Fig. 5: B_EM interaction with antigen on SCS macrophages.

a Intravital observation of interactions of eGFP⁺ B_EM (green) and SCS macrophages (CD169 turquois). b Intravital Ca²⁺ levels in B_EM. Left: Surface rendering of CD169-stained macrophages (purple) and GC (orange, CD21/35^Atto590), B1-8^hi TN-XXL⁺ B cells (green). Right: FRET intensity in B_EM of same frame with color-coded mean FRET intensity. Arrowheads: clusters of FRET-positive B_EM in contact with SCS macrophages. Animation in movie 6. Scale bar 50 µm. c Intravital observation (clockwise from top left) of B_EM (green) in SCS containing CD169⁺ material (blue, arrowhead). d drLN from an Cγ1Cre mTmG mouse 10 min after foot injection with Alexa647 labeled immune complex (IC). Open Arrow heads: eGFP⁺ B_EM in SCS. Closed arrow heads: Alexa647-IC colocalizing with eGFP⁺ B_EM in follicle. Representative image of three lymph nodes. e Left: Alexa647-IC on CD11b⁺ CD169⁺ SCS macrophage (green), eGFP⁺ B_EM (red), naïve B cells (blue), or naïve B cells without Alexa647 injection (gray). Right: Percent Alexa647-IC positive B_EM or macrophages compared to naïve B cells. Overton subtraction from two independent experiments with three mice each. SCS M⏀: CD169⁺ subcapsular sinus macrophages. Two-tailed unpaired Student’s t test, ****p = 1.34 × 10⁻⁷. f Light-sheet microscopy of live explanted drLN, showing S1PR2^tdTom B_EM (red) in located between GC and SCS (closed arrowheads). SCS macrophages (blue). Some B_EM are seen in SCS above the SCS layer (open arrowheads). See also movie 8. g Same as (f) showing Alexa488 labeled IC (green) in the SCS and S1pr2 expression dependent tdTomato positive B_EM (red) between GC and SCS (closed arrowheads) or in contact with IC in SCS (open arrowheads). Representative image of four lymph nodes. See also Suppl. movies 9, 10.