Fig. 7

LEC apoptosis causes increased antigen presentation during LN contraction. a Schematic of experimental design for b, e–g. b Mice were infected weekly for 5 weeks with vaccinia virus (1 × 10⁴ colony forming units (CFU)) per foot and the number of LEC from the popliteal lymph nodes was determined to evaluate the increase in LECs over time. c Wild-type mice were immunized subcutaneously with ovalbumin conjugated to Alexafluor 488 (ova488) (10 μg) poly(I:C)/αCD40 (2 μg each) or not and killed 2.5 weeks later. Lymph nodes were removed and imbedded in optimal cutting temperature compound, sectioned, and stained with LEC marker Lyve-1 (red), cleaved caspase 3 (white), and dapi (blue), antigen-ova488 is visible by green fluorescence where indicated. Arrows in slide are as follows: white-antigen⁺ caspase⁺lyve1⁺, yellow-caspase⁺lyve1⁺, pink-lyve-1⁺caspase⁺. Scale bar is 100 μm in top panels and 15 μm in zoomed panels. d Quantification of lymph nodes from c. Experiment was repeated thrice with five lymph nodes evaluated. An unpaired t-test was used to calculate the p-values for differences from naive which were p = 0.046 for LEC⁺Ag⁺ and p = 0.0076 for LEC⁺Ag⁺Caspase⁺. e Mice infected weekly as in Fig. 1 with ova488 + vaccinia virus were killed and the percentage of antigen-positive LECs was calculated using flow cytometry. f OT1 T cells were transferred into mice infected weekly, as in c, 3 days prior to killing and the percent antigen-specific T cells divided was calculated. g The ratio of percent divided to number antigen positive was calculated from e and f. All experiments were performed at least twice with at least three mice per group per time point unless otherwise indicated. Error bars are mean ± standard error of the mean