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.

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.

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/K^b 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/K^b-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/K^b 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/K^b complexes.

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 CD11b^bright 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%.