DCs require CD4⁺ T cell–derived stimuli to escape apoptosis. (A-C) Assessment of DC survival. (A) Unperturbed distribution and regulation of MHC class II in transfected DCs. pIRES2-EGFP–transfected (left column) and nontransfected (right column) DCs were cultured in the absence (top row) or presence (bottom row) of CD40L. DCs immobilized onto adhesion slides were fixed, permeabilized, and stained with anti–HLA-DRβ mAbs followed by TRITC-labeled secondary reagent. Fluorescence was analyzed by laser scanning microscopy, as described.¹⁸  (B) Discrimination of viable and apoptotic DCs. DCs were transfected with pIRES2-EGFP and FACS sorted to a purity of 98%. EGFP⁺ DCs were cultured for 3 days after transfection, stained with annexin V–PE, and analyzed using FACS. Two major DC populations can be distinguished on the basis of EGFP (x-axis) and annexin V–PE binding (y-axis). EGFP^low/annexin V–PE⁺ cells correspond to dead/apoptotic DCs, and EGFP^high/annexin V–PE^– cells correspond to viable DCs. Dead (red gate) and viable (green gate) DCs are also clearly distinguished by their nonoverlapping FSC histograms (transparent inset: dead DCs, red line; viable DCs, green line). (C) Enumeration of viable DCs in DC–T-cell cocultures. FACS-sorted pIRES2-EGFP–transfected DCs and purified allogeneic CD4⁺ T cells were cocultured for 3 days. Cell conjugates were dissociated, and cells were stained with annexin V–PE and anti–CD3–PerCP and analyzed by FACS. CD3⁺ T cells were gated and are shown as orange dots. Four cell populations are resolved: EGFP^–/annexin V^– cells (viable T cells), EGFP^–/annexin V⁺ cells (dead T cells), EGFP^low/annexin V-PE⁺ cells (dead DCs), and EGFP^high/annexin V–PE^– cells (viable DCs). Again, viable DCs (green gate) can be distinguished from dead T cells and dead DCs (red gate) by nonoverlapping FSC histograms, as shown in the transparent inset (green and red lines, respectively). Fewer than 3% of DCs remained conjugated to T cells (orange dots in green gate). (D-E) Comparable basal and T cell–dependent survival of nontransfected DCs (D) and EGFP-transfected sorted DCs (E). DCs were cultured in the absence (▪) or presence (▧) of allogeneic CD4⁺ T cells at a DC/T-cell ratio of 1:100 until day 16. Cells were harvested at the indicated time points, and numbers of viable DCs were determined by FACS analysis. (F) T-cell–dependent DC survival requires cognate DC–T-cell interaction. DCs were pulsed with 1 μg/mL TSST1 sAg or were left untreated. sAg-loaded or nonpulsed DCs were cultured alone or cocultured with autologous CD4⁺ T cells with or without 10 μg/mL anti-CD40L mAbs. ▥ indicates medium; ▧, autologous CD4⁺ T cells alone; ▪, with sAg; and □, with sAg and anti-CD40L mAbs. (G) DCs were cultured in the absence (▪) or presence (▧) of CD40L. (D-G) Results are presented as percentage of viable DCs after culture relative to the number of input DCs (mean ± SD of triplicate determinations). Results are representative of at least 3 independent experiments.