Intracellular trafficking of HA-HLA-B*35 during HIV infection. (A) HIV infection: CEM cells expressing HA-HLA-B*3501 and HA-HLA-B*3503 were infected with HIV-1 encoding placental alkaline phosphatase (PLAP). Percentage infected cells are indicated. (B) CEM cells from part A or uninfected cells were pulse la(beled, chased for the indicated time in minutes, immunoprecipitated with W6/32, followed by a secondary immunoprecipitation with anti-HA, and then digested with Endo H or left undigested, and analyzed by SDS-PAGE and phosphorimaging analyses. (C) Percentage Endo H resistant heavy chains were quantified from the gels shown in the (B). Similar trends as in C were observed in a second experiment which had a lower percentage of infected cells (~50%).

Surface expression of indiacted HA-HLA-B allotypes in the tapasin deficient cell line M553. (A) M553 cells expressing the indicated HLA-B allotypes were stained with anti-HA (top panels) or with mAb W6/32 (lower panels) followed by FITC conjugated secondary antibody staining and analyzed by FACS.

Intrinsic peptide loading efficiencies of soluble HLA-B*3501 and HLA-B*3503 in insect cells or as purified proteins. Sf21 cells were infected with baculoviruses encoding the indicated soluble HLA-B*35/β₂m constructs, metabolically labeled, and thermostability analyses conducted in the absence (−) or presence of (A) a randomized nonamer library at the indicated concentrations, (B) randomized nonamer library with a proline at position 2 at the indicated concentrations, (C) randomized 11mer library with a proline at position 2 and methionine at position 11 at the indicated concentrations, (D) 0, 10, or 100 μM LPSSADEVF as indicated, (E) 0, 10 or 100 μM LPSSADEVFCL as indicated, (F) 0 or 100 μM YPLHEQHGM, or YFLHEQHGM as indicated. Lysates were incubated at 4° or 37° degrees for 12 minutes, followed by immunoprecipitation analyses with w6/32. Samples were separated by SDS-PAGE and proteins visualized by phosphorimaging analyses. (Upper panels) Representative images of heavy chains recovered at 4 °C and 37 °C in the absence or presence of peptide, following SDS-PAGE. (Lower panels) Quantifications of ratios of heavy chains recovered at 37 °C relative to 4°C under the indicated condition. Error bars represent the standard error of the mean (SEM) percentages. Data are the average of 3, 2, 4, 2, 2, and 2 experiments respectively for (A–F). Purified HLA-B*35 (2 μM) and (G) LPSC_FADVEF (0.2 μM) or (H) YPLK_FEQHGM (H; 0.2 μM) or (I and J) XPXK_FXXXXM at the indicated concentrations were incubated at 37 °C for (I) 1 or (J) 2 hr. Mixtures were separated by native-PAGE and protein-peptide complexes were quantified by fluorimaging analyses of gels. Representative fluorescent scans of native gels are shown in the upper panels and lower panels display quantifications of fluorescent bands as percentage fluorescence relative to the maximum fluorescence within each experiment. Data is the average of two independent analyses and the error bars represent the standard error of the mean percentage fluorescence.

Effect of HLA Genotypes (HLA-B*5701, HLA-B*3501, HLA-B*3503 and HLA-B*4402) on progression to four AIDS-Related End Points. For each indicated outcome, relative hazards (HR), confidence intervals (CI), and significance were calculated for a dominant model as previously described (Gao et al. 2001; O’Brien et al. 2001). The ranges in sample size and p values were as follows: B*5701 (N = 75–78), p = 0.00003–0.003 for the four outcomes indicated (significantly associated with slow AIDS progression for all AIDS outcomes); B*4402 (N = 128–137), p = 0.22–0.62 for the four outcomes indicated; B*3501 (N = 117–123), p = 0.07 (CD4<200), 0.13 (AIDS 87), 0.03 (AIDS 93) and 0.13 (Death) (significantly associated with rapid AIDS progression only by the AIDS 93 criteria); B*3503 (N = 40–43) p = 0.004 (CD4<200), 0.001 (AIDS 87), 0.01 (AIDS 93) and 0.004 (Death) (significantly associated with rapid AIDS progression for all four AIDS outcomes). HLA-B*4405 is a very rare allele in the study cohort, and its effect on AIDS progression could not be studied.

Maturation rates of folded MHC class I heterodimers. CEM T cells were metabolically labeled for 10 minutes and chased for the indicated times in fresh media. Lysates were immunoprecipitated with mAb W6/32 followed by a secondary anti-HA immunoprecipitation then digested with Endo H or left undigested, and analyzed by SDS-PAGE and phosphorimaging analyses. Upper panels: Representative gels for indicated HA-HLA-B trafficking. Lower panels: Percent Endo H resistant heavy chain bands (Endo H resistant band/Endo H resistant + Endo H sensitive) were quantified from gels such as those shown in the upper panels. Data is the average of three independent measurements for HA-HLA-B*4402 and two independent measurements for HA-HLA-B*4405, HA-HLA-B*3501, HA-HLA-B*3503 and HA-HLA-B*5701. R indicates Endo H resistant MHC class I heavy chain band, and S indicates Endo H sensitive MHC class I heavy chain band.

TAP binding profiles of HA-HLA-B allotypes in CEM cells. Lysates from CEM cells expressing the indicated class I were directly loaded (top panel), or proteins first immunoprecipitated with anti-TAP1 antisera (lower panel), separated by SDS-PAGE, and immunoblotting analyses undertaken with an anti-HA antibody. NS denotes a non-specific band. Data is representative of at least two independent sets of analyses for each class I. The dotted line indicates where different lanes from the immunoblots have been juxtaposed. The solid line indicates where different immunoblots were juxtaposed.

Tapasin dependence of the cell surface expression of HA-HLA-B allotypes. (A and C) M553 cells expressing the indicated class I molecules alone, co-expressing both tapasin and the indicated class I molecule or uninfected M553 control cells were stained with anti-HA and FITC-conjugated secondary antibodies followed by flow cytometric analyses to assess MHC class I cell surface expression. (A) Cell surface expression of indicated class I in the absence of tapasin were represented as ratios of mean fluorescence values relative to the parent uninfected M553 cells (following staining with anti-HA and FACS analyses). Data are the average of 8 independent analyses for HA-HLA-B*4405 from two separate infections, 5 independent analyses of a single infection for HA-HLA-B*4402, 8 independent analyses of three separate infections for HA-HLA-B*3501 and HA-HLA-B*3503, and 5 separate analyses from a single infection for HA-HLA-B*5701. (B) Cells used for the analyses in panels A and C were lysed and immunoblotting analyses was undertaken with anti-HA in the top panel and anti-tapasin in the lower panel. For the anti-HA immunoblots all cells were compared in the same set of blots; for the anti-tapasin analyses, separate blots are indicated by spaces. The arrow indicates the location of a non-specific band. (C) Tapasin induced expression of the indicated class I were represented as ratios of the mean fluorescence of anti-HA staining in the presence of tapasin/anti-HA staining in the absence of tapasin. Data are the average of 5 independent analyses from two separate infections for HA-HA-HLA-B*4405, HA-HLA-B*4402, HA-HLA-B*3501, and HA-HLA-B*3503, and two independent analyses from two separate infections for HA-HLA-B*5701. In A and C, error bars represent the standard error of the mean ratios.

Peptide loading efficiencies of HA-HLA-B*3501 and HA-HLA-B*3503 in CEM cells: Upper panel: CEM cells expressing HA-HLA-B*3501 or HA-HLA-B*3503 were metabolically labeled for 10 minutes and, lysed immediately or chased with fresh media for 4 hours, and lysates incubated at 4°, 37°, or 50° degrees for 12 minutes, followed by sequential immunoprecipitations with primary W6/32 and secondary anti-HA antibodies. Samples were separated by SDS-PAGE and proteins visualized by phosphorimaging analyses. Lower panel represents the quantification of percentage heavy chains recovered at 37 °C or 50 °C relative to 4 °C. Data are averages of 2 independent analyses with error bars representing the standard error of the mean.