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Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells - PubMed

  • ️Thu Jan 01 1998

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells

M Rescigno et al. Proc Natl Acad Sci U S A. 1998.

Erratum in

  • Proc Natl Acad Sci U S A 1999 Aug 17;96(17):9666

Abstract

Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 10(6) times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.

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Figures

Figure 1
Figure 1

DC internalize S. gordonii via conventional phagocytosis. DC were incubated with bacteria for different time periods at a ratio of 10 bacteria to 1 DC, then washed and processed for transmission electron microscopy. (A) Thirty minutes after infection, bacteria were contacting the cell membrane and inducing local thickening of plasma membrane (arrows) (magnification, ×39,000; bar represents 3.9 μm). (B) Four hours after infection, bacteria were found in phagolysosomes in a partially degraded form (arrow) at a magnification of ×28,700; bar = 2.9 μm.

Figure 2
Figure 2

Phenotypical maturation of D1 cells. FACS profiles of surface markers after incubation with S. gordonii at a ratio of 10 bacteria to 1 DC (Left) or latex beads at a ratio of 100 beads to 1 DC (Right). Histograms represent the following: filled, cells after treatment; open, untreated cells; dashed, isotype controls.

Figure 3
Figure 3

S. gordonii-treated DC secrete large amounts of inflammatory cytokines. Culture supernatants from untreated cells (hatched bars), S. gordonii-treated D1 (black bars), or R1 culture medium (white bars) were tested for cytokine release by ELISA. Recombinant cytokines were used as standards in each ELISA.

Figure 4
Figure 4

Bacterial infection induces de novo synthesis and stabilization of MHC class I molecules. (A) DC were metabolically labeled before (t = 0) or at different times (t = 1–36 h) after bacterial infection. HCm, heavy chain mature form; HCi, heavy chain immature form; a, actin; β2 m, β2 microglobulin. The graph shows the quantification by Phosphorimager of the radioactivity associated with MHC I heavy chain. A.U., arbitrary units. (B) Stability of MHC I molecules synthesized by untreated cells (Top) or by cells treated for 18 h with bacteria (Bottom). Cells were biosynthetically labeled and chased for 0–48 h in an excess of cold methionine. Abbreviations are as in A. The graph represents the quantification by

bio-1d

software of the radioactivity associated with MHC I molecules in treated and untreated DC chased in the absence of bacteria.

Figure 5
Figure 5

Bacterial infection induces de novo synthesis and stabilization of MHC class II molecules. (A) DC were metabolically labeled before (t = 0) or at different times (t = 1–36 h) after bacterial infection. α + Ii, alpha chain + invariant chain; β, beta chain. The graph shows the quantification by Phosphorimager of the radioactivity associated with the α-chains. (B) Stability of mature MHC class II molecules synthesized by untreated cells (Top) or cells treated for 18 h with bacteria (Bottom). α, alpha chain; β, beta chain. The graph represents the quantification by

bio-1d

software of the radioactivity associated with β-chains in DC incubated or not with bacteria and then chased in the absence of bacteria.

Figure 6
Figure 6

DC can process bacterial antigens for MHC I presentation very efficiently. DC were incubated with recombinant OVA-gordonii (GP1252, ○), wild-type gordonii (GP204) + exogenous OVA (◊), or OVA alone (•) for 16 h and then washed and fixed. The ratio of bacteria to DC ranges from 500:1 to 0.3:1. Ratio of 500 bacteria to 1 DC corresponds to 1 μg/ml of OVA. IL-2 production by B3Z T cell hybridoma was measured as [3H]thymidine incorporation by IL-2-dependent CTL.

Figure 7
Figure 7

MHC class I presentation of bacterial antigens is TAP dependent. The B3Z hybridoma response to BM-derived TAP−/− DC (A and C) or to D1 TAP+/+ (B) incubated with the following is shown: (A and B) OVA-gordonii (GP1252, •) or wild-type gordonii (GP204, ○); (C) soluble OVA (○) or SIINFEKL peptide. (•).

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