Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A) Flow cytometry analysis of tetrameric FGL-1 FD::streptavidin Phycoerythrin (SA-PE) binding to LAG-3+ Jurkat cells across a concentration range (1 – 16 µM). (B) Comparison of FGL-1 FD tetramer binding to LAG-3+ Jurkat cells versus wild-type (WT) Jurkat cells (LAG-3 low) (n = 3). Data are presented as median fluorescent intensity (MFI), normalized to LAG-3+ Jurkat cells MFI at each concentration. Error bars represent standard deviation (SD). (C) NF-κB::eGFP induction and (D) IL-2 production in LAG-3+ Jurkat T cells stimulated with anti-CD3/CD28-coated beads +/− FGL-1 FD or mouse IgG1 isotype control (mIgG1) for 24 hours (n = 4, n = 3, respectively). NF-κB::eGFP induction data is shown as percent MFI normalized to anti-CD3/CD28 stimulation (set to 100%). Error bars represent standard deviation (SD). (E) Expression of T cell activation markers: PD-1, CD69, CD25, ICOS, 4-1BB and OX40 in LAG-3+ Jurkat T cells, either unstimulated or stimulated with anti-CD3/CD28 +/− FGL-1 FD (n = 4). Data are shown as MFI ± SEM, normalized to the anti-CD3/CD28 condition. (F) Control expression of activation markers in WT (LAG-3 low) Jurkat T cells under identical conditions (n = 4). Data represent MFI ± SEM from triplicate wells across two independent experiments. Statistical significance was performed using one or two-way ANOVA, followed by Tukey or Šídák’s post hoc tests, respectively. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant p > 0.05.

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Binding Assay, Concentration Assay, Comparison, Standard Deviation, Control, Expressing, Activation Assay

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A, B) Label-free LC–MS/MS analysis of phosphotyrosine (pY) peptides in LAG-3+ Jurkat cells, either unstimulated or stimulated for 5 minutes with anti-CD3/CD28 +/− FGL-1 FD (n = 4), (A) number of unique pY peptides identified per condition (B) Log 2 intensity distribution of detected pY peptides. Statistical analysis performed using two-way ANOVA with Tukey’s post hoc tests. Solid black line = median; dashed lines = interquartile range. **** p < 0.0001, ns = not significant (p > 0.05) (C) Venn diagram showing overlap of pY peptides detected in cells stimulated with anti-CD3/anti-CD28 versus anti-CD3/anti-CD28 + FGL-1 FD from one representative experiment. (D – F) Volcano plots illustrating differential log 2 fold changes (log 2 FC) in pY intensity differences, across the three conditions: (D) anti-CD3/CD28 vs. unstimulated; (E) anti-CD3/CD28 + FGL-1 FD vs. unstimulated; (F) anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. Red = significantly upregulated phosphosites (log 2 FC ≥ 1.5); blue = significantly downregulated phosphosites, (log 2 FC ≤ 1.5); grey = unchanged phosphosites (log 2 FC) < 1.5).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Functional enrichment analysis of proteins with upregulated pY sites (log 2 FC ≥ 1.5, red), and downregulated pY (log 2 FC ≤ 1.5; blue) in response to anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation, relative to unstimulated cells and stimulation without FGL-1 FD. Color intensity indicates –Log 10 (p-value) of pathway enrichment; X denotes non-enriched pathways. ( B ) Log 2 fold change (log 2 FC) analysis of TCR signaling-associated pY peptides following anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation compared to unstimulated cells. ( C ) Heatmap of log 2 FC phosphorylation differences in TCR signaling-related pY peptides across three conditions: anti-CD3/CD28 vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. ( D ) Bar graphs showing log 2 FC values of TCR signaling pY sites in CD3 ITAMS, ZAP-70, LAT, ERK1/2, TEC and distal molecules upon FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( E ) Time-course western blot analysis of phosphorylation at CD3ζ Y142, ZAP70 Y319, and ERK 1/2 Y204/Y187 in LAG-3+ Jurkat cells unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Quantification represents phospho/total protein signal intensity, normalized to unstimulated (US) values (n = 3). ( F ) Bar graphs showing log 2 FC values of CD28 signaling pY sites: CD28 pY191, pY209, and PI3K P85A pY467, in response to FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( G ) Western blot analysis of CD28 Y191 phosphorylation in LAG-3+ cell lysates: unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Band intensities were quantified and normalized to total CD28 and unstimulated control (n = 5). Statistical significance was determined using a two-way ANOVA with Šídák’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Functional Assay, Phospho-proteomics, Western Blot, Control

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD3 (green) and LAG-3 (red) in unstimulated and anti-CD3/CD28-stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Representative confocal images depicting colocalization of CD28 (green) and LAG-3 (red) under the same conditions: unstimulated and anti-CD3/CD28. Scale bar: 5 µm. (C) Violin plots of Manders’ Overlap Coefficient (MOC) quantifying colocalization of CD3 and CD28 with LAG-3 in unstimulated and stimulated cells (n = 66). Median values are indicated by solid black lines; interquartile ranges by dashed black lines. Statistical significance was assessed using Kruskal-Wallis ANOVA, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD28 (red) and Lck (pY394, cyan) in unstimulated, anti-CD3/CD28-stimulated, and anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Violin plots quantifying CD28 and Lck pY394 colocalization using Manders’ overlap coefficient (MOC) and Pearson’s correlation coefficient (PCC) (n ≥ 50). (C) Representative confocal images showing colocalization of CD28 (green) and total Lck (red) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (D) Colocalization analysis of CD28 and total Lck, measured by MOC and PCC (n ≥ 60). (E) Representative confocal images of CD28 (red) and Lck pY394 (cyan) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated Jurkat cells. Scale bar: 5 µm. (F) Colocalization analysis of CD28 and Lck pY394, determined by MOC and PCC (n ≥ 75). (G) Comparison of MOC and PCC values for CD28 and Lck pY394 colocalization between unstimulated and stimulated LAG-3+ Jurkat cells compared to WT (LAG-3 low) Jurkat cells (n ≥ 75). Median values are shown in solid black lines; interquartile ranges as dashed black lines. Statistical analysis was performed using Kruskal-Wallis ANOVA test, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: WT Jurkat (Clone E6-1) cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis

doi: 10.1172/JCI159348

Figure Lengend Snippet: ( A ) Photographs of the index patient at presentation at the NIH in 2004 (left) and following antifungal treatment and secondary prophylaxis in 2018 (right). ( B ) Grocott’s methenamine silver–stained (GMS-stained, left) and hematoxylin and eosin–stained (H&E-stained, right) section of soft tissue biopsy demonstrating C . cassiicola being engulfed but not destroyed in macrophages (black arrowheads) within granulomas (×100 magnification shown in ). Scale bars: 20 μm. ( C ) Chromatograms from CLEC7A sequencing on healthy control and our patient, over the site of mutation in each allele. ( D ) Representative FACS histograms showing Dectin-1 surface expression in our patient and 2 healthy control patients. Histograms were gated on CD14 + monocytes isolated from peripheral blood. ( E ) Representative protein immunoblot images of Dectin-1 expression in PBMCs from our patient and a healthy volunteer (HV). β-Actin was used as loading control. ( F ) TNF-α production by PBMCs after 48 hours of stimulation with either purified particulate β-glucan or α-mannan. Each data point represents an individual well; at least 2 separate blood draws were analyzed in the Dectin-1–deficient patient (each tested in 2–3 technical replicates) and compared to 2 different healthy controls (each tested in 2–3 technical replicates). A single blood draw from each of the CARD9-deficient patients was analyzed in 3–5 technical replicates. Data in panel F were analyzed by 2-way ANOVA with Bonferroni’s correction. *** P < 0.005, **** P < 0.0001. NS, not significant. ( G ) Photograph of a previously reported CARD9-deficient patient (CARD9.02) at presentation at the NIH (age 12). ( H ) Volume rendering of computed tomography data of patient CARD9.02 at age 12 emphasizing bone, which reveals erosions of the frontal bone (black arrows) and loss of maxillofacial structures (white arrow), including the hard palate, resulting in a common oronasal cavity. ( I ) Parasagittal T1-weighted magnetic resonance imaging of patient CARD9.02 at age 12 obtained following i.v. gadolinium-based contrast agent administration, which reveals epidural abscess with adjacent cerebritis (black arrow) and tissue loss (white arrow) resulting in a common cavity encompassing the nasopharynx, oropharynx, nasal cavity, oral cavity, and portions of the paranasal sinuses. ( J ) GMS- (upper) and H&E-stained (lower) section of soft tissue biopsy demonstrating granulomatous inflammation with C . cassiicola engulfed within macrophages (accompanying images from brain biopsy shown in ). Both images are from consecutive cuts of the same biopsy sample. Scale bars: 20 μm.

Article Snippet: WT Dectin-1 expression plasmid (Origene, clone SC307610, NM_197947) was used for site-directed mutagenesis to generate the patient variant, c.714T>G; p.Y238*.

Techniques: Staining, Sequencing, Mutagenesis, Expressing, Isolation, Western Blot, Purification, Computed Tomography, Magnetic Resonance Imaging

Journal: The Journal of Clinical Investigation

Article Title: Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis

doi: 10.1172/JCI159348

Figure Lengend Snippet: ( A ) Representative images of staining for Dectin-1–binding pathogen-associated molecular patterns using soluble Dectin-1 (Dectin-1 recognition domain fused with human Fc fragment) on C . cassiicola . Anti–human Fc–PE (visualized in DS-Red channel) was used as the secondary antibody or was used alone as negative control. Scale bars: 50 μm (upper panel) and 25 μm (enlarged images of C . cassiicola overlay in lower panel). ( B ) Cytokine production by PBMCs stimulated ex vivo with C . cassiicola in healthy controls ( n = 2 donors, each tested in 3–4 technical replicates) and our patient ( n = 2 different blood draws, tested in 3–4 technical replicates). ( C ) Shows similar experiments using PBMCs from 2 CARD9-deficient patients (1 blood draw per patient tested in 3–4 technical replicates), compared to 1 healthy donor tested in 4 technical replicates. Data in panels B and C were analyzed by 2-way ANOVA with Bonferroni’s correction. ** P < 0.01, **** P < 0.0001.

Article Snippet: WT Dectin-1 expression plasmid (Origene, clone SC307610, NM_197947) was used for site-directed mutagenesis to generate the patient variant, c.714T>G; p.Y238*.

Techniques: Staining, Binding Assay, Negative Control, Ex Vivo

Journal: The Journal of Clinical Investigation

Article Title: Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis

doi: 10.1172/JCI159348

Figure Lengend Snippet: ( A ) Cytokine analysis in the infected footpad homogenates (WT n = 12 mice, Clec7a –/– n = 12 mice, Card9 –/– n = 7 mice) on day 3 after infection. Each data point represents an individual mouse; data were pooled from 2 independent experiments. ( B ) Footpad swelling in Il1r –/– (day 5: WT n = 24, KO n = 23, 3 pooled experiments; day 10: WT n = 16, KO n = 20, 4 pooled experiments), Il1b –/– (WT n = 7, KO n = 5, 1 experiment), and Tnfa –/– (day 5: WT n = 13, KO n = 15, 2 pooled experiments; day 10: WT n = 5, KO n = 15, 2 pooled experiments) mice, relative to WT controls. ( C ) Total numbers of neutrophils (live CD45 + CD11b + Ly6G + ) and macrophages (live CD45 + CD11b + MHCII hi F4/80 + ) in the infected footpad on day 3 after infection ( n = 6 mice per group), measured using flow cytometry. ( D ) IL-1β and TNF-α production within footpad macrophages on day 3 after infection in Clec7a –/– ( n = 7) and Card9 –/– mice ( n = 6), normalized to the WT controls ( n = 6). Each data point represents an individual mouse; data were pooled from 2 independent experiments and analyzed by 1-way ANOVA with Dunnett’s correction. ( E ) Results of an in vitro C . cassiicola killing assay with bone marrow–derived macrophages, prestimulated for 24 hours with TNF-α, IL-1β, or both. Killing was determined by measuring β-D-glucan levels in the culture supernatant. Bar graph shows the mean ± SEM for 2 independent experiments; overlaid dot plot shows technical replicates from one of these experiments. ( F ) Schematic representation of the proposed model of anti– C . cassiicola immunity in the footpad of WT, Dectin-1–deficient, and CARD9-deficient mice. Data in panels A and B were analyzed by 2-way ANOVA with Bonferroni’s correction. Data in panel E were analyzed by unpaired, 2-tailed t test. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001.

Article Snippet: WT Dectin-1 expression plasmid (Origene, clone SC307610, NM_197947) was used for site-directed mutagenesis to generate the patient variant, c.714T>G; p.Y238*.

Techniques: Infection, Flow Cytometry, In Vitro, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis

doi: 10.1172/JCI159348

Figure Lengend Snippet: ( A ) Relative frequency of deleterious CLEC7A mutations with high CADD scores (>20) at the population level (calculated using data from 1000 Genomes) and in the index patient and 17 additional unrelated patients with severe forms of phaeohyphomycosis who were enrolled consecutively over an 8-year period at the NIH (see for details). Data were analyzed by Fisher’s exact test. **** P < 0.0001. ( B ) Mean fluorescence intensity (MFI) values for Dectin-1 surface expression in CD14 + monocytes isolated from 8 healthy donors (black dots, see for details), 5 phaeohyphomycosis patients without CLEC7A mutations (gray dots), and 6 phaeohyphomycosis patients carrying CLEC7A mutations (green, red, and blue dots as indicated). FACS staining was performed using an antibody that targets the C-terminus where the CLEC7A mutations reside. No PBMCs were available for testing in the other patients with severe phaeohyphomycosis, including the one carrying the c.547C>T (p.Leu183Phe) variant . FACS histograms show representative Dectin-1 staining for healthy control (black line), a patient homozygous for p.Y238* (red line), and patients heterozygous for p.Y238* (green line) or p.I223S (blue line) relative to isotype staining control (filled gray histogram). ( C ) TNF-α production by PBMCs stimulated with particulate β-glucan for 48 hours, from the same patients as shown in panel B . One blood draw was tested per healthy donor or patient in 3–12 technical replicates and the mean value per individual is depicted. Data were analyzed by 1-way ANOVA with Dunnett’s correction. * P < 0.05, ** P < 0.01. NS, not significant.

Article Snippet: WT Dectin-1 expression plasmid (Origene, clone SC307610, NM_197947) was used for site-directed mutagenesis to generate the patient variant, c.714T>G; p.Y238*.

Techniques: Fluorescence, Expressing, Isolation, Staining, Variant Assay