Fig. 2

ANXA2m enhances dendritic cell maturation and cross-priming in a TLR2-dependent manner. a Splenocytes from TLR2 ^+/+ mice were pulsed with the OVA_248–274 peptide SIIFNEKL. SIINFEKL cross-presentation was detected 24 h later by flow cytometry using the 25-D1.16 antibody (specific for the SIINFEKL/H-2K^b complex). Data represent the median fluorescence intensity of cells within a CD11c⁺MHCII⁺ gate. Bars signify p <0.002 by unpaired t-test. The experiment was repeated three times with similar results. The average MFIs for the three experiments were 1,236 and 274 for peptide-pulsed CD11c⁺MHCII⁺ splenocytes with or without human ANXA2m, respectively. b Same experimental design as (a) except the splenocytes were from TLR2 ^−/− mice. In all three replicates there were no significant differences among the treatment groups. c BMDC cell cultures were pulsed with 2.5 μg/ml human ANXA2m or medium. Cells were harvested and stained after 2 days. Graphs indicate the MFI for the CD11c⁺MHCII^hi gated population. The average MFIs from three replicate experiments were 12,736 and 7,577 for CD80 and CD86, respectively. d Supernatants from (c) were collected and assayed using a cytometric bead array. The averages from the three replicate experiments were 106 and 7,529 pg/ml for IL12p70 and TNFα, respectively. Error bars are ± SEM, and lines indicate P <0.01 by unpaired t-test. e Naïve mice were vaccinated with four sequential daily injections of OVA (days 1–4), with or without recombinant human ANXA2m and boosted once on day 7. Staining of peripheral blood was performed on day 8. Graph represents the percentage of peripheral blood cells staining positive for the SIINFEKL/K^b dextramer within the CD8α⁺ lymphocyte gate. The experiment was replicated twice with similar results with n = 4 per group (the average frequencies of dextramer-binding CD8α + cells were 7.17 and 2.3 % in the wildtype and TLR2 ^−/− mice, respectively). Bars denote p <0.05 by unpaired t-test. f Monocyte-derived dendritic cells from CMV seropositive and seronegative HLA-A0201⁺ donors were pulsed with the pp65_495–503 peptide, with or without human ANXA2m. After a day cells were washed and autologous PBMCs were added for an additional 2 days. Soluble INF-γ was measured by bead array. The experiment was repeated once with similar results. The averages of the two experiments were 4003 and 114 pg/ml IFN-γ for pulsed PBMCs from CMV seropositive and seronegative individuals, respectively, and 399 pg/ml for unpulsed PBMCs from a CMV seropositive individual. Error bars are ± SEM, and lines show p <0.01 by unpaired t-test