Presentation of fungal antigen (calnexin) to transgenic Tg1807 T cells by PMN-DCs generated <i>in vitro</i> from ER-HoxB8 GMP cells.

<p>(<b>A</b>–<b>D</b>) PMN-DCs or bone-marrow DCs (BMDCs) were incubated overnight with or without recombinant fungal calnexin, and the next day 5 x 10⁴ cells were injected subcutaneously into CD90.2 mice that had received adoptive transfer of Tg1807 cells (CD90.1⁺, calnexin-specific CD4⁺ T cells). One week later skin draining lymph nodes were harvested. <b>(A)</b> IL-6 in supernatants from overnight culture of PMN-DCs or BMDCs with or without calnexin, n.d,:not detected. <b>(B)</b> Experimental design of delivering calnexin-loaded APCs into mice that had received congenic Tg1807 cells. <b>(C)</b> Proportion of Tg1807 cells activated in lymph nodes (indicated by CD44⁺ and CD62L^-). <b>(D)</b> The absolute number of Tg1807 cells and activated Tg1807 cells for each treatment group (N = 5–6 mice). (<b>E</b>) Lymph node cells (as shown in C-D) were stimulated <i>ex vivo</i> for 3 days with calnexin, and IFN-γ and IL-17 assayed in culture supernatants, Ag: antigen pulsed. (<b>F-G</b>) PMN-DCs or BMDCs were incubated overnight with fungal calnexin or heat-killed <i>Blastomyces</i> yeast before enriched CD4⁺ Tg1807 cells were added. (F) Presence of IFN-γ and IL-17 in supernatants collected after 3 days was determined by ELISA. (G) Expression of MHC class II from PMN-DCs at the end of the assay (Day 11) shown with comparison of MHC class II expression before mixing with T cells (Day 7) and before PMN-DC differentiation (Day 0), MFI indicated (±SEM). Both <i>in vivo</i> and <i>in vitro</i> experiments were completed independently twice. Statistics: for C-E, statistical significance between calnexin-loaded PMN-DCs and no antigen controls; for F-G, statistical significance between samples with fungal antigen and unstimulated.