Journal: bioRxiv

Article Title: B-cell SIGLEC-5 engages T-cell components of the elastin receptor complex (ERC) to suppress inflammatory T-cell cytokines

doi: 10.64898/2026.06.24.734204

Figure Lengend Snippet: a Immunoblots of SIGLEC-5 and SIGLEC-14 expression in B-cells from SIGLEC-14 homozygous positive ( S5 +/+ S14 +/+ ) and SIGLEC-14 null individuals ( S5 F/F S14 -/- ); F denotes the fusion allele between the SIGLEC-5 and SIGLEC-14 genes. Ratio of SIGLEC-5 to SIGLEC-14 band intensity in healthy individuals for both SIGLEC-14 bands (n=9). b, SIGLEC-14 +/+ B-cells were split into sub-cellular fractions and assessed by immunoblots for SIGLEC-5, SIGLEC-14, CD40, VIMENTIN and HSP90. c, Immunoblots of SIGLEC-5 and SIGLEC-14 after pull-down of biotinylated ex vivo B-cell surface proteins. d, B-cells were isolated from healthy controls and either left unstimulated or stimulated with 1μM CpG-C or 1μg/ml mega-CD40L for the indicated time points. The lysates were assessed by immunoblot for SIGLEC-5 and SIGLEC-14. e, Immunoblots of SIGLEC-5 and SIGLEC-14 after pull-down of biotinylated surface proteins from 72h mCD40L stimulated B-cells. f, Supernatants were taken from CD40L stimulated SIGLEC-14 null ( S5 F/F S14 -/- ) B-cells and CpG-C, CpG-C+anti-Ig or CD40L stimulated SIGLEC-14 + B-cells and were assessed for SIGLEC-14 expression by ELISA (n=3 and n=6). g, B-cells were activated for 24h with 1μg/ml megaCD40L and then incubated for a further 18h in the presence of either Brefeldin A (5μg/ml) or O-propargyl-puromycin (OPP; 50μM). Supernatants were assessed for the levels of SIGLEC-14 by ELISA (n=3). h , Schematic illustrating the SIGLEC-5 signalling pathway. i, Immunoblots of SIGLEC-5 and SHP1 in 72h mCD40L stimulated B-cells, after immunoprecipitation with a IgG1 control antibody or a SIGLEC-5 antibody (1A5). j, Immunoblots of SIGLEC-5 and SHP1 in 72h αCD3 stimulated CD4 + T-cell and B-cell co-cultures, after immunoprecipitation with a IgG1 control antibody or a SIGLEC5 antibody (1A5). For immunoblots, β-ACTIN was used as a loading control. For subcellular fractions CD40, VIMENTIN and HSP90 were used as fractionation controls (membrane, cytoskeletal and cytoplasmic respectively). All data are representative of at least two independent experiments. All values represent the mean ± SEM; one-way ANOVA for g .

Article Snippet: For analysis of SIGLEC-5 and SIGLEC-14 in B-cell supernatants by immunoblotting, cells were stimulated for 72h with 1μg/ml CD40L (Invivogen) and the supernatants were concentrated by spinning for 30 mins at 16,000g for 30 minutes in Amicon® 10kDA MWCO ultra centrifuge filter tubes (Merck).

Techniques: Western Blot, Expressing, Ex Vivo, Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Immunoprecipitation, Control, Fractionation, Membrane

Journal: bioRxiv

Article Title: Cognate T-cell labeling by conventional and unconventional antigen-presenting cells in vivo using a modular SrtA-based toolkit

doi: 10.64898/2026.09.17.752294

Figure Lengend Snippet: Scheme of the LIPSTIC approach ( A ) and flow cytometry analysis ( B ) showing biotin staining in SrtA-CD40 + (D; donor) and CD40L-G5 + (A; acceptor) 291PC cells. ( C ) Intercellular labeling of CD40L-G5 + 291PC cells pre-treated or not with a blocking αCD40L or isotype antibody. Scheme of the LIPSTIC approach ( D ) and flow cytometry analysis ( E ) showing biotin staining in SrtA-hCD58 + (D) and G5-hCD2 + (A) 291PC cells. ( F ) Quantification of the LIPSTIC labeling using SrtA-hCD58/G5-hCD2 and SrtA-CD40/CD40L-G5 expressing 291PC cells. Quantification of the LIPSTIC labeling using SrtA-CD40 ( G ) or SrtA-hCD58 ( H ) and G5-hCD2 or CD40L-G5 expressing 291PCs. Mean values ± SEM of three independent experiments. Statistical significance was determined by one-way ANOVA, followed by Tukeys multiple-comparisons test. p>0.05 (ns; not significant) and p<0.01 (**).

Article Snippet: Some CD4 + T cells were incubated with an αCD40L blocking Ab (BE0017-1, Bio X Cell) or an isotype Ab (BE0091, Bio X Cell) for 30 min before the beginning of co-culture at a final concentration of 150 μg/mL.

Techniques: Flow Cytometry, Analysis, Staining, Labeling, Blocking Assay, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: (A) Schematic representation of the E. coli display system used for selections. The surface-expressed fusion protein consists of an albumin-binding domain for expression normalization and an affibody library member. Cells displaying affibody variants capable of binding biotinylated CD40L were isolated by fluorescent-activated cell sorting (FACS). The gate used for sorting is indicated in green. The x-axis shows fluorescence intensity corresponding to surface display (561/543–627 nm), and the y-axis shows fluorescence intensity corresponding to hCD40L binding (638/650–670 nm). (B) The protein structure of an affibody molecule with diversified positions indicated in yellow. (C) Flow cytometry dot plots from analysis of the naive library and the output libraries from successive selection rounds, comprising three rounds of MACS, followed by three rounds of FACS. Each plot represents one library, with axes corresponding to surface display (x-axis) and hCD40L binding (y-axis)

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Binding Assay, Expressing, Isolation, FACS, Fluorescence, Flow Cytometry, Selection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: (A) Representative flow cytometry dot plots from high-throughput screening of unique affibody clones in the E. coli display format. Each dot plot corresponds to a unique clone, with axes representing surface display (x-axis) and hCD40L binding (y-axis) as described above. (B) SPR sensorgrams from soluble protein candidates, indicating their different abilities to bind to recombinant hCD40L immobilized on the chip surface. (C) Flow cytometry analysis showing binding of candidate or control affibodies and an anti-CD40L antibody to CD3 + CD4 + T cells from human peripheral blood following in vitro stimulation with phorbol myristate acetate (PMA) and ionomycin

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Flow Cytometry, High Throughput Screening Assay, Clone Assay, Binding Assay, Recombinant, Control, In Vitro

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: Biophysical characterization and binding kinetics of monomeric CD40L-binding affibody candidates. (A) Circular dichroism (CD) spectra of the affibody candidates D10 and G6 recorded at room temperature before (solid lines) and after (dashed red lines) variable temperature measurements (VTM). (B) Melting curves from the VTMs of D10 (blue) and G6 (orange) with indicated melting temperatures. (C) Sensorgrams from kinetic assessment of D10 and G6 with recombinant hCD40L immobilized on the chip surface. The respective kinetic constants are shown in the bottom table with values representing four independent measurements

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Binding Assay, Circular Dichroism, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: (A) Crystal structures of the NC1 domain from human collagen XV (3N3f.pdb) and human collagen XVIII (3HON.pdb) . Their respective structures are highly similar, as illustrated by the superimposition of their monomers to the right. (B) Suggested interaction geometry of hCD40L (beige) and the fusion protein candidate colXVIII-D10 (pink), superimposed with a co-crystal structure of human CD40 (green) and CD40L (3QD6.pdb) to the right. (C) Construct design for fusion proteins with NC1 domains. H 6 denotes a hexahistidine tag, Z an affibody molecule, (G 4 S) 4 the glycine-serine spacer, and colXV and colXVIII the respective NC1 domains. (D) Chromatograms from analytical SEC of indicated fusion protein candidates or controls harboring the NC1 domain in the N-terminal (left) and C-terminal (right). Z-ABD was used as an arbitrary monomer of similar molecular size to a monomeric fusion protein candidate

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Construct

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: (A) Sensorgrams from the kinetic measurements of the N-terminal fusion protein candidates. The apparent kinetic constants were generated using a 1:1 Langmuir curve fitting model and are shown in the table. (B) Sensorgrams from two independent measurements of the CD40 blocking SPR assay. CD40 was immobilized on the chip surface and hCD40L, pre-incubated with the respective candidates or controls, was injected over the surface. The y-axis shows the normalized response units (RU) at equilibrium for each tested concentration of candidate, and the x-axis shows the molar ratio of candidate to CD40L

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Generated, Blocking Assay, SPR Assay, Incubation, Injection, Concentration Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Trimerization of CD40L-specific affibody molecules using collagen domains enhances target binding and CD40 blockade

doi: 10.1007/s00018-026-06301-2

Figure Lengend Snippet: (A) Surface expression of CD40L, represented by the median fluorescent intensity (MFI), on Jurkat D1.1 cells and Jurkat wild-type (wt) cells following staining with a fluorescently labeled CD40L-specific monoclonal antibody. The data points represent duplicate values from two independent measurements. (B) Jurkat D1.1 cell binding (MFI) of monomeric and trimeric candidates or negative controls following labeling with the indicated concentrations. The data points represent duplicate values from three independent measurements. (C) Overlay of histograms displaying fluorescence intensity normalized to each population’s maximum signal (% of max) for the indicated candidates and negative controls at 100 nM, measured using the 638/655–665 nm laser/filter combination. (D) Inhibition curves from the NF-κB reporter assay showing CD40 blockade by monomeric D10 and colXVIII-D10, with colXVIII-Zwt included as a negative control

Article Snippet: HEK-BlueTM CD40L cells (cat.no. hkb-cd40, Invivogen) were grown according to manufacturer’s recommendations.

Techniques: Expressing, Staining, Labeling, Binding Assay, Fluorescence, Inhibition, Reporter Assay, Negative Control