FIGURE 2.
Characterization of apoptotic cell uptake and its effects on phenotype and Ag presentation of monocyte-derived cells. A, LCLs were CFSE labeled, irradiated, and incorporated into collagen beneath HUVEC monolayers. The capture and presence of CFSE+ LCL-derived material (bold lines) by reverse-transmigrated (R/T) and subendothelial (S/E) monocyte-derived cells, respectively, were analyzed. Shaded profiles indicate the fluorescence associated with reverse-transmigrated (top) or subendothelial cells (bottom) from cultures wherein LCLs were incorporated in the collagen without CFSE labeling. In the subendothelial population, monocyte-derived cells were distinguished from LCLs by CD14 counterstaining. B, Cell surface expression of HLA-DR and CD86 on reverse-transmigrated monocyte-derived cells after some of these cells phagocytized apoptotic, autologous LCLs (LCLs, no Flu) or influenza A virus-infected LCLs (LCL/Flu) incorporated within collagen, or after they were directly infected with 0.5 MOI live influenza A virus PR/8 (Direct Flu) or were only mock infected (No LCLs, no Flu) before addition to endothelium. C, ELISPOT to quantify the induction of IFN-γ during a CTL assay by the HLA-A201-restricted MP58–66 peptide (GILGFVFTL)-specific CD8+ T cell line primed by HLA-A201+ reverse-transmigrated cells derived from HUVEC/collagen cultures containing LCLs without influenza MP Ag (background reactivity control, open bar) or cultures in which HLA-A201− LCLs expressed MP with or without flu infection (black bars), or from reverse-transmigrated cells derived from monocytes directly infected with flu (gray bars). Spontaneous activity in the absence of APC activation is shown by the stippled bar. Data shown are from one experiment, representative of four conducted. D, HLA-A201+ monocyte-derived reverse-transmigrated cells derived from the conditions described in C were cocultured with autologous CD45RA− memory T cells for 7 days. IFN-γ-producing CD8+ CTLs were assayed by ELISPOT using T2 cells (TAP−/−, HLA-A201+) pulsed with influenza A virus MP58–66 peptide as target cells. The ratio of T cells to target cells was 1:1. Data shown are representative of five independent experiments. E, Flow cytometric cell sorting of reversetransmigrated and subendothelial monocyte-derived cells derived from HUVEC/collagen cultures containing flu-infected LCLs was done to compare their relative efficacy in stimulating CTL responses by cross-presentation. The open bar shows background reactivity observed with reverse-transmigrated cells when T2 cells were pulsed with control HIV Gag peptide rather than with MP peptide. Measurement by ELISA of TNF-α (F) and IFN-α (G) concentrations in conditioned medium collected from cultures 48 h after monocytes were added to the endothelium. Mo + flu indicates monocytes directly infected with influenza A; LCL + flu, LCLs infected with flu and loaded in collagen. Where specified, media was collected without addition of PBMCs as a source of monocytes. Otherwise, monocytes were present in the cultures for 48 h at the time of supernatant collection. In some such cultures, LPS was added after monocyte transmigration across the endothelium, as described (12). Bar sets to the right of each graph show cultures wherein TLR agonists ssRNA, poly(I:C), or both were added to the culture medium (open bars) or LCLs were loaded with the ligands by electroporation (filled bars). Concentrations of these cytokines were measured in three independent experiments; one representative experiment is shown.