Mechanisms determining TAP-independent HLA-B surface expression.

<p>The TAP1 dependencies (A) and TAP2 dependencies (B) of HLA-B allotypes are partly correlated with their tapasin dependencies. The MFI ratios of HLA-B allotypes (derived from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1A</a> or <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">1B</a>) were used as indexes of TAP1 and TAP2 dependencies and MFI ratios of HLA-B allotypes (derived from Fig 1A of Ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.ref022" target="_blank">22</a>) were used as index of tapasin dependencies. HLA-B supertypes are color-coded, B7, blue; B44, pink; B62, green; B58, orange. (C and D) The anchor residue preferences at P₂ based on peptide sequences mined from two recent mass spectrometric studies (Ref. 42 and 43 respectively). In A-D, alleles belonging to the same supertype are color-coded. (E) Frequencies of indicated amino acids at the N-termini (excluding the last 6 residues at the C-terminus which cannot be P₂ for any epitope) of known human signal peptide sequences obtained from <a href="http://www.signalpeptide.de/" target="_blank">www.signalpeptide.de</a>. (F) Signal peptide sequences were used to predict potential 9-mer epitopes for the indicated HLA-B, using NetMHC 4.0. For each allele, the number of peptides with predicted IC₅₀ values < 500 nM and 50 nM are shown. (G and H) Similar to E and F, but using human transmembrane domain sequences obtained from <a target="_blank">ftp://ftp.ncbi.nih.gov/repository/TMbase/</a>, to estimate the frequencies of occurrence of indicated amino acids, excluding the last 6 residues at the C-terminus (G) and the predicted number of epitopes for each indicated HLA-B (H).</p

Allele-dependent variations in permissive HLA-B antigen presentation pathways.

In the conventional pathway operative in normal cells (A), TAP-dependent peptides are presented by the majority of HLA-B allotypes, although members of the B7 supertype may also present optimal and suboptimal peptides from TAP-independent sources, due to mismatch between their peptide-binding preferences and TAP transport specificity. While expression levels of many allotypes are strongly reduced by tapasin or TAP deficiency, some allotypes are relatively resistant to the deficiency of these factors (B and C). In particular, B*18:01, B*44:05, B*40:01, B*35:01, B*35:03 and B*15:01 are detectable at the highest levels in tapasin-deficient cells (B), based on higher stabilities of the peptide-deficient forms and/or high peptide-loading efficiencies (Ref. 22). B*57:03, B*35:03, B*15:01 and B*35:01 (RIT-HLA-B) are detected at the highest levels under TAP-deficiency conditions (C). The high expression of RIT-HLA-B under TAP-deficiency conditions is mediated by the synergy between their high intrinsic stability, high peptide loading efficiency and generally broader peptide repertoires, particularly for peptides present within unconventional sources such as signal sequences. The strong reduction in the ER peptide levels under TAP-deficiency conditions contributes to the suboptimal loading and cell surface peptide receptivity of RIT-HLA-B. The ER quality control system for the retrieval of suboptimally loaded RIT-HLA-B molecules is imperfect, and alternative (non-Golgi) pathways may exist for transport of suboptimally loaded RIT-HLA-B molecules to the cell surface. RIT-HLA-B allotypes are expected to induce stronger CD8+ T cell responses in the context of viruses that inhibit TAP, but weaker NK responses. Conversely, other non-RIT HLA-B are expected to induce stronger NK responses but weaker CD8+ T cell responses, following infections with viruses that inhibit TAP.</p

Selected HLA-B allotypes are resistant to inhibition or deficiency of the transporter associated with antigen processing (TAP)

<div><p>Major histocompatibility complex class I (MHC-I) molecules present antigenic peptides to CD8⁺ T cells, and are also important for natural killer (NK) cell immune surveillance against infections and cancers. MHC-I molecules are assembled via a complex assembly pathway in the endoplasmic reticulum (ER) of cells. Peptides present in the cytosol of cells are transported into the ER via the transporter associated with antigen processing (TAP). In the ER, peptides are assembled with MHC-I molecules via the peptide-loading complex (PLC). Components of the MHC-I assembly pathway are frequently targeted by viruses, in order to evade host immunity. Many viruses encode inhibitors of TAP, which is thought to be a central source of peptides for the assembly of MHC-I molecules. However, human MHC-I (HLA-I) genes are highly polymorphic, and it is conceivable that several variants can acquire peptides via TAP-independent pathways, thereby conferring resistance to pathogen-derived inhibitors of TAP. To broadly assess TAP-independent expression within the HLA-B locus, expression levels of 27 frequent HLA-B alleles were tested in cells with deficiencies in TAP. Approximately 15% of tested HLA-B allotypes are expressed at relatively high levels on the surface of TAP1 or TAP2-deficient cells and occur in partially peptide-receptive forms and Endoglycosidase H sensitive forms on the cell surface. Synergy between high peptide loading efficiency, broad specificity for peptides prevalent within unconventional sources and high intrinsic stability of the empty form allows for deviations from the conventional HLA-I assembly pathway for some HLA-B*35, HLA-B*57 and HLA-B*15 alleles. Allotypes that display higher expression in TAP-deficient cells are more resistant to viral TAP inhibitor-induced HLA-I down-modulation, and HLA-I down-modulation-induced NK cell activation. Conversely, the same allotypes are expected to mediate stronger CD8⁺ T cell responses under TAP-inhibited conditions. Thus, the degree of resistance to TAP inhibition functionally separates specific HLA-B allotypes.</p></div

RIT HLA-B allotypes are more resistant to TAP inhibition than non-RIT HLA-B allotypes.

<p>(A and B) CEM cells or (C and D) K562 cells expressing different exogenous HLA-B allotypes were infected with a HA-tagged BNLF2a-encoding retrovirus or retrovirus lacking BNLF2a (vector). BNLF2a expression levels were assessed by flow cytometry after intracellular staining with monoclonal anti-HA antibody and normalized to MFI values obtained from CEM or K562 cells lacking exogenous HLA-B (labeled vec) infected with the BNLF2a-encoding retrovirus (A and C). Cell surface HLA-B was measured by flow cytometry after staining with W6/32. The MFI ratios in cells expressing or lacking BNLF2a were calculated (BNLF2a/vector) (n = 3 measurements for each HLA-B expressing CEM cells or K562 cells from one infection with BNLF2a-encoding retrovirus) (B and D).</p

Allotype-dependent variations in cell surface HLA-B expression in TAP-deficient cells.

<p>(A and B) Cell surface HLA-B levels in SK19 (TAP1-deficient) or STF1 (TAP2-deficient) cells infected with retroviral constructs encoding indicated HLA-B were expressed as mean fluorescence intensity (MFI) ratios relative to those obtained for infections with an empty retroviral vector lacking HLA-B (vec). Data are derived from 4–13 (A) or 2–9 (B) flow cytometric measurements with the W6/32 antibody following 2–5 (A) or 1–4 (B) separate retroviral infections. (C) The MFI ratios for HLA-B allotypes from SK19 cells (Panel A) correlate with those from STF1 cells (Panel B). (D) SK19 cells were also infected with retroviruses encoding HA-tagged versions of HLA-B (HA-HLA-B). Cell surface expression levels of HLA-B molecules were tested by flow cytometry after staining with anti-HA. Data are derived from 3 measurements following one 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.</p

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_(+peptide)–MFI HLA-I_(-peptide)) / MFI HLA-I_(+peptide)*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="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" 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="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g002" 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

RIT HLA-B allotypes of the Bw4 group are more efficient in inhibiting KIR3DL1⁺ NK cell activation in the presence of the viral TAP inhibitor BNLF2a.

<p>HLA-I deficient K562 cells infected with retrovirus encoding exogenous HLA-B*44:03 and HLA-B*57:03 or retrovirus lacking HLA-B (vector) were chosen and further infected with a BNLF2a-encoding retrovirus or retrovirus lacking BNLF2a (vector). Intracellular BNLF2a expression levels (A) and cell surface expression of HLA-B were assessed by flow cytometry (B). HLA-B*44:03 expression is more strongly reduced by BNLF2a than HLA-B*57:03 (one representative experiment of three measurements is shown). (C) K562 cell based NK cell activation assay was performed with PBMCs from three different donors (D187, D136 and D215). CD3^-CD56⁺KIR3DL1⁺ cells were gated and NK cell activation was assessed by quantifying IFN-γ expressing population. One representative dataset from two experiments is shown.</p

TAP1-dependencies of HLA-B expression.

<p>(A) Cells from one infection from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1A</a> were subsequently infected with a TAP1-encoding retrovirus. The MFI ratios in the presence and absence of TAP1 (+TAP1/-TAP1) were calculated for each HLA-B–expressing cell line (n = 4 analyses from one infection). (B) Correlation between surface HLA-B expression in SK19 cells (calculated from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1A</a>) and their +TAP1/-TAP1 MFI ratios (calculated from Fig 2A). (C) Cells from the infections shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1D</a> were subsequently infected with a TAP1-encoding retrovirus. The MFI ratios in the presence and absence of TAP1 (+TAP1/-TAP1) were calculated for each HA-HLA-B–expressing cell line (n = 4 analyses from one infection). (D) Parental and TAP1-knockdown Hela cells (Hela-TAP1-KD) were assessed by immunoblotting with the anti-TAP1 antibody 148.3 (inset panel; 5, 10 or 20 μg of cell lysate was loaded in each lane) and infected with retroviruses encoding HLA-B allotypes or a control retrovirus (vector). TAP1 expression levels were measured by flow cytometry after intracellular staining with 148.3 antibody. (E) HLA-B expression levels at the surface of Hela or Hela-TAP1-KD cells were measured after W6/32 staining. The MFI ratios (Hela-TAP1-KD/Hela) were calculated for each HLA-B–expressing cell line (n = 6–7 measurements from three separate infections of Hela or Hela-TAP1-KD cells with retroviruses encoding indicated HLA-B). 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.</p

Fig 2

Fig

Cross-presentation of a peptide antigen.

<p>(A) 10 µM calreticulin (CRT) or bovine serum albumin (BSA) were incubated with 10 µM peptide (QLESIINFEKLTE-FITC). Free peptide was removed using a centrifugal filter device at 4°C, and peptide still in complex with CRT or BSA was measured (left panel). CRT- or BSA-peptide complexes were incubated with BMDC and B3Z cells. IL-2 production was determined by ELISA of the supernatants after 24 hours (right). Peptide concentration is indicated; CRT or BSA were present at a final concentration of 1 µM. (B) Cross-presentation of free peptide or CRT-peptide complexes was measured as in <i>A</i>. Data are representative of two independent analyses for both <i>A</i> (right panel) and <i>B</i>. Mean ± s.e.m. are shown in A and B.</p