Peptide presentation is reduced by Nef expression. HeLa-CIITA cells were transfected with CD8stop in combination with the indicated plasmids. CD8⁺ sorted cells were assayed for presentation of the HA peptide to the THA1.7 T cell line specific for HLA-DR1/HA complexes. T cell responses, reflected by proliferation of the CTL-L2 cells, are plotted as a function of peptide concentration. Error bars are calculated for triplicates of one representative experiment.

Characterization of Nef effects on MHC II and Ii surface expression. (A) HeLa-CIITA cells transfected with Nef-FT (red lines) or Nef-mock (blue lines) and pEGFP were either untreated (thin lines) or exposed to 20 nM ConB overnight (bold lines) and stained for the indicated markers. Analysis of GFP⁺ cells was performed as in Fig. 2. (B) Nef-FT dose-response curves. HeLa-CIITA cells transfected with pEGFP and increasing amounts of the Nef-FT plasmid were stained with anti-Ii and anti-mature MHC II mAbs. MFI values of GFP⁺ gated cells were determined for surface Ii and mature MHC II in FL-2. The background MFI value (obtained with a control mAb, PIN1) was subtracted from these MFI values, which were plotted as a function of the amount of the Nef-FT plasmid transfected. (C) HeLa-CIITA cells transfected with pEGFP and Nef-A01 (bold black line), Nef-G2A (orange line), or Nef-mock (blue line) were stained and analyzed by flow cytometry as in Fig. 2. The 9E10 mAb was used as a negative control (dotted lines).

Mutational analysis of Nef. (A) HeLa-CIITA cells were cotransfected with the plasmids encoding Nef-wt, Nef-mock, or the different Nef mutants in combination with the plasmids encoding CD4 and EGFP. Cells were processed for cytofluorimetry, and GFP⁺ cells were analyzed as in Fig. 2. Immature MHC II complexes were followed with the anti-Ii mAb LN2 and mature MHC II complexes with the mAb L243. Relative activities of Nef mutants on CD4, MHC I, and mature and immature MHC II were calculated as percentage of Nef-wt activity in comparison to the activity of the Nef-mock control plasmid (0%). Nef from the LAI strain (FT-wt) was used as reference (100% of effect) for Nef_LL165GG, as this mutant has been derived from this allele. The other three mutants (Nef_WL58AA, Nef_EEEE65AAAA, and Nef_PP75/78AA) have been derived from the Nef allele HXB2, thus HXB2-wt was taken as reference (100% of effect) for these mutants. Nef_WL58AA induced a higher down-modulation of surface MHC I than Nef-wt (181%) as previously reported (16). The figure shows mean values and their standard deviations calculated from the data generated in four independent experiments. (B) Western blot analysis of lysates of cells transfected with the indicated wild type or mutants of Nef. The equivalent of 1 × 10⁵ cells was loaded per lane.

Nef down-modulates surface-mature MHC II and induces an increase of surface Ii expression. (A) FACS analysis of HeLa-CIITA cells transfected with Nef-FT or Nef-mock in combination with the plasmids encoding CD4 and EGFP. MFI values of phycoerythrin fluorescence of GFP⁺ cells were subtracted of the negative control value and plotted for each indicated marker. The data shown represent mean values of four independent experiments. (B) FACS profiles of HeLa-CIITA or MelJuSo cells transiently transfected with the indicated plasmids, each in combination with the pEGFP plasmid (see Materials and Methods). Flow cytometry histograms of GFP⁺ gated cells are presented for phycoerythrin fluorescence. An irrelevant mAb, 9E10, was used as a negative control (dotted lines). (C) Localization of Ii and mature MHC II by confocal microscopy of HeLa-CIITA cells transfected with the Nef-FT and pEGFP plasmids. Transfected cells are identified by GFP expression.

Biochemical characterization of surface MHC II complexes. Cell-surface biotinylation followed by parallel and sequential immunoprecipitations were performed with HeLa-CIITA cells transfected with Nef-FT, Nef-mock, or nontransfected cells exposed to 20 nM ConB overnight. Immunoprecipitates performed with mAbs specific for the indicated proteins were eluted at 95°C (except RT, which means eluted at room temperature) and revealed with streptavidine–peroxidase by Western blot. Below the Ii panel, a longer exposure of the same blot is presented. The Ii* panel corresponds to immunoprecipitates performed with the anti-Ii mAb PIN1 on lysates that had been precleared with the anti-HLA-DR mAb TU36. A short exposure of the MHC II panel obtained by elution at room temperature of immunoprecipitations performed with TU36 is presented to allow visualization of the different levels of SDS-stable complexes, but Ii bands clearly appeared on longer exposures (not shown). Total lysates from the same cells were analyzed by Western blot by using either the anti-HLA-DRα mAb 1B5 or the anti-Nef mAb MATG 020.