Fig 2: 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.