Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Diagram depicting the culture model used to generate time course samples from healthy human CD4+ memory T cells. Cells were stimulated with CD3/CD28 Dynabeads for 12 days, with fresh beads plus full media change every other day (fresh media alone added on alternate days). Samples were collected on days highlighted in bold. (B) Multiomics workflow (see for details). Experiments were performed in biological triplicate, with proteomics, phosphoproteomics, and metabolomics done on same samples. (C) ELISA-based quantification of secreted cytokines (IL-2, IFN-γ, TNF-α) on days 0, 6, 10, and 12, 3 replicates per time point. (D) Cell counts were taken each time point, and average cell division compared to previous time point calculated. Cell viability remained stable throughout at ~75%–85%. (E) Flow cytometric analysis of inhibitory receptors PD-1, TIM-3, and TIGIT at resting, day 6, and day 12 time points. Data normalized to mode for accurate population comparisons to account for differences in number of cells analyzed. The data underlying can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: Mean fluorescence intensity of PD-1 in CD4+ T cells from both HIV+ and HIV- patients (grouped due to observing same effect in both) for CD4+ cells that are CD276+ or FLT-1+. The data underlying this figure can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Fluorescence, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Causal network analysis leveraging annotated interactions (transcription factor-target, kinase-substrate, phosphatase-substrate) as a framework to evaluate protein expression changes (late exhaustion (day 12) versus resting, taking into account previous time point comparisons) to predict key nodes (regulators) impacting most changes seen across the data set. (B) Pruned version of a large subnetwork with p300 (black outline) at main hub. Orange circles mark proteins of interest mentioned in main text. (C) Depiction of experimental design of p300 inhibition studies. (D) Flow cytometric analysis of PD-1 and TIM-3 showing shifts in fluorescent intensity of cell populations in treated versus untreated (cell number normalized to mode). Cell numbers shown for each plot are 20,312 for p300 treated and 30,438 for DMSO, which corresponds to all CD4+ T cells from the sample (excluding doublets and debris, see for gating), corresponding to >95% of sample. (E) Bar chart of mean fluorescent intensity showing significant changes ( T test) in PD-1 and TIM-3 during p300 inhibition versus controls (antigen removed for 2 days, or treatment with T-cell activation inhibitor Dasatinib); *, p -value <0.05, ***, p -value <0.005. (F) Heatmap showing recruitment of cofactors (p300, class I HDACs, KAT2B) to transcription factor family DNA motifs as measured using CASCADE protein-binding microarrays. The data underlying this figure can be found in and Data (A, B), supplemental flow cytometry files uploaded to flowrepositroy.org (D), and the (E, F). Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Expressing, Inhibition, Activation Assay, Protein Binding, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: UMAPs of CD4+ subtypes and CD8+ T exhausted tumor lymphocytes showing gene expression of (A) exhaustion-associated genes, and (B) gene expression of proteins identified in the model presented by Lawton and colleagues. UMAPs made using Single Cell Portal, Tarhan and colleagues. The data from this figure and tool used to make it can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP1039/a-single-cell-and-spatially-resolved-atlas-of-human-breast-cancers . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Alirocumab attenuated plaque inflammation and PCSK9-induced proinflammatory signalling in M1 macrophages independently of lipid lowering

doi: 10.1101/2025.11.20.689631

Figure Lengend Snippet: By qPCR, levels of proinflammatory CXCL9 , CXCL10, TNF-α, IL-1β and IL-6 and anti-inflammatory CCL17, TGM2, IL-10 and TGF-β1 cytokines in M1 macrophages stimulated with PCSK9. TNF-α was used as a positive control. *p<0.05, **p<0.01, and ***p<0.001 vs. unstimulated cells, by One-sample t test, control value set to 1. N=4-5 independent biological replicates.

Article Snippet: The human taqman assay IDs were as follows: CXCL9 (Hs00171065_m1), CXCL10 (Hs00171042_m1), IL-1β (Hs01555410_m1), TNF-α (Hs00174128_m1), IL-6 (Hs0174131_m1), CCL17 (Hs00171074_m1), TGM2 (Hs01096681_m1), IL-10 (Hs00961622_m1), TGF-β1 (Hs00998133_m1), and TLR4 (Hs00152939_m1) serving 18S rRNA (Hs99999901_s1) as the endogenous control.

Techniques: Positive Control, Control

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 1. Colonies of proliferating CD4þ T-cells during in vitro human TH17 cell differentiation. For stimulation and expansion of the T-cells, polyclonal stimulators (includ- ing anti-CD3 and anti-CD28 antibodies) were used (see ‘‘Methods’’ section). Representative micro-images are shown with different magnifications.

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: In Vitro, Cell Differentiation

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 2. IL-1b, IL-6, and IL-23 – but not TGFb – are required for optimal in vitro differentiation of human CD4þ TH17 cells. (A) Flow cytometric dot-plot analysis of intra- cellular IL-17 expression by CD4þ T-cells. Representative data of five independent experiments (i.e. PBMC from five different donors) are shown. (B) Columns represent mean percentage (±SEM) of T-cells expressing intracellular IL-17 in each culture condition. ns: Non-significant (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: In Vitro, Expressing

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 3. IFNc production by human T-cells differentiated in presence of IL-1b, IL-6, and IL-23 – with or without TGFb as well. (A) Flow cytometric analysis of intracellu- lar expression of IFNc by TH17 cells derived from CD4þ T-cells. (B) Mean expression levels (±SEM) from five independent experiments (i.e. PBMC from five different donors) using various culture conditions (i.e. different TGFb concentration and inhibitors of TGFb signaling pathways) are shown. ns: Non-significant (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Expressing, Derivative Assay, Concentration Assay, Protein-Protein interactions

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 4. TGFb effects on Foxp3 expression by CD4þ T-cells differentiated under TH17 cell-promoting conditions. (A) Intracellular staining of Foxp3. (B) Columns are mean (±SEM) Foxp3 expression levels in five independent experiments (i.e. PBMC from five different donors) (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Expressing, Staining

Journal: Journal of Immunotoxicology

Article Title: Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

doi: 10.1080/1547691x.2016.1193574

Figure Lengend Snippet: Figure 6. TH17 cell-derived cytokines; modulation by TGFb and blocking of TGFb signaling. Cytokine levels in supernatants of CD4þ T-cells differentiated toward TH17 states in the presence or absence of TGFb and/or inhibitors of signaling pathways. Data showed means± SEM from five independent experiments (i.e. PBMC from five different donors). ND: not detected (*p < 0.05).

Article Snippet: From each sample, CD4þ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions.

Techniques: Derivative Assay, Blocking Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in various tissues of 10-week-old C57BL/6 mice. (B) Procedure used for flow cytometry of CD4 + -gated TCRβ + IL-17A + Th17 cells from ex vivo Th17 differentiation culture. (C) qPCR of various PLA 2 enzymes in splenic CD4 + T cells cultured for 3 days under Th0 and Th17 differentiation conditions ( n = 3). (D) Strategy for gene targeting of Pla2g12a . Positions of PCR primers for genotyping are indicated by arrows. (E) PCR genotyping of Pla2g12a +/+ (+/+), Pla2g12a +/– (+/–), and Pla2g12a -/- (–/–) mice. (F) qPCR of Pla2g12a in the skin and spleen of Pla2g12a +/+ and Pla2g12a -/- mice. Values are mean ± s.e.m.. Representative data of two experiments (C) and combined results of two experiments (A, F) are shown. Statistical analysis was performed by Mann-Whitney U test (B, F, G). **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Flow Cytometry, Ex Vivo, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for ex vivo culture of splenic naïve CD4 + T cells with (Th17) or without (Th0) IL-1β, IL-6, IL-23 and TGF-β on anti-CD3ε/CD28-coated plates. (B) qPCR of Pla2g12a in splenic CD4 + T cells cultured for various periods under Th0 and Th17 differentiation conditions. (C) FACS of Th17 (CD4 + -gated TCRβ + IL-17A + ) cells after culture of naïve T cells prepared from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (D-F) Scatter plots (D) and heatmap (E) of genes expressed in CD4 + T cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days under Th0 and Th17 differentiation conditions. Cells from 6-7 mice of each genotype were pooled and subjected to microarray analysis. Colors in (E) show the z-score. (F) Pathway enrichment analysis of genes decreased (>2-fold) in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. (G) Heatmap of Th17-related genes in Th17 cells from Pla2g12a -/- mice relative to those from Pla2g12a +/+ mice. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (H) qPCR of Th17 signature genes in Th0 and Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice. (I) Heatmap of lipid-related genes in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (J) FACS of non-pathogenic Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days with IL-6 and TGF-β. Values are mean ± s.e.m.. Representative data of three experiments (C) and results of one experiment (B, D–I) are shown. Statistical analysis was performed by one way ANOVA with Tukey’s multiple comparisons test (C, J), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H), and two-way ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Ex Vivo, Cell Culture, Microarray

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in the whole spleen and isolated CD4 - and CD4 + cells of Pla2g12a fl/fl (fl/fl) and Pla2g12a fl/fl Cd4 cre (T-KO) mice. (B) FACS of Th17 cells after culture of naïve T cells from fl/fl and T-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (C) IMQ-induced ear swelling in fl/fl and T-KO mice over 6 days ( n = 20–30). (D) HE staining of ear skin sections from IMQ-treated fl/fl and T-KO mice on day 6. Scale bar, 100 μm. (E, F) FACS of Th17 cells in the spleen (E) and Th17 and γδ T cells in the skin (F) of IMQ-treated fl/fl and T-KO mice on day 6. (G) qPCR of Il17a in the skin of IMQ-treated fl/fl and T-KO mice on day 6. (H) qPCR of Pla2g12a in the whole LNs and isolated CD45 + hematopoietic cells and CD140a + fibroblasts of fl/fl and Pla2g12a fl/fl Col1a2 cre (F-KO) mice. (I) FACS of Th17 cells after culture of naïve T cells from fl/fl and F-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (J) IMQ-induced ear swelling in fl/fl and F-KO mice over 6 days ( n = 4–6). (K) FACS of splenic Th17 cells in IMQ-treated fl/fl and F-KO mice on day 6. A representative histogram ( left ) and the number of Th17 cells ( right ) are shown. Values are mean ± s.e.m.. Representative data of two (A, F) or three (E) experiments, results of one experiment (B, I–K), and combined results of two (C, G, H) are shown. Statistical analysis was performed using unpaired t test (G, K), Mann-Whitney test (A, E, F, H), one-way ANOVA with Tukey’s multiple comparisons test (B, I), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (C, J). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Isolation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) FACS of Th17 cells after culture of naïve T cells for 3 days in the presence of the indicated concentrations of the LPA 1/3 antagonist Ki16425. (B) qPCR of Lpar1 in Th0 and Th17 cells after culture for the indicated periods. (C) qPCR of Lpar1 in the LNs of Lpar1 +/+ and Lpar1 -/- mice. (D, E) IMQ-induced ear swelling over 6 days ( n = 8–12) (D) and ear histology on day 6 (E) of Lpar1 +/+ and Lpar1 -/- mice. (F, G) IMQ-induced ear swelling over 6 days ( n = 6) (F) and the number of splenic Th17 cells (G) of Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells. (H, I) FACS of splenic CDε+ T cells in Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells (H) or in Pla2g12a +/+ mice that had been transferred with Pla2g12a +/+ or Pla2g12a -/- BM cells (I). Representative FACS profiles ( left ) and the proportion of CD3ε + T cells ( right ) are shown. (J) FACS of CD4 + Th0 cells from Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with those from respective control mice. Representative FACS profiles ( upper ) and the proportion of CD4 + T cells ( lower ) are shown. (K) FACS of splenic CD4 + -gated CD44 - CD62L + naïve T cells and CD44 + CD62L - effector memory T cells in Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with WT mice. Values are mean ± s.e.m.. Representative data of two experiments (B, J) and results of one experiment (A, C–I, K) are shown. Statistical analysis was performed using unpaired t test (C, I, J), Mann-Whitney U test (G, H), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A, K), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (B), and two-way ANOVA with Sidak’s multiple comparisons test (D, F). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Comparison, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for antibody treatment in IMQ-induced psoriasis. (B) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A, -IL-17A, or control IgG 1 antibody over 6 days. Antibody treatment started on day 0 (arrow). (C) FACS of splenic Th17 cells in (B) on day 6. (D) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice treated with anti-PLA2G12A or control antibody over 6 days. Antibody treatment started on day 0 (arrow). (E) FACS of splenic Th17 cells in (D) on day 6. (F) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A or control antibody. Antibody treatment started on day 3 (arrow). (G) FACS of splenic Th17 cells in (F) on day 6. (H) Schematic representation of the procedure used for antibody treatment in CIA. (I, J) Paw thickness (I) and clinical sore (J) in WT mice treated with anti-PLA2G12A, - IL-17A, or control antibody over 42 days. (K) FACS of splenic Th17 cells in (I, J) on day 42. (L) Schematic diagram showing PLA2G12A-driven regulation of Th17 differentiation. PLA2G12A is induced in CD4 + T cells after TCR stimulation and is also supplied from stromal fibroblasts. EV secretion is facilitated by Th17 differentiation driven by Th17-inducing cytokines. PLA2G12A hydrolyzes PE and PC to generate LPE and LPC in EV membranes. The EVs are taken up by Th17-differentiating cells and deliver LPE(1-18:1) intracellularly, which then transactivates RORγt. LPE and LPC are converted to LPA by ATX, which is supplied from Th17 cells, stromal cells ( e.g ., fibroblasts and endothelial cells), and serum. LPA acts mainly on LPA 2 (and to a lesser extent LPA 1 ) on Th17-differentiating cells to augment IL-6/IL23-mediated STAT3 phosphorylation via G 12/13 -Rho-ROCK2 signaling, assisting pathogenic Th17 differentiation in autocrine and paracrine fashions. Gene disruption of PLA2G12A dampens this lipid-orchestrated amplification loop and decreases EV secretion, uptake, and transfer of lipid, miRNA, and protein cargos. Accordingly, antibody-mediated neutralization of PLA2G12A efficiently attenuates psoriasis, arthritis, and probably other Th17-related pathologies such as EAE. Values are mean ± s.e.m.. Results are from one experiment (B–G, I–K). Statistical analysis was performed using unpaired t test (G), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, E, K), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, D, F, I, J). *, P < 0.05; **, P < 0.01 versus control IgG 1 .

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Control, Disruption, Amplification, Neutralization

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) PLA 2 enzyme assay using human and mouse PLA2G12A proteins after incubation with several anti-PLA2G12A antibody clones (100 ng/ml), with the activity in the presence of a control antibody being regarded as 1. *, P < 0.05; **, P < 0.01 versus control. (B) FACS of Th17 cells after culture of WT naïve T cells for 3 days with several anti-PLA2G12A antibody clones (100 ng/ml). *, P < 0.05; **, P < 0.01 versus control. (C) Immunoblotting of human and mouse PLA2G12As and human PLA2G3 (50 ng/lane for each) with an anti-PLA2G12A antibody (clone #44). (D) ELISA of various sPLA 2 s with an anti-PLA2G12A antibody (clone #44). hIIA, human PLA2G2A; hIID, human PLA2G2D; hIIE, human PLA2G2E; hIIF, human PLA2G2F; hIII, human PLA2G3; hV, human PLA2G5; hX, human PLA2G10; hXIIA, human PLA2G12A; mXIIA, mouse PLA2G12A. *, P < 0.05; **, P < 0.01. (E) Immunohistochemistry of mouse skin sections with or without IMQ treatment with an anti-PLA2G12A antibody (clone #44). Red, PLA2G12A; green, CD4 + T cells. Scale bar, 100 µm. Values are mean ± s.e.m.. Results of one experiment is shown (A–D). Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (A) and ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B, D).

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Enzymatic Assay, Incubation, Clone Assay, Activity Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Inhibition assay of sCD4 and CD4-derived protein molecules against pseudotyped and infectious Ebola viruses (A) sCD4 (2D), soluble first two-domain CD4; sCD4 (4D), soluble four-domain CD4; CD4-Ig, first two-domain CD4 linked with human Fc region (Ig domain) ; CD4-IgG2, first two-domain CD4 with human Fc region (Ig domain); EBOV, Zaire Ebola virus; BDBV, Bundibugyo ebolavirus. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments. (B) sCD4 (4D) inhibits infectious Ebola virus infection. This experiment was conducted in BSL-4 containment in triplicates, and the error bars represent standard deviations.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Inhibition, Derivative Assay, Virus, Infection

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Inhibition assay of CD4-mimetic small molecules against pseudotyped Ebola viruses (A) NBD-556 and analogs inhibiting EBOV infection. (B) Newly designed and synthesized compounds with phenyl ring substituted molecules in the region-I inhibiting EBOV. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Inhibition, Infection, Synthesized

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Specificity assay of CD4-IgG-2 and JRC-II-191 against pseudo-typed HIV, EBOV, VSV, and A-MLV CD4-mimetic molecules CD4-IgG2 and JRC-II-191 were evaluated against four different viruses: HIV (YU2), EBOV, VSV (vesicular stomatitis virus), and A-MLV (amphitropic murine leukemia virus). All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Virus

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Binding affinity and kinetics assay of sCD4 (2D) and NBD-556 binding to EBOV receptor binding domain by biolayer interferometry Two-domain CD4 (sCD4-2D) (A) and NBD-556 (B) at two pH conditions, pH7.4 and pH6.1. Open circle in (B) was not included in the fit of the data. (See the BLI section for details).

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Binding competition assay of sCD4 and CD4-mimetic compounds with receptor NPC1 (A) sCD4, NBD-556, JRC-II-191, and DY-III-228 competing with NPC1 receptor. (B) Dose response (0, 10, 20, and 40 μM) of NBD-556 in binding competition with NPC1 receptor. (C) Comparison of wild-type (EGPDCM-WT) and mutant (EGPDCM-mut) (WF/AA, 86W/A, and 88F/A) receptor binding domain (RBD) of EBOV binding to the NPC1 receptor. The mutant (WF/AA) surface expression level was confirmed to be comparable to the wild type (see ). Representative of 3 experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay, Competitive Binding Assay, Comparison, Mutagenesis, Expressing

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Molecular docking analysis of sCD4 and CD4mcs (A) Molecular docking analysis of sCD4 and CD4mcs binding to the EBOV-GP. sCD4 docking using HDOCK program. The docking energy is −150.41 kcal/mol. CD4mcs docking using AutoDock program. (a) sCD4-RBD ribbon model; (b) sCD4-RBD surface binding model; (c) sCD4-RBD interactions: hydrogen bond: GP T83:OG1-CD4 L44:N (green) and salt bridge: GP K84:NZ-CD4 D56:OD2 (brown) ; (d) NBD-556 docking ribbon model (−6.477 kcal/mol); (e) NBD-556 docking surface model; and (f), superimposed of NBD-556 (yellow), JRC-II-191 (magenta), and DY-III-228 (cyan). (B) Comparisons of two receptor binding sites of CD4bs and NPC1-bs. (a) CD4bs , of HIV gp120 (based on PDB 1G9N , YU2 strain). (b) NPC1bs , of EBOV-GP (based on PDB 5F1B , Zaire EBOV). Showing the accommodation of CD4-mimetic compound NBD-556 in the red dash circle. Hydrophobic surface in gray; CD4bs of HIV gp120 in magenta dash circle; NPC1bs of EBOV-GP in red dash circle.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses

doi: 10.1084/jem.20082460

Figure Lengend Snippet: Immunological and biochemical characterization of ESP from S. mansoni eggs. (A) Monocyte-derived DCs pulsed for 48 h with the different antigen preparations in the absence (top) or presence (bottom) of 100 ng/ml LPS as a maturation factor were co-cultured with allogeneic naive CD4 + T cells for 12 d in the presence of staphylococcal enterotoxin B and IL-2. Intracellular cytokine production was assayed by FACS 6 h after the stimulation of primed T cells with PMA and ionomycin. The frequencies of each population are indicated as percentages in the plot. One representative result from three independent experiments is shown. (B) 5 µg/cm ESP was separated under nonreducing conditions by SDS-PAGE and silver stained. (C) The presence of omega-1 and IPSE/alpha-1 was confirmed on Western blots by staining with specific anti–IPSE/alpha-1 and anti–omega-1 monoclonal antibodies.

Article Snippet: To determine T cell polarization, 5 × 10 3 48-h-pulsed DCs were co-cultured with 2 × 10 4 naive T cells that were purified using a human CD4 + /CD45RO − column kit (R&D Systems) in the presence of 100 pg/ml staphylococcal enterotoxin B (Sigma-Aldrich) in 96-well flat-bottom plates (Costar).

Techniques: Derivative Assay, Cell Culture, SDS Page, Staining, Western Blot, Bioprocessing

Journal: The Journal of Experimental Medicine

Article Title: Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses

doi: 10.1084/jem.20082460

Figure Lengend Snippet: Omega-1 is sufficient to drive Th2 polarization in vivo, independently of IL-4R signaling. (A) 4get/KN2 IL-4 dual reporter mice were injected s.c. with 20 µg SEA, 2 µg omega-1, or 2 µg IPSE/alpha-1 into the footpad. After 7 d, the frequency of GFP + and huCD2 + within the CD4 + CD44 high effector T cell population was determined by flow cytometry in the draining popliteal lymph nodes. (B) 4get/KN2 mice were injected with recombinant proteins and analyzed as in A. (C) IL-4Rα −/− 4get/KN2 mice were treated and analyzed as in A. In all panels, representative plots are shown with the frequencies of each population indicated as percentages. Graphs depict the combined data of three to four individual mice per group and show one of at least two independent experiments. Error bars represent the mean ± SD. * P < 0.05; **, P < 0.01; ***, P < 0.001 for values significantly different from the PBS control (two-sided paired Student's t test).

Article Snippet: To determine T cell polarization, 5 × 10 3 48-h-pulsed DCs were co-cultured with 2 × 10 4 naive T cells that were purified using a human CD4 + /CD45RO − column kit (R&D Systems) in the presence of 100 pg/ml staphylococcal enterotoxin B (Sigma-Aldrich) in 96-well flat-bottom plates (Costar).

Techniques: In Vivo, Injection, Flow Cytometry, Recombinant, Control