 (clone SK3) (1:50 dilution) , Fluidigm , Cat#3174004B.

Techniques:

Journal: STAR Protocols

Article Title: CyTOF mass cytometry analysis of human memory CD4 + T cells and memory B cells

doi: 10.1016/j.xpro.2022.101269

Figure Lengend Snippet:

Article Snippet: 174 Yb-conjugated anti-human CD4 (clone SK3) (1:50 dilution) , Fluidigm , Cat#3174004B.

Techniques: Recombinant, Saline, Staining, Software, Cytometry