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. 2000 Jun 5;191(11):1957-64.
doi: 10.1084/jem.191.11.1957.

Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways

F Castellino  1 , P E Boucher, K Eichelberg, M Mayhew, J E Rothman, A N Houghton, R N Germain
Affiliations

Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways

F Castellino et al. J Exp Med. .

Abstract

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen-specific CD8(+) T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we show that HSP 70 binds to the surface of antigen presenting cells by a mechanism with the characteristics of a saturable receptor system. After this membrane interaction, processing and MHC class I presentation of the HSP-associated antigen can occur via either a cytosolic (transporter associated with antigen processing [TAP] and proteasome-dependent) or an endosomal (TAP and proteasome-independent) route, with the preferred pathway determined by the sequence context of the optimal antigenic peptide within the HSP-associated material. These findings not only characterize two highly efficient, specific pathways leading to the conversion of HSP-associated antigens into ligands for CD8(+) T cells, they also imply the existence of a mechanism for receptor-facilitated transmembrane transport of HSP or HSP-associated ligands from the plasma membrane or lumen of endosomes into the cytosol.

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Figures

Figure 1
Figure 1
Antigenic peptides derived from material bound to HSP 70 in vitro can be presented by MHC class I molecules of normal macrophages. C57BL/6 macrophages were cultured overnight in 1 the absence of any antigen, 2 the presence of 1 nM SIINFEKL peptide, 3 2 μg of unloaded HSP 70, 4 20 nM OVA-BiP, 5 2 μg HSP70/OVA-BiP, 6 20 nM BiP-OVA, or 7 2 μg HSP 70/BiP-OVA complexes and 5 × 104 B3Z. IL-2 accumulation in the supernatant at 16 h was measured as an indication of T cell activation. Comparable results were obtained in >10 similar experiments.
Figure 2
Figure 2
Presentation of SIINFEKL from HSP/OVA-BiP but not HSP/BiP-OVA is dependent on proteasomal proteases and TAP. (A) The effect of 25 μM lactacystin on the presentation of the indicated antigens to either the B3Z or the 3A9 T cell hybridoma cells was assayed. In bars 1–7, the presentation to B3Z hybridoma cells was analyzed in 1 the absence of any antigen, 2 the presence of 1 nM SIINFEKL peptide, 3 10 nM SIINFEKL peptide, 4 20 nM OVA-BiP, 5 2 μg HSP70/OVA-BiP, 6 20 nM BiP-OVA, or 7 2 μg HSP 70/BiP-OVA complexes. In four independent experiments, the presentation of HSP 70/BiP-OVA was not affected by lactacystin, whereas the inhibition of the presentation of HSP70/OVA-BiP ranged between 70 and 90%. The effect of lactacystin on the presentation of HEL-46-61 to 3A9 hybridoma cells was tested 8 in the absence of any antigen, or 9 in the presence of 1 mg/ml hen egg lysosyme. (B) 105 TAP-1−/− macrophages (C57BL/6 × 129) were grown at 26°C for 24 h and transferred to 37°C when incubation with the indicated antigens and the T cells was started. The presentation to B3Z hybridoma cells was analyzed in 1 the absence of any antigen, 2 the presence of 1 nM SIINFEKL peptide, 3 10 nM SIINFEKL peptide, 4 20 nM OVA-BiP, 5 2 μg HSP70/OVA-BiP, 6 20 nM BiP-OVA, or 7 2 μg HSP 70/BiP-OVA complexes. Similar results were obtained in three independent experiments.
Figure 3
Figure 3
Direct visualization of two distinct intracellular sites of SIINFEKL association with MHC class I molecules after antigen delivery via HSP 70. C57BL/6 macrophages were incubated for 6 h at 37°C with either 100 μg/ml of HSP/BiP-OVA (A, C–E) or HSP/OVA-BiP (B, F–H). During the last 30 min of incubation, FITC-OVA was added to some of the samples. A, B, C, and F show the red channel fluorescence of cells stained with biotinylated 25-D1.16 followed by Streptavidin–Texas red. C shows the red channel (SIINFEKL/Kb complexes) for one cell, demonstrating the predominant vesicular location of these complexes; D shows the green channel (FITC-OVA) for the same high-power field; and E shows the colocalization of the two signals (yellow), indicating the endosomal nature of at least a fraction of the SIINFEKL/Kb-containing structures. In F–H, cells were double stained with biotinylated 25-D1.16 followed by Streptavidin–Texas red as well as with rabbit anticalnexin followed by FITC-conjugated anti–rabbit Ig. F shows the red channel (SIINFEKL/Kb complexes), revealing primarily reticular cytoplasmic staining along with a few stained vesicles. G shows the green channel (calnexin) for the same cell. Colocalization of the two signals (yellow) is seen in H, consistent with an ER location for the bulk of the SIINFEKL/Kb complexes.
Figure 4
Figure 4
A subset of normal macrophages expresses a surface receptor for HSP 70 that mediates internalization into endosomes. (A) C57BL/6 macrophages were incubated with 100 μg/ml of biotinylated HSP 70 for 30 min at 4°C; (B–D) after incubation as in A, the excess HSP was washed away and the macrophages were incubated for an additional 30 min at 37°C in the presence of FITC-OVA. All the samples were washed and then stained with Streptavidin–Texas red either without permeabilization (A), or after permeabilization with 0.1% Brij (B–D). B shows the red channel (HSP 70), and C shows the green channel (FITC-OVA). D shows the partial colocalization of the two signals (yellow). (E) 3 × 105 macrophages were incubated at 4°C with 50 μg/ml of biotinylated BSA (gray histogram), with 50 μg/ml of biotinylated HSP 70 (thick solid line), or with 500 μg/ml of unlabeled HSP 70 for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (thin solid line), or with 500 μg/ml of unlabeled BSA for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (dotted line). Cells were stained with FITC-anti-CD11b and streptavidin-PE. Only propidium iodide–negative CD11b+ cells are shown. (F) 5 × 105 macrophages were incubated at 4°C with increasing concentrations of biotinylated HSP 70 or biotinylated BSA. Cells were washed and stained with FITC–anti-CD11b and Streptavidin-PE. Only propidium iodide–negative CD11b+ cells are included in the analysis. The mean fluorescence of the CD11bbright subpopulation of macrophages that shows high binding to HSP 70 is plotted. (G) Macrophages were cultured overnight in the presence or absence of the indicated antigens and 5 × 104 B3Z cells. Where indicated “competitor,” macrophages were preincubated for 30 min with 2 or 20 μg of unloaded HSP 70 in 100 μl of medium, followed without washing by addition of 2 μg of antigen-loaded HSP or SIINFEKL and B3Z cells. In four independent experiments, the inhibition of the presentation of either HSP70/OVA-BiP or HSP 70/BiP-OVA by 20 μg of competitor averaged 50%.
Figure 4
Figure 4
A subset of normal macrophages expresses a surface receptor for HSP 70 that mediates internalization into endosomes. (A) C57BL/6 macrophages were incubated with 100 μg/ml of biotinylated HSP 70 for 30 min at 4°C; (B–D) after incubation as in A, the excess HSP was washed away and the macrophages were incubated for an additional 30 min at 37°C in the presence of FITC-OVA. All the samples were washed and then stained with Streptavidin–Texas red either without permeabilization (A), or after permeabilization with 0.1% Brij (B–D). B shows the red channel (HSP 70), and C shows the green channel (FITC-OVA). D shows the partial colocalization of the two signals (yellow). (E) 3 × 105 macrophages were incubated at 4°C with 50 μg/ml of biotinylated BSA (gray histogram), with 50 μg/ml of biotinylated HSP 70 (thick solid line), or with 500 μg/ml of unlabeled HSP 70 for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (thin solid line), or with 500 μg/ml of unlabeled BSA for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (dotted line). Cells were stained with FITC-anti-CD11b and streptavidin-PE. Only propidium iodide–negative CD11b+ cells are shown. (F) 5 × 105 macrophages were incubated at 4°C with increasing concentrations of biotinylated HSP 70 or biotinylated BSA. Cells were washed and stained with FITC–anti-CD11b and Streptavidin-PE. Only propidium iodide–negative CD11b+ cells are included in the analysis. The mean fluorescence of the CD11bbright subpopulation of macrophages that shows high binding to HSP 70 is plotted. (G) Macrophages were cultured overnight in the presence or absence of the indicated antigens and 5 × 104 B3Z cells. Where indicated “competitor,” macrophages were preincubated for 30 min with 2 or 20 μg of unloaded HSP 70 in 100 μl of medium, followed without washing by addition of 2 μg of antigen-loaded HSP or SIINFEKL and B3Z cells. In four independent experiments, the inhibition of the presentation of either HSP70/OVA-BiP or HSP 70/BiP-OVA by 20 μg of competitor averaged 50%.
Figure 4
Figure 4
A subset of normal macrophages expresses a surface receptor for HSP 70 that mediates internalization into endosomes. (A) C57BL/6 macrophages were incubated with 100 μg/ml of biotinylated HSP 70 for 30 min at 4°C; (B–D) after incubation as in A, the excess HSP was washed away and the macrophages were incubated for an additional 30 min at 37°C in the presence of FITC-OVA. All the samples were washed and then stained with Streptavidin–Texas red either without permeabilization (A), or after permeabilization with 0.1% Brij (B–D). B shows the red channel (HSP 70), and C shows the green channel (FITC-OVA). D shows the partial colocalization of the two signals (yellow). (E) 3 × 105 macrophages were incubated at 4°C with 50 μg/ml of biotinylated BSA (gray histogram), with 50 μg/ml of biotinylated HSP 70 (thick solid line), or with 500 μg/ml of unlabeled HSP 70 for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (thin solid line), or with 500 μg/ml of unlabeled BSA for 30 min, followed without washing by addition of 50 μg/ml of biotinylated HSP 70 and incubation for an additional 30 min (dotted line). Cells were stained with FITC-anti-CD11b and streptavidin-PE. Only propidium iodide–negative CD11b+ cells are shown. (F) 5 × 105 macrophages were incubated at 4°C with increasing concentrations of biotinylated HSP 70 or biotinylated BSA. Cells were washed and stained with FITC–anti-CD11b and Streptavidin-PE. Only propidium iodide–negative CD11b+ cells are included in the analysis. The mean fluorescence of the CD11bbright subpopulation of macrophages that shows high binding to HSP 70 is plotted. (G) Macrophages were cultured overnight in the presence or absence of the indicated antigens and 5 × 104 B3Z cells. Where indicated “competitor,” macrophages were preincubated for 30 min with 2 or 20 μg of unloaded HSP 70 in 100 μl of medium, followed without washing by addition of 2 μg of antigen-loaded HSP or SIINFEKL and B3Z cells. In four independent experiments, the inhibition of the presentation of either HSP70/OVA-BiP or HSP 70/BiP-OVA by 20 μg of competitor averaged 50%.
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