Journal: Frontiers in Immunology

Article Title: Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections

doi: 10.3389/fimmu.2021.698420

Figure Lengend Snippet: No influence of Hck in pDCs on the IFN-α production and reduced in vitro migration of Siglec-H ko pDCs to CCL25. (A) Strategy to sort pDCs from B16Flt3L-treated wt and Siglec-H ko spleens to use in transwell-migration assays (purity more than 90%). (B) In vitro generated pDCs from wt, Siglec-H ko and HCK ko or HFL KO mice were stimulated for 24 hrs with LPS (c=1 µg/ml), R848 (c=4 µg/ml) or CpG A (c=2,5 µg/ml) (two separate experiments are shown). The amount of IFN-α was measured by ELISA. White bars: wt, black bars: Siglec-H ko, grey bars: HCK ko (left), HFL ko (right). n.d.: not detectable. N=3 mice per genotype. Error bars are mean ± SD values. (C) Sorted pDCs were used in a transwell-migration assay to evaluate migratory capacity towards CCL25. CCL25 or CXCL13 were given in the lower chamber of the transwell and sorted pDCs were given into the upper chamber. pDCs could then migrate towards CCL25 or CXCL13 for 2 hrs. Migrated cells were counted in the lower chamber and a ratio in relation to the sample with no-chemokine in the lower chamber was calculated. As a negative control CXCL13 was used. CCL25 c= 125nM, CXCL13 c= 100nM. Ratio of migrated cells to non-chemokine control is displayed. Summary of three independent experiments with N=5 mice per genotype. Mice were aged 10-16 weeks. Mann-Whitney test, *p < 0,05. Error bars are mean ± SD values. (D) Leukocytes from the small intestine and bowel were isolated and stained as shown in . pDC percentages are shown as % of living leukocytes. Mice were aged 10-16 weeks, N=3 mice per genotype.

Article Snippet: The lower chamber was filled with 600 µl of medium including the specific chemokine ligand: CCL25 (125 nM, Biolegend) or CXCL13 (100 nM, Biolegend).

Techniques: In Vitro, Migration, Generated, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Negative Control, MANN-WHITNEY, Isolation, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

doi: 10.1016/j.omtn.2023.04.017

Figure Lengend Snippet: Fas-CD40 activates NF-κB and induces strong proliferation upon FasL binding (A) Left: upon binding FasL, Fas trimerization recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) overnight and NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) Human T cells (5 × 10 4 ) expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) for 5 days, at which point cell counts were analyzed by flow cytometry. Due to the large list of Fas-TNFR chimeras, they were tested over two separate experiments (screens 1 and 2) with the data being compiled onto one graph. The conditions were identical between screens having the same 19-ζ and FasΔDD controls. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilized recombinant FasL (20 μg/mL) for 3 days, at which point RNA was extracted and analyzed using the nCounter NanoString platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were normalized to 19-ζ alone, and the number of significantly (p < 0.05) upregulated differentially expressed genes (DEGs) were categorized by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from the experiment described in (F). (H) Volcano plot from the experiment described in (F) of Fas-CD40-19-ζ cells compared with 19-ζ alone after incubation with immobilized FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from the experiment described in (F). (J and K) 19-ζ cells were cultured in the presence or absence of immobilized FasL (20 μg/mL) for 5 days and then stained for CCR8, ICOSL, and ICOS expression by flow cytometry (I), or the cell culture supernatant analyzed for CCL1, CXCL10, and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA. (L) Top: TCF7 expression from the experiment described in (F). Bottom left: 19-ζ cells were stained for TCF-1, representative flow cytometry plots from one donor. Bottom right: TCF-1 expression from three independent donors, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA.

Article Snippet: Cytokine concentrations in cell culture supernatants were measured by ELISA using kits to detect IFN-γ (BioLegend, 430104), IL-2 (BioLegend, 431804), CCL1 (R&D Systems, DY272), CXCL10 (R&D Systems, DY266), and CXCL13 (R&D Systems, DY801) according to the manufacturer’s instructions using a Multiskan FC microplate photometer (Thermo Scientific).

Techniques: Binding Assay, Cell Culture, Recombinant, Activity Assay, Expressing, Flow Cytometry, Incubation, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

doi: 10.1016/j.omtn.2023.04.017

Figure Lengend Snippet: Upregulated DEGs specific to Fas-CD40, Fas-Fn14, and Fas-BCMA expression

Article Snippet: Cytokine concentrations in cell culture supernatants were measured by ELISA using kits to detect IFN-γ (BioLegend, 430104), IL-2 (BioLegend, 431804), CCL1 (R&D Systems, DY272), CXCL10 (R&D Systems, DY266), and CXCL13 (R&D Systems, DY801) according to the manufacturer’s instructions using a Multiskan FC microplate photometer (Thermo Scientific).

Techniques: Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: CD69 + CD103 + CD8 + tissue-resident memory T cells possess stronger anti-tumor activity and predict better prognosis in colorectal cancer

doi: 10.1186/s12964-024-01990-3

Figure Lengend Snippet: Relationship of different CD8 + TRM subset with B cells and TLS in CRC tumor tissues. ( A ) Volcano plot showing differentially expressed genes between DP CD8 + TRM subsets and SP CD8 + TRM subsets of the GSE108989 dataset. ( B ) The FFPE CRC tumor tissue slides from patients with CRC were stained with DAPI (blue), CD20 (green), CD8 (yellow), CD103 (orange), and CD69 (red). ( C ) Relationship between the proportion of different CD8 + TRM cells and the proportion of B cells in CRC tumor tissues detected by flow cytometry ( n = 40) and relationship between the proportion of different CD8 + TRM cells and the count of B cells and the count and area of TLS in CRC tumor tissues detected by mIHC ( n = 64). Statistical analysis was performed by Pearson correlation analysis. ( D ) Schematic overview of the transwell migration assay design. ( E ) B cell migration rate when cultured either alone or with different CD8 + TRM subsets ( n = 10) and B cell migration rate when cultured with DP CD8 + TRM cells with or without anti-CXCL13 neutralizing antibody ( n = 7). Statistical analysis was performed by non-parametric Wilcoxon’s matched-pairs tests and Friedman test with Dunn’s multiple comparisons test. (* p < 0.05; ** p < 0.01)

Article Snippet: To disrupt CXCL13/CXCR5 interactions during chemotaxis, neutralizing antibody against CXCL13 (2 µg/mL, MAB801, R&D Systems) was added to the culture medium.

Techniques: Staining, Flow Cytometry, Transwell Migration Assay, Migration, Cell Culture

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: The depletion of hMENA 11a down-regulates LTβR and up-regulates FN1. In node-negative patients with NSCLC, hMENA 11a expression associates with intratumoral TLS . a. Volcano plots showing differentially expressed genes (q-value <0.05, n = 1096, GSE217451) in Si-hMENA 11a versus control H1650 NSCLC cells (n = 3). Reported are the negative log10-transformed adjusted P values plotted against the log2 fold changes. Dots represent individual genes. b . qRT-PCR analysis of LTβR mRNA expression in the indicated cell lines transfected with control (Si-CNTR), and hMENA 11a pool SiRNAs (Si-hMENA 11a ) or with plasmid control (CNTR) and hMENA 11a expressing vector (hMENA 11a ). The control of hMENA 11a expression in the transfected cells by WB is reported in Supplementary Figure S3a . Data are reported as the mean ± SD of technical triplicates which are representative of two independent experiments. P value was calculated by 2-tailed Student's t test. c. qRT-PCR analysis showed that LTβR and hMENA 11a mRNA expression level correlate in NSCLC cell lines. The value of Spearman correlation is reported. d. Consecutive sections of a representative NSCLC primary tumor hMENA 11a positive, showing low stromal FN1, TLS IT localization and high CXCL13. Magnification 8×. Scale bar 300 μm. Right, histograms relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a positive cases more frequently show TLS within the tumor area (TLS IT). e. Consecutive sections of a representative case of lung adenocarcinoma hMENA 11a negative, showed high stromal FN1, TLS PT and low CXCL13. Right, histograms are relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a negative (hMENA 11a low /hMENA (t) high ) cases more frequently show peritumoral TLS (TLS PT). P value was estimated with Fisher Exact test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Control, Transformation Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA 11a isoform in tumor cells affects CXCL13 production by T RM cells . a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si-hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student's t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey's multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA 11a (Si-hMENA 11a ), or control (Si-CNTR). Results within CD8 + T RM (CD103 + CD69 + , left panel) or CD4 + T RM (CD103 + CD69 + , right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Flow Cytometry, Ex Vivo, Isolation

Journal: Frontiers in Immunology

Article Title: GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity

doi: 10.3389/fimmu.2023.1242531

Figure Lengend Snippet:

Article Snippet: Anti-mCXCL13 , 143614 , 1:1000 , RnD Systems , MAB470.

Techniques: Concentration Assay

Journal: Advanced Science

Article Title: Tertiary Lymphoid Structure in Dental Pulp: The Role in Combating Bacterial Infections

doi: 10.1002/advs.202406684

Figure Lengend Snippet: Multiplex immunofluorescence staining of TLS in human dental pulp. a) Distribution of CD20 + cells, CD34 + cells, CCL3 + cells, and CXCL13 + cells in healthy dental pulp (Healthy 1 and 2). Colors were indicated as follows: Red (CD34), Yellow (CD20), Magenta (CCL3), Green (CXCL13), and Blue (DAPI). Od, odontoblast. Scale bars = 50 µm. b) Distribution of TLS marked by CD20 + cells, CD34 + cells, CCL3 + cells, and CXCL13 + cells in inflamed dental pulp (Pulpitis 1, 2, 3, and 4). Color coding as above. The area indicated by the green arrow represented the CCL3 expression. Od, odontoblast. Scale bars = 50 µm.

Article Snippet: Anti‐CXCL13 (NBP3‐07988) antibody was purchased from Novus Biologicals (Colorado, CA, USA).

Techniques: Multiplex Assay, Immunofluorescence, Staining, Expressing

Journal: Nature materials

Article Title: Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

doi: 10.1038/nmat4866

Figure Lengend Snippet: CSF1R inhibition prevents the entire (innate and adaptive) immune response to implanted biomaterials. Phase contrast images showing that fibrosis, as compared to wild type (WT) vehicle-treated control, was partially eliminated by CXCL13 neutralization, and completely eliminated with continuous CSF1R inhibition (inh.; 160 mg/kg BW GW2580 SC, daily) over a 14-day implant period ( a ). Scale bar = 2000 μm. Fibrosis was reduced the same extent as that of the B cell knockout (B KO) shown in . ( b ) Flow analysis for responding macrophages, neutrophils, and B cells dissociated directly from spheres (100 μl material in all cases) 14 days post-intraperitoneal (IP) implant, showing partial loss of cell presence with CXCL13 neutralization, and complete loss following CSF1R inhibition. ( c ) Brightfield images showing that fibrosis, compared to vehicle control, was completely eliminated by both macrophage depletion (- Mϕs) and CSF1R inhibition. Scale bar = 1000 μm. ( d ) Flow analysis for responding host innate immune macrophages and neutrophils dissociated directly from spheres (100 μl of each material) 14 days post-IP implantation, showing the loss of immune adhesion with loss of fibrosis due to either macrophage depletion (- Mϕs) or CSF1R inhibition. ( e ) NanoString expression analysis for all known cytokine and cytokine receptors (see for complete data set, excerpted here), showing similar unique factors increased across all material (hydrogel alginate, ceramic glass, and polymer polystyrene (PS)) groups. ( f ) Confocal for DAPI (cellular nuclei), macrophage marker CD68 (green), B cell marker CD19 (magenta), fibrosis-associated myofibroblast marker alpha smooth muscle actin (αSMactin, myofibroblasts, red), overlay, and brightfield imaging, showing that CXCL13 neutralization resulted in loss of B cell recruitment. Scale bar = 200 μm. ( g ) qPCR expression analysis of α-SMactin directly on retrieved spheres from Vehicle-treated (WT), B KO, and CXCL13-neutralized WT mice, plotted relative to expression levels on spheres from WT mice. Statistical analysis: one-way ANOVA with Bonferroni multiple comparison correction ***: p < 0.0001, vs Vehicle. n = 5 mice/group. Experiments repeated at least 2–3 times.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected IP in sterile 1X PBS at a dose of 100 μg/mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Inhibition, Control, Neutralization, Knock-Out, Expressing, Polymer, Marker, Imaging, Comparison

Journal: Nature materials

Article Title: Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

doi: 10.1038/nmat4866

Figure Lengend Snippet: 0.5 mm-sized SLG20 hydrogel spheres of were implanted into either the intraperitoneal (IP) or subcutaneous (SC) dorsal region of cynomolgus monkeys and retrieved by laparoscopy-guided tissue excision or biopsy punch after 28 days . ( a ) Laparoscopy images taken on days 0 and 28 (initial implantation and retrieval from the IP space). ( b ) H&E and Masson’s Trichrome stained histological sections of excised IP omentum tissue at 28 days for non-fibrosed fat-laden (No material, Mock) or heavily collagen-deposited and sphere-embedded (Implanted) omental tissue. Scale bars = 400 μm. ( c ) Main figure: Confocal staining showing DAPI (cellular nuclei), innate immune macrophage marker CD68 (green), and fibrosis-associated activated myofibroblast alpha smooth muscle actin (αSMactin) staining (red), showing cellular infiltration around and fibrosis deposition on an embedded 500 μm alginate sphere; Inset: additional confocal for CSF1R (green), showing positive staining on not only more distant macrophages but also material-proximal and fused foreign body giant cells (FBGCs); both 20x magnification. White scale bars: both 200 μm for each respective image. ( d ) Flow analysis showing similar host innate immune macrophage (CD68 + CD11b + , top right quadrants) and remaining neutrophil/myeloid (CD68 − CD11b + , bottom right quadrants) cells across C57BL/6 mice and cynomolgus monkeys, dissociated directly from fibrosed spheres and adjacent fibrosed omentum tissue (as percent composition) 28 days post-IP implantation. While the prominence of CD11b seems to be inverted in C57BL/6 mice vs cynomolgus monkeys, population response percentages are similar 28 days post-IP implantation. ( e ) NanoString analysis for immune markers and cytokines, originally identified in C57BL/6 mice (Note: CD66b is used here as a neutrophil marker, as Ly6g/Gr1 does not exist in NHPs or humans). Significant increases are observed for macrophage markers, as well as CSF1R and CXCL13 in both peritoneal and subcutaneous implant sites, as compared to mock (saline-injected) controls (there was no difference between SC and IP mock controls). N = 2 for IP-implanted groups; N = 4 for subcutaneous (SC) treatment groups. These experiments were performed once for SC and twice for IP delivery.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected IP in sterile 1X PBS at a dose of 100 μg/mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Staining, Marker, Saline, Injection