CD8α⁺ dendritic cells (DC) selectively capture apoptotic cells and cross-present cell-associated antigen on major histocompatibility complex (MHC) class I in vitro. (a) Sorted CD4⁺ (open bars) or CD8α⁺ (filled bars) DC subsets (C57BL/6) were co-cultured with OT-I T cells in the presence of irradiated, ovalbumin (OVA)-loaded allogeneic (BALB/c) splenocytes. Results represent the mean [³H]thymidine uptake of triplicate wells. All error bars are shown and represent 1 SD from the mean. c.p.m., counts /min. (b)–(d) Flow cytometry analysis of 3-hr cultures containing prestained, CD11c-enriched cells and PKH26-labelled RAW264.7 cells, as indicated. (b) Ultraviolet (UV)-treated RAW cells were added to DC cultures in the presence or absence of sodium azide (top) or were first cultured and then sorted into annexin V⁺ or annexin V⁻ (AxV⁺, AxV⁻, respectively) fractions and fixed in paraformaldehyde (PFA), as described in the Materials and methods (bottom). Dot-plots represent CD8α expression versus PKH26 fluorescence for DC gated on the basis of high CD11c expression. Numbers show the percentage of cells within each quadrant gate. (c) Histograms showing PKH26 profile for CD8α⁺ (top), CD4⁺ (middle) and double negative (DN, bottom) DC subsets from cultures with (thick line) or without (thin line) PKH26-labelled UV-treated RAW cells. (d) Frequencies of PKH26⁺ DC subsets after incubation with increasing doses of UV-treated, PKH26-labelled RAW cells. Data are representative of (a) six experiments, (b) three experiments, or (c) and (d) two experiments.

Visualization of dead cell uptake by CD8α⁺ dendritic cells (DC). CD8α⁺ DC purified by cell sorting were co-cultured with ultraviolet (UV)-treated, PKH26-labelled RAW cells. Images were acquired at 15-second intervals for a period of 3 hr. The image is a composite of four stills taken from a film and is shown as a superimposed phase-contrast and PKH26 fluorescence image; DC appear as phase-dense, unlabelled, irregularly shaped cells, whereas the dying RAW cells appear as round cells labelled red. The DC in this composite is indicated by the white arrowhead in the first frame. Note that labelled material is associated with the DC as it moves away from the cluster of RAW cells. The time in seconds is indicated in each frame; the time on the first frame was arbitrarily set at 0. Excerpts from the films can be seen on the world wide web as supplementary material to the publication of this article on the web.

CD4⁺ dendritic cells (DC) can phagocytose Escherichia coli and latex beads and present exogenous ovalbumin (OVA) on major histocompatibility complex (MHC) class I. (a) Flow cytometric analysis of 3-hr cultures of DC-enriched splenocytes and PKH26-labelled E. coli (left) or yellow green (YG) latex beads (right). Dot-plots represent gated CD11c^bright cells. Numbers show the percentage of cells within each quadrant gate. (b) Quantification of E. coli uptake by double negative (DN), CD8α⁺ and CD4⁺ DC subsets. (c) Sorted CD4⁺ DC (open bars) and CD8α⁺ DC (filled bars) were cultured with OT-I T cells in the presence of two doses of OVA-E. coli or OVA/LLO-E.coli, as indicated. c.p.m., counts /min. (d) Sorted CD4⁺ (circles) and CD8α⁺ (triangles) DC subsets (3 × 10⁴/well) were cultured with OT-I T cells and different doses of OVA-peptide in the absence (open symbols) or presence (filled symbols) of paraformaldehyde (PFA)-treated E. coli (10⁶/well). Results in (c) and (d) represent the mean [³H]thymidine uptake of triplicate wells. All error bars are shown and represent 1 SD from the mean. Results are representative of (a) and (b) three experiments, (c) two experiments, or (d) one experiment, with a full peptide dose range and an additional experiment using selected peptide doses.

Major histocompatibility complex (MHC) class I presentation of ovalbumin (OVA)-bearing splenocytes and OVA-Escherichia coli by dendritic cells (DC) requires proteasomal activity. CD11c-enriched splenocytes and OT-I T cells were cultured with either OVA-loaded splenocytes (5 × 10⁵/well; open bars), OVA-E. coli (1 × 10⁶/well; grey bars) or OVA-peptide (25 pm, filled bars) in the presence or absence of the indicated doses of the proteasome-specific inhibitor lactacystin. Results represent the mean [³H]thymidine uptake of triplicate wells. All error bars are shown and represent 1 SD from the mean. Results are representative of four experiments.