Peptide receptive RIT HLA-B molecules are prevalent on the cell surface of TAP-deficient cells and traffic to the cell surface via conventional and unconventional pathways.

<p>(A-D) Peptide binding studies suggest that a significant amount of cell surface RIT HLA-B (B*35:01, B*57:03, B*15:01 and B*44:05) in TAP-deficient cell line SK19 is receptive to exogenous peptides (n = 3 replicates). Peptide receptivity of cell surface HLA-B was assessed as described in methods. Briefly, cells cultured at 26°C or 37°C were incubated with indicated peptides at 26°C for 2h and then incubated in the presence of BFA at 37°C for an additional 2h. The HLA-B signals were quantified by flow cytometry and signals from cells infected with retrovirus lacking HLA-B were subtracted. Peptide receptive HLA-I was quantified as (MFI HLA-I<sub>(+peptide)</sub>–MFI HLA-I<sub>(-peptide)</sub>) / MFI HLA-I<sub>(+peptide)</sub>*100. (E-F) Peptide receptivity of B*15:01 or B*44:05 was assessed following expression in a TAP-sufficient cell line, CEM, as described in methods. Cell surface B*15:01 and B*44:05 are mostly unreceptive to exogenous peptides in these cells. (G) Some cell surface HLA-B is empty as assessed by flow cytometry with HC10, an antibody specific for open HLA class I conformations. HC10-based flow cytometric analysis of selected HLA-B-expressing SK19 cells (obtained as described in <a href="" target="_blank">Fig 1A</a>, but using the HC10 antibody (n = 5 measurements from a single infection)). Significant differences are indicated (with an asterisk) on the graph (<i>P</i><0.05). Statistical significance is based on an ordinary one-way ANOVA analysis with Fisher’s LSD test. (H) Comparative staining of indicated TAP1-deficient or TAP1-reconstituted SK19 cells (obtained as described in <a href="" target="_blank">Fig 2A</a>) with W6/32 and HC10 (n = 2 measurements from a single infection). Compared with TAP1-reconstituted SK19 cells, TAP1-deficient SK19 cells expressing RIT HLA-B allotypes showed high HC10 / W6/32 ratios. (I) Cell surface (upper panel) or total HLA-I molecules (lower panel) from SK19-HLA-B or CEM-B*35:01 cells were digested with Endo-H or left undigested, and analyzed by SDS-PAGE and immunoblot described in the method section. R indicates Endo-H resistant HLA-I heavy chain band, and S indicates Endo-H sensitive HLA-I heavy chain band. One representative set of blots from two experiments is shown.</p>