Fig 2: Tetravalent anti-hCD27 hIgG1 V11 induces greater cluster formation, with FcγRIIB engagement polarizing hCD27 clusters and reducing internalization.A, B Jurkat cells expressing hCD27-GFP+ fusion protein were stimulated with the specified antibodies at the indicated concentrations, before PFA fixation and confocal imaging. A Representative images at 100 nM (white corresponds to hCD27-GFP fluorescence; clustered areas of the signal are indicated by green arrows) and B quantification of the number of clusters per cell section, performed using ImageJ. Data shown are mean ± SEM of CD27 clusters per cell section from 2 independent experiments (n = 54 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. C, D hCD27-GFP+ Jurkat cells were stimulated with 1 nM tetra anti-hCD27 hIgG1 V11 and either WT CHO cells or hFcγRIIB+ CHO cells for the specified time points before PFA fixation and confocal imaging using Oxford Nanoimager. C Representative images of hCD27-GFP+ Jurkat cells with WT CHO (upper panels) or hFcγRIIB+ CHO (lower panels) for the specified time points. Dashed blue and green lines (determined by brightfield and confocal images) indicate the plasma membrane of CHO and Jurkat cells, respectively. D Quantification of intracellular GFP MFI of individual cell sections of hCD27-GFP+ Jurkat cells after stimulation. Data are the mean ± SEM from 2 independent experiments (n = 24 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. E, F hCD27+ Jurkat NF-κB GFP reporter cells were stimulated with the indicated concentrations of either bivalent anti-hCD27 hIgG1 V11 (left panel) or Tetra anti-hCD27 hIgG1 V11 (right panel) in the presence of WT CHO cells (WT CHO), WT CHO cells and a goat anti-hFc f(ab)2 (WT CHO + Xlink) or hFcγRIIB+ CHO cells (hFcγRIIB+ CHO). GFP reporter production was determined by flow cytometry and presented as E mean fluorescence intensity (MFI) of GFP+ cells and F percentage of GFP+ cells. Data points are the mean ± SEM (n = 3) from 3 independent experiments. Statistical significance between hFcγRIIB+ CHO and WT CHO + Xlink was determined by one-way ANOVA on AUC values, with significance values indicated in the figure. Source data are provided as a Source data file.

Fig 3: Dendritic cells form distinct synapse morphologies dictated by CD70 signaling. (A) Schematic of the pseudo-synapse model using total internal reflection fluorescence (TIRF) microscopy. (B) Representative TIRF images BMDCs forming either pancake or firework synapses on anti-MHCI-coated glass, stained for F-actin and quantification of the prevalence of each phenotype (n=4 biological replicates, n1 = 127 cells, n2 = 155 cells, n3 = 24 cells, n4 = 168 cells). (C) Time-lapse TIRF imaging of a live BMDC stained with CellTracker Deep Red, showing the formation and stability of either pancake or firework synapse. Image acquisition was initiated immediately after the stained BMDCs were added to the imaging chambers. Images were acquired every minute for 3 hours at 37 °C in a 5% CO2 atmosphere. Time stamp is hh:mm. (D) Representative TIRF images showing CD70 and actin expression in pancake vs. firework synapses on MHCI-coated glass. (E) Quantification of membrane-proximal CD70 mean fluorescence intensity (MFI) at the synapse interface (measured by TIRF microscopy) in each synapse phenotype on glass coated with anti-MHCI (aMHCI) alone or with CD27. MFI is normalized to the background (n1 = 117 cells, n2 = 109 cells, n3 = 104 cells). (F) Representative confocal images with orthogonal side-views (G) showing CD70 localization in BMDCs. The arrow indicates CD70 recruitment to the synapse upon CD27 engagement. (G) Quantification of CD70 recruitment to the synapse measured measure by confocal microscopy. Briefly, CD70 mean fluorescence intensity (MFI) was quantified at the synapse and normalized by the CD70 MFI in the whole cell. Cells were classified by their synapse morphologies, namely fireworks or pancakes (n1 = 41 cells, n2 = 44 cells, n3 = 39 cells). (H) Quantification of CD70 recruitment to the synapse measured measure by confocal microscopy. Briefly, CD70 mean fluorescence intensity (MFI) was quantified at the synapse and normalized by the CD70 MFI in the whole cell. Cells were classified by their total CD70 expression. To define CD70high and CD70low, the median fluorescence intensity was calculated across all cells pooled from three independent experiments, with cells above the median classified as CD70high and cells below as CD70low (n1 = 41 cells, n2 = 44 cells, n3 = 39 cells). (I) Representative confocal images showing CD70 distribution in BMDCs. Images are representative of 3 independent experiments. (J) Frequency of firework synapses in BMDCs, with or without pre-treatment with anti-CD40 (aCD40), on surfaces coated with aMHCI alone or with CD27 (n1 = 547 cells, n2 = 629 cells, n3 = 1028 cells). Data are shown as mean ± SD (n=3 biological replicates). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.00; ns, non-significant. Main scale bars: 20 µm, inlets scale bars = 10µm. Statistical significance was determined using two-way ANOVA. Note: These images illustrate the two distinct morphological states initially observed in LPS-matured BMDCs. The dendritic cell identity and subpopulation characteristics corresponding to these morphologies are validated by marker analysis (including Zbtb46) in Figures 2D, E.