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. 2000 Mar 20;191(6):927-36.
doi: 10.1084/jem.191.6.927.

The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli

K Inaba  1 , S Turley, T Iyoda, F Yamaide, S Shimoyama, C Reis e Sousa, R N Germain, I Mellman, R M Steinman
Affiliations

The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli

K Inaba et al. J Exp Med. .

Abstract

During their final differentiation or maturation, dendritic cells (DCs) redistribute their major histocompatibility complex (MHC) class II products from intracellular compartments to the plasma membrane. Using cells arrested in the immature state, we now find that DCs also regulate the initial intracellular formation of immunogenic MHC class II-peptide complexes. Immature DCs internalize the protein antigen, hen egg lysozyme (HEL), into late endosomes and lysosomes rich in MHC class II molecules. There, despite extensive colocalization of HEL protein and MHC class II products, MHC class II-peptide complexes do not form unless the DCs are exposed to inflammatory mediators such as tumor necrosis factor alpha, CD40 ligand, or lipoplolysaccharide. The control of T cell receptor (TCR) ligand formation was observed using the C4H3 monoclonal antibody to detect MHC class II-HEL peptide complexes by flow cytometry and confocal microscopy, and with HEL-specific 3A9 transgenic T cells to detect downregulation of the TCR upon MHC-peptide encounter. Even the binding of preprocessed HEL peptide to MHC class II is blocked in immature DCs, including the formation of C4H3 epitope in MHC class II compartments, suggesting an arrest to antigen presentation at the peptide-loading step, rather than an enhanced degradation of MHC class II-peptide complexes at the cell surface, as described in previous work. Therefore, the capacity of late endosomes and lysosomes to produce MHC class II-peptide complexes can be strictly controlled during DC differentiation, helping to coordinate antigen acquisition and inflammatory stimuli with formation of TCR ligands. The increased ability of maturing DCs to load MHC class II molecules with antigenic cargo contributes to the >100-fold enhancement of the subsequent primary immune response observed when immature and mature DCs are compared as immune adjuvants in culture and in mice.

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Figures

Figure 1
Figure 1
Formation of MHC–peptide complexes by bone marrow–derived DCs. (A) DCs in bone marrow cultures were pulsed with 3,000 μg/ml of HEL for the times indicated on the x-axis, washed, and assayed immediately for C4H3 staining (pulse) or chased until 21 h of total culture (pulse–chase). The y-axis displays increases in mean fluorescence index (MFI) for I-E+ DCs relative to cultures in the absence of HEL protein. (B) A maturation stimulus is present in commercial preparations of HEL. Immature 6-d marrow cultures were maintained for one additional day in the absence of another stimulus or in the presence of the stimuli listed on the top, including transfer to a new vessel (right). Maturation was monitored at the level of surface MHC class II expression (y-axis) and surface CD40 and CD86 (x-axis), with anti-CD8 antibody used as a nonreactive isotype control.
Figure 1
Figure 1
Formation of MHC–peptide complexes by bone marrow–derived DCs. (A) DCs in bone marrow cultures were pulsed with 3,000 μg/ml of HEL for the times indicated on the x-axis, washed, and assayed immediately for C4H3 staining (pulse) or chased until 21 h of total culture (pulse–chase). The y-axis displays increases in mean fluorescence index (MFI) for I-E+ DCs relative to cultures in the absence of HEL protein. (B) A maturation stimulus is present in commercial preparations of HEL. Immature 6-d marrow cultures were maintained for one additional day in the absence of another stimulus or in the presence of the stimuli listed on the top, including transfer to a new vessel (right). Maturation was monitored at the level of surface MHC class II expression (y-axis) and surface CD40 and CD86 (x-axis), with anti-CD8 antibody used as a nonreactive isotype control.
Figure 2
Figure 2
Antigen and a maturation stimulus synergize to form MHC–peptide complexes. (A) Day 6 bone marrow cultures from CBA/J mice were cultured for 24 h in the absence of HEL or LPS, with either HEL (1,000 μg/ml) or LPS (5 ng/ml), or with both. The HEL had been passed over a Kuttsuclean™ adsorbent. Left: anti-CD8 isotype control. Middle: CD86 marker for DC maturation, with the percentage of mature CD86-rich cells indicated. Right: mean fluorescent index (MFI) of the C4H3 signal on I-E+ DCs. Similar results were obtained using C3H/HeJ DCs matured with CD40L in three experiments. (B) Cells were pulsed with HEL protein for 3 h, and then washed before adding the maturation stimulus where indicated for a chase period of 21 h. (C) Cells were pulsed with preprocessed HEL peptide for 3 h, and then washed before adding LPS where indicated for a chase period of 21 h.
Figure 3
Figure 3
Localization of HEL protein and MHC class II–peptide complexes in DCs. The procedure was the same as described in the legend to Fig. 2, but here the formation of MHC class II–peptide was monitored at 3 and 24 h by immunofluorescence confocal microscopy. On the left are CBA DCs cultured in HEL only, and on the right are cells cultured with HEL + LPS. Representative cells are shown following double labeling in green for the HEL antigen, either HEL protein with 1B12 antibody or I-Ak/HEL MHC-peptide complexes with C4H3. Red stain identifies the lysosomal membrane glycoprotein lysosomal-associated membrane protein 2. Identical red staining is seen with H-2M or MHC class II. The results are representative of >10 experiments, and of experiments with both HEL protein and preprocessed HEL peptide. The maturation-induced C4H3 signal at 24 h is shown for a well-spread cell, but in optical sections through thicker cells, C4H3 stain is almost entirely along the cell perimeter.
Figure 3
Figure 3
Localization of HEL protein and MHC class II–peptide complexes in DCs. The procedure was the same as described in the legend to Fig. 2, but here the formation of MHC class II–peptide was monitored at 3 and 24 h by immunofluorescence confocal microscopy. On the left are CBA DCs cultured in HEL only, and on the right are cells cultured with HEL + LPS. Representative cells are shown following double labeling in green for the HEL antigen, either HEL protein with 1B12 antibody or I-Ak/HEL MHC-peptide complexes with C4H3. Red stain identifies the lysosomal membrane glycoprotein lysosomal-associated membrane protein 2. Identical red staining is seen with H-2M or MHC class II. The results are representative of >10 experiments, and of experiments with both HEL protein and preprocessed HEL peptide. The maturation-induced C4H3 signal at 24 h is shown for a well-spread cell, but in optical sections through thicker cells, C4H3 stain is almost entirely along the cell perimeter.
Figure 4
Figure 4
Formation of MHC–peptide complexes in immature DCs in situ. (A) Explants of CBA ear skin were bathed in 3,000 μg/ml LPS containing HEL for 5 or 22 h. At each time point, epidermal sheets were prepared and double labeled for the H-2M marker of MIICs (green) and for C4H3 epitope (red). The sheets were examined by two-color immunofluoresence confocal microscopy. The effect of a maturation stimulus could not be examined in vivo, because simple injection of PBS could induce some DCs to mature. (B) Explants of ear skin were cultured in the absence or presence of HEL for 48 h, and then the emigrated cells were doubled labeled for CD86 to identify the DCs (arrows) and C4H3. Some of the cells that emigrated in the absence of HEL were then cultured for 2 d in the presence of HEL before similar FACS® studies. The MFI for CD86+ cells is given in each panel.
Figure 4
Figure 4
Formation of MHC–peptide complexes in immature DCs in situ. (A) Explants of CBA ear skin were bathed in 3,000 μg/ml LPS containing HEL for 5 or 22 h. At each time point, epidermal sheets were prepared and double labeled for the H-2M marker of MIICs (green) and for C4H3 epitope (red). The sheets were examined by two-color immunofluoresence confocal microscopy. The effect of a maturation stimulus could not be examined in vivo, because simple injection of PBS could induce some DCs to mature. (B) Explants of ear skin were cultured in the absence or presence of HEL for 48 h, and then the emigrated cells were doubled labeled for CD86 to identify the DCs (arrows) and C4H3. Some of the cells that emigrated in the absence of HEL were then cultured for 2 d in the presence of HEL before similar FACS® studies. The MFI for CD86+ cells is given in each panel.
Figure 5
Figure 5
Antigen presenting activity of DCs in vitro after culture with graded doses of HEL, minus or plus LPS or CD40L as a maturation stimulus. (A) Day 6 bone marrow DCs from CBA mice (top) or C3H/HeJ mice (bottom) were cultured for 20 h with graded doses of HEL (x-axis) that had been endotoxin depleted with Kuttsuclean™. One group of cultures was matured by simultaneous addition of LPS or CD40L (closed symbols), and the other was unstimulated (open symbols). After 20 h, the cells were harvested, washed, and fixed in paraformaldehyde to block further processing and maturation. MHC–peptide complexes were quantified in terms of C4H3 staining, and are shown as mean fluorescence indices on the left. The fixed DCs were added in graded doses (top) to 250,000 CD4+ T cells from 3A9 TCR transgenic mice (specific for the same complex of I-Ak + HEL peptide as C4H3 antibody). 3H-TdR uptake was measured at 30–42 h. One of three similar experiments. (B) The display of TCR ligands by fixed DCs was monitored at 5 h on T cells (gated away from DCs by light scattering) by the criteria of TCR downregulation (anti-Vβ8, y-axis) and CD69 upregulation (x-axis). The DC/T cell ratio was 1:3 in the data that are shown, and the percentage of CD69+ cells is indicated in each dot plot (upper right).
Figure 5
Figure 5
Antigen presenting activity of DCs in vitro after culture with graded doses of HEL, minus or plus LPS or CD40L as a maturation stimulus. (A) Day 6 bone marrow DCs from CBA mice (top) or C3H/HeJ mice (bottom) were cultured for 20 h with graded doses of HEL (x-axis) that had been endotoxin depleted with Kuttsuclean™. One group of cultures was matured by simultaneous addition of LPS or CD40L (closed symbols), and the other was unstimulated (open symbols). After 20 h, the cells were harvested, washed, and fixed in paraformaldehyde to block further processing and maturation. MHC–peptide complexes were quantified in terms of C4H3 staining, and are shown as mean fluorescence indices on the left. The fixed DCs were added in graded doses (top) to 250,000 CD4+ T cells from 3A9 TCR transgenic mice (specific for the same complex of I-Ak + HEL peptide as C4H3 antibody). 3H-TdR uptake was measured at 30–42 h. One of three similar experiments. (B) The display of TCR ligands by fixed DCs was monitored at 5 h on T cells (gated away from DCs by light scattering) by the criteria of TCR downregulation (anti-Vβ8, y-axis) and CD69 upregulation (x-axis). The DC/T cell ratio was 1:3 in the data that are shown, and the percentage of CD69+ cells is indicated in each dot plot (upper right).
Figure 6
Figure 6
Antigen presenting activity of DCs in vivo after culture with graded doses of HEL, minus (open symbols) or plus (closed symbols) CD40L as a maturation stimulus. As described in the legend to Fig. 5, graded doses of antigen (top) were used to pulse C3H/He DCs overnight. The washed, unfixed cells were injected subcutaneously into syngeneic mice, 2 × 105 per paw. 5 d later, cultured draining lymph node cells were challenged with different doses of HEL protein (x-axis), and 3H-TdR uptake measured at 42–48 h (y-axis). Data are shown as stimulation relative to the background cpm in lymph nodes cultured without antigen. One of three similar experiments.
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