Fig 2: CD40 receptor organization is modified during maturation of BMDCs, and its engagement impacts synapse architecture. A Schematic of the pseudo-synapse model for TIRF microscopy. B Representative TIRF images of CD40 on LPS-matured BMDCs seeded on glass coated with either Fibronectin + CCL21 (Fn + CCL21) or anti-MHCII (aMHCII). White boxes indicate the regions of interest (ROI) used for subsequent dSTORM acquisition. Scale bar = 10 µm (n = 3). C Reconstructed dSTORM images showing CD40 localization within 3 µm × 3 µm ROIs from cells shown in (B). D-F Quantification of CD40 nanocluster parameters derived from dSTORM analysis: (D) cluster area, (E) cluster number per µm2, and (F) CD40 molecular density within clusters (each dot represents one cell, n = 3, unpaired Student’s t-test). G Representative TIRF images of F-actin (phalloidin stain) in BMDCs after 30 min on glass coated with aMHCII or aMHCII + CD40L. Scale bar = 20 µm. H Quantification of the BMDC spreading area from (G) (unpaired Student’s t-test, n = 3, n1 = 37 cells, n2 = 140 cells, n3 = 154 cells). I Schematic of the bead-cell conjugation assay. J Quantification of bead–cell conjugates after 5 or 35 min of interaction, with or without CD40 engagement (Two-way ANOVA, n = 3). Bars indicate mean ± SD. *p < 0.05, **p < 0.01; ns, not significant

Fig 3: CD40 concentration at the synapse depends on actomyosin cytoskeleton activity. A Representative TIRF images of F-actin (phalloidin) in LPS-matured BMDCs on glass coated with anti-MHCII (aMHCII) or aMHCII + CD40L. B Quantification of radial actin mean fluorescence intensity (MFI), based on (A) (Two-way ANOVA, n1 = 82 cells, n2 = 97 cells, n3 = 98 cells). C Representative TIRF image of LifeAct-eGFP-BMDC on an aMHCII + CD40L-coated surface. The red line indicates the region used for the kymograph in (D). D Kymograph showing retrograde actin flow (indicated by arrows) from the region in (C). E Quantification of actin flow speed in the center versus the synapse’s periphery (Two-way ANOVA, n1 = 27 cells, n2 = 39 cells, n3 = 41 cells). F Representative TIRF images of α-actinin-4 on glass coated with Fibronectin and CCL21 (Fn + CCL21), aMHCII or aMHCII + CD40L, showing sarcomeric patterning in the ROIs. G Representative TIRF image showing co-localization of α-actinin-4 (grey) and CD40-mGFP (false-colored, fire LUT) on an aMHCII + CD40L-coated surface. H Representative TIRF images of phosphorylated myosin light chain (pMLC) staining. I Quantification of pMLC mean fluorescence intensity (MFI) (Unpaired Student’s t-test, n1 = 179 cells, n2 = 161 cells, n3 = 199 cells). J Representative TIRF images of α-actinin-4 in BMDCs treated with DMSO (vehicle) or 50 µM blebbistatin (Blebb). Insets show that blebbistatin treatment disrupts the sarcomeric pattern. K Quantification of the percentage of cells displaying a sarcomeric α-actinin-4 pattern on aMHCII + CD40L coated glass (mean of 3 blinded counters, Unpaired Student’s t-test, n1 = 251 cells, n2 = 267 cells, n3 = 239 cells). L Representative TIRF images of CD40 after DMSO or 50 µM blebbistatin treatment on aMHCII + CD40L coated glass. M Quantification of radial CD40 mean fluorescence intensity (MFI) after treatment (Two-way ANOVA, n1 = 45 cells, n2 = 49 cells, n3 = 46 cells). For all graphs, data are from three independent experiments (n = 3); bars represent mean ± SD. Main scale bar = 20 µm; inset scale bar = 10 µm. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

Fig 4: CD40 concentration at the synapse is required for fine-tuning of CD40-mediated dendritic cell licensing. A Schematic of CD40 downstream signaling upon CD40L ligation in licensing. B Representative TIRF images of TRAF2 after DMSO or 50 µM blebbistatin treatment on aMHCII + CD40L coated glass. Scale bar = 20 µm. C Quantification of radial TRAF2 mean fluorescence intensity (MFI) after treatment (Two-way ANOVA, n1 = 90 cells, n2 = 60 cells, n3 = 62 cells). D Experimental setup for in vitro recreation of CD40 signaling in BMDCs. LPS-matured BMDCs were pretreated with 50 µM blebbistatin or DMSO (vehicle) for 1 h at 37 °C, then stimulated with anti-CD40 (10 µg/mL) for 5 min to induce signaling. Cells were analyzed by western blot. E Representative western blots showing phosphorylation of ERK, JNK, p38 and NF-κB upon activation as described in (D) and β-actin as loading control. F Quantification of the western blots shown in (E) relative to β-actin (One-way ANOVA, symbols represent data from the same experiment). G Experimental setup to assess CD40 licensing function. LPS-matured BMDCs were treated with 50 µM blebbistatin or DMSO (control) and stimulated with anti-CD40 (10 µg/ml) for 6 h at 37 °C. H Quantification of CD70 and OX40L expression (median fluorescence intensity, MFI) by flow cytometry after stimulation as described in (G) (Two-way ANOVA). I Experimental setup to assess CD40 licensing function. LPS-matured BMDCs were treated with 50 µM blebbistatin or DMSO (control) and stimulated with anti-CD40 (10 µg/mL) for 6 h at 37 °C. Cells were washed and cultured for 48 h before assessing IL-12 release and cell viability. J Quantification of IL-12 release in the 48 h culture supernatant (Two-way ANOVA). K Quantification of BMDC survival assessed using Sytox Blue live/dead staining (Two-way ANOVA). For all graphs, data represent mean ± SD from three or four independent experiments (n = 3 and n = 4 for 5E,F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Fig 5: CD40 mobility is restricted by ligand engagement at the immunological synapse. A Representative time-lapse FRAP-TIRF images of a CD40-mGFP-expressing BMDC on an aMHCII + CD40L-coated surface, showing fluorescence before bleaching, immediately after bleaching, and at 2 and 8 min of recovery. Scale bar = 20 µm. B Schematic defining the peripheral and central regions of interest (ROIs) used for FRAP analysis. C-G FRAP analysis of CD40-mGFP mobility. C Fluorescence recovery time-course for CD40-mGFP on aMHCII-coated glass. D Mobile fraction of CD40-mGFP on aMHCII at 15 min post-bleach (n1 = 14 cells, n2 = 19 cells, n3 = 12 cells). E Fluorescence recovery time-course for CD40-mGFP on aMHCII + CD40L-coated glass. F Mobile fraction of CD40-mGFP on aMHCII + CD40L at 15 min post-bleach (n1 = 9 cells, n2 = 15 cells, n3 = 13 cells). G Restriction index for CD40 mobility, calculated from the mobile fractions in (D) and (F). H–L FRAP analysis of a fluorescent plasma membrane marker as a control. H Recovery time-course on aMHCII-coated glass. I Mobile fraction on aMHCII at 5 min post-bleach (n1 = 18 cells, n2 = 20 cells, n3 = 20 cells, n4 = 20 cells). J Recovery time-course on aMHCII + CD40L-coated glass. K Mobile fraction on aMHCII + CD40L at 5 min post-bleach (n1 = 21 cells, n2 = 18 cells, n3 = 21 cells). L Restriction index for the plasma membrane marker. For all graphs, data represent mean ± SD from three or four independent experiments (n = 3 and n = 4 for 3H,I) and statistical significance was determined using an unpaired Student’s t-test. *p < 0.05, **p < 0.01; ns, not significant