Regulation of Class II Expression in Monocytic Cells after HIV-1 Infection

Human macrophage hybridomas

Human macrophage hybridomas were obtained by fusing macrophages (obtained by allowing monocytes to mature into macrophages in Teflon bag cultures) with a hypoxanthine-guanine phosphoribosyltransferase-deficient promonocytic line (U937), as previously described (21). We have uniformly infected and characterized one clone, 43, with different strains of HIV-1 (43_HIV) (25).

Monocyte isolation

Mononuclear cells were separated from buffy coats obtained from normal healthy volunteers by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation. The cells were washed three times with sterile PBS and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Life Technologies), 2 mM l-glutamine, and 1% penicillin/streptomycin (Life Technologies), henceforth called complete medium (CM). Freshly isolated PBMCs were incubated at 37°C in CM and allowed to adhere for 45 min. The nonadherent cells were removed, and adherent cells were washed with sterile PBS, harvested with a rubber policeman, and stained with monocyte-specific anti-CD14 mAbs to assess the purity of the preparation. Ninety percent of the isolated cells expressed CD14 (21).

HIV-1 infection

Monocytes or clone 43 cells were infected with HIV-1_ADA, HIV-1_87.9, HIV-1_BaL, and HIV-1_IIIB, respectively, as previously described (25). These reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Bethesda, MD). Dilutions of HIV-1-containing supernatant standardized to contain reverse-transcriptase activity (80,000 cpm/ml) were incubated for 90 min, followed by three washes with PBS.

DNA-binding studies

Oligonucleotides were synthesized for the W/Z/S, X-1, X-2, and Y box-binding studies by Integrated DNA Technologies (Coralville, IA) and labeled with ³²P dCTP, dATP, dGTP, and dTTP (ICN Pharmaceuticals, Irvine, CA) using DNA polymerase I (Sigma, St. Louis, MO) (32). Nuclear protein was extracted from 43 and 43_HIV, and monocytes were infected with HIV-1_BaL that retained (HLA-DR⁺) and lost (HLA-DR⁻) HLA-DR expression, according to the protocol of Dingham (33). Ten million cells were resuspended in 100 mM pepstatin A (Sigma), 10 mg/ml leupeptin (Sigma), 100 mM PMSF (Sigma), and 10 mg/ml aprotinin (Sigma). The cells were lysed with 20 mM HEPES (Sigma), pH 7.9, 1.5 mM MgCl₂ (Sigma), 10 mM KCl (Sigma), and 1 mM DDT (Sigma), and then centrifuged at 10,000 rpm for 30 s. The nuclei containing supernatant were added to 20 mM HEPES (Sigma), pH 7.9, 1.5 mM MgCl₂ (Sigma), 0.2 mM EDTA (Sigma), 20% glycerol (Sigma), 0.42 mM KCl (Sigma), and 1 mM DTT (Sigma); incubated on ice for 30 min; and then centrifuged for 5 min at 15,000 rpm. The nuclei were then added to the DNA oligonucleotides in Murphy’s binding buffer (BD PharMingen, San Diego, CA) for 2 h at room temperature. After binding, the mixture was electrophoresed on a 10% polyacrylamide gel, which was exposed to x-ray films (Kodak, Renseassler, NY) overnight and developed. In some experiments, the specificity of the binding was determined by adding 10 μg unlabeled oligonucleotide probes.

Surface and intracytoplasmic immunofluorescence

The HIV-1_BaL-infected monocytes, 43, and 43_HIV cells were stained by indirect methods, as previously described, with various mAbs (see below) or with isotype-matched controls, followed by affinity-purified F(ab′)₂ FITC-conjugated goat anti-mouse Ig Ab (Tago Scientific, Burlingame, CA), and analyzed by flow cytometry gating on live cells (21). The anti-class I Abs (W6/32) were obtained from the American Type Culture Collection (ATCC; Manassas, VA); anti-DR Abs, anti-class II-associated Ii peptide (CLIP), anti-CD80, and anti-CD86 Abs were purchased from BD Biosciences (Mountain View, CA); and the human polyclonal anti-HIV-1 Abs were a kind gift of A. Pinter (New York University, New York, NY). For intracytoplasmic staining, cells were fixed and permeabilized with 70% ethanol for 30 min at 4°C. The cells were then washed three times with PBS, and polyclonal anti-HIV-1 Abs or noninfected control Abs were added for 30 min at 4°C, followed by affinity-purified FITC-conjugated goat anti-human Ig Ab (Tago Scientific), and analyzed as described above (25).

RT-PCR for CIITA, RFX-5, hX-2BP, NF-YA, NF-YB, Ii, HLA-DMα, HLA-DRβ, and actin

Sequences for oligonucleotide primers used for PCR amplification and the size of the predicted PCR products are as follows: CIITA predicted size PCR product, 5′-CAACTCCCTGAAGGATGTGGA-3′, 350 bp, and 5′-ACGTCCATCACCCGGAGGGAC-3′; RFX-5, 5′-CGAGAATTCAGCTGTATCTCTACCTTC-3′, 586 bp, and 5′-GTCGAATTCAGGGAAGATCTCTCTGATG-3′; hX-2BP, 5′-ACCCCTAAAGTTCTGCTTCTGTCG-3′, 738 bp, and 5′-CATTAATGGCTTCCAGCTTGGCTG-3′; NF-YA,5′-GCAATAGTTCCACAGAGCAGATCG-3′, 929 bp, and 5′-CTAGGGATTTCTGCAGACTACATCGG-3′; NF-YB, 5′-ACTCGGATGATCTGTGTTCATGGCT-3′, 490 bp, and 5′-AGCCAGCTGGTAACTGGTTAGTGA-3′; Ii, 5′-TCCCAAGCCTGTGAGCAAGATG-3′, 410 bp, and 5′-CCAGTTCCAGTGACTCTTTCG-3′; HLA-DM-α, 5′-ACTTTTCCCAGAACACTCGG-3′, 341 bp, and 5′-CTGGAAGCTGAGTCCATC-3′; HLA-DM-β, 5′-ACAGCCACCTCAACCAAAAAGA-3′, 321 bp, and 5′-GGGGTTAAGGCTAAATGGGA-3′; and actin, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′, 661 bp, and 5′-CTAGAAGCATTGCGGTGGACGATGGAGGG-3′.

These primer sets have been previously published (27) and were synthesized by Integrated DNA Technologies. RNA was extracted from 43 and 43_HIV, HLA-DR⁺, and HLA-DR⁻ HIV-1_BaL-infected monocytes using acid guanidium thiocynate/phenol/chloroform, as described previously (RNAzol, Linnai, Dallas, TX) (34). Known quantities of RNA were mixed with 1 μg total cellular RNA and reverse transcribed at 37°C for 60 min in 20 μl buffer containing 10 mM Tris (Sigma) (pH 8.3); 50 mM KCl (Sigma); 5 mM MgCl₂ (Sigma); 1 mM each of dATP, dCTP, dGTP, and dTTP (Sigma); 20 U RNase inhibitor (Promega, Milwaukee, WI); 0.1 μg oligo(dT)₁₅ (Boehringer Mannheim, Indianapolis, IN); and 50 U murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Bethesda, MD). The PCR for RFX-5, hX-2BP, NF-YA, NF-YB, HLA-DM-α, HLA-DM-β, Ii, and actin was performed for 40 cycles at 94°C for 1 min, at 50°C for 1 min, and at 68°C for 3 min, with a 20-s elongation step per cycle (27). The amplification cycles were performed in the same tube for all of the X and Y box-binding proteins and for HLA-DM-α, HLA-DM-β, and actin. Reactions were stopped by heat inactivation for 10 min at 95°C, annealed for 2.5 min, and extended at 65°C. Negative controls were performed omitting RNA from the cDNA synthesis and specific amplification. PCR products were separated in a 2% NuSieve agarose (FMC, Rockland, ME) or a 5% polyacrylamide gel (23).

Transfection with NF-YA

The 43_HIV cells were transiently transfected with a bacterial plasmid containing NF-YA obtained through the ATCC using a DEAE-dextran method (35). The bacteria containing the NF-YA plasmid was cultured, ethanol precipitated, extracted, and then centrifuged and resuspended in TBS. The resuspended DNA was added to 10 mg/ml DEAE-dextran and incubated with the 43_HIV and 63_HIV cells for 4 h at 37°C. After aspirating the DEAE-dextran, the cells were shocked by adding 5 ml 10% DMSO (Sigma) in PBS for 1 min at room temperature, washed with sterile PBS, and resuspended in CM for 48 h at 37°C. In some experiments, the 43_HIV cells were treated with 10 mg/ml DEAE-dextran alone without the NF-YA DNA, while in other experiments the 43_HIV cells were treated with DEAE-dextran and jellyfish green fluorescent protein (Promega) to assess the efficiency of transfection.

Tetanus toxoid (TT)-induced T cell proliferation

Uninfected, HIV-1-infected (43_HIV), or HIV-1-infected NF-YA-transfected 43 and 63 cells (43_HIV + T) were used as accessory cells in TT-induced T cell proliferation assays. In these experiments, T cells were obtained from an HLA-matched donor (HLA-DR2⁺) and were monocyte depleted using a nylon wool column. The PBMC were incubated for 45 min and eluted. T cells obtained in this manner failed to proliferate to TT in the absence of accessory cells (23). Monocyte-depleted PBMC (10⁵) were cultured with varying concentrations of 43 and 43_HIV, as well as 43_HIV transfected with NF-YA (10³–10⁵) and TT (0.4–40 μg/ml). The cells were maintained in 0.2 ml CM in triplicate round-bottom plates (Linbro, Oxnard, CA) at 37°C in a 5% CO₂ incubator for 5 days. Eighteen hours before harvesting, 1 μCi [³H]thymidine (ICN Pharmaceuticals) was added to each well. The cells were harvested onto glass fiber filters, and incorporated radiolabel was measured by scintillation counting (23).

Annexin V

FITC-labeled annexin V, a phospholipid-binding protein of the annexin family, was used to measure apoptosis using a commercially available kit (Beckman Coulter, Hialeah, FL). After incubating 43 and 43_HIV with T cells, the cell samples were washed with ice-cold PBS, followed by centrifugation at 500 × g at 4°C. The cells were stained simultaneously with annexin V FITC/PE-labeled anti-CD3 mAb and incubated at room temperature for 10 min in the dark. The cells were then analyzed by flow cytometry to measure costaining of the CD3⁺ and the annexin V⁺ population, gating on the live cells (36).

Western blot

The 43 and 43_HIV cells were lysed using buffer containing PMSF (100 mM), aprotonin (10 mg/ml), leupeptin (10 mg/ml), iodoacetamide (1.8 mg/ml), and 0.1% Triton X. The lysates were resolved on a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, blocked with 5% milk in PBS at room temperature for 60 min, then incubated with the PIN.1 mAb (37) at 4°C overnight. A secondary horseradish-labeled goat anti-mouse Ig (Tago Scientific) was then added at 25°C for 2 h, and the blot was developed by chemiluminescence using a commercially available kit (DuPont Pharmaceuticals, Wilmington, DE).

Vaccinia virus infection

Vaccinia virus-containing vectors for HIV-1 envelope, gag, pol, and nef proteins were obtained from the AIDS Research and Reference Laboratory. Vaccinia virus was added to cultures of either uninfected 43 or 63 cells at a virus to cell ratio of 10⁸:1 that has been described previously (38). Successful infection was determined by intracytoplasmic immunofluorescence, as described above.

Colocalization studies

To colocalize HLA-DR in different subcellular compartments, we used mouse anti-cathepsin D (early endosomes) Ab (Accurate Antibodies, Westbury, NY), rabbit polyclonal anti-M6PR (late endosomes; provided by R. Dunn, University of Florida, Gainesville, FL), and mouse anti-HLA-DR (BD Biosciences). The 43 and 43_HIV cells and 43 cells infected with vaccinia virus containing different HIV-1 proteins were fixed with methanol/acetic acid (3/1) for 5 min at 4°C. Fixed cells were then blocked with PBS containing 5% goat serum and 0.2% Triton X-100 (Sigma) for 45 min before incubating with the primary Ab for 1 h. The cells were rinsed with PBS three times before incubating with Texas Red-conjugated Abs directed against either rabbit or mouse Ig for 30 min. Following this incubation, the cells were again washed three times with PBS and incubated with an FITC-labeled murine anti-HLA-DR Ab. The cells were rinsed with PBS, prepared as a cytospin, and mounted with Immu-mount (Shandon, Pittsburgh, PA) before being viewed by a Leica fluorovert laser-scanning confocal microscope (Leica, Deerfield, IL) at a step position of 1 μm on the x-y-axis or x-z-axis. To localize with 3-(2,4-dinitroanilino)-3′-N-methyldipropylamine) (DAMP), the HIV-1-infected and uninfected cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were then washed twice with 50 mM NH₄Cl (Sigma) for 5 min each before permeabilization with 0.02% Triton X-100 (Sigma) in PBS for 30 min. The cells were then incubated with rabbit polyclonal rabbit anti-DAMP Abs (Molecular Probes, Eugene, OR) for 60 min at room temperature before mounting with Immu-mount (Shandon) and viewing by confocal microscopy (25).

Decreased X and Y binding in the HIV-1-infected cells

Our previous studies demonstrated a loss of HLA-DR expression in the HIV-1-infected human macrophage hybridomas that could be overcome by transfecting HLA-DR-α and HLA-DR-β genes driven by a nonphysiologic CMV promoter (25). These findings suggested that HIV-1 infection had an effect on the regulation of HLA-DR mRNA production. Class II and HLA-DR mRNA production is regulated by different DNA-binding proteins that interact with a compact, conserved, multicomponent motif that contains four subelements, the W/Z/S box, the X-1 and X-2 boxes, and the Y box (39, 40, 41, 42, 43). We first investigated whether HIV-1 infection had any effect on the binding of these regulatory proteins by synthesizing oligonucleotides corresponding to the W/Z/S, X-1, X-2, and Y binding sites to use in gel-shift assays comparing HIV-1-infected with uninfected human macrophage hybridomas. We used one human macrophage hybridoma cell line (clone 43) for these experiments that lost HLA-DR expression after HIV-1 infection (22, 25). Nuclear protein extracts from 43 and 43_HIV were electrophoresed on a 5% polyacrylamide gel with ³²P-labeled or unlabeled oligoprobes (to assess the specificity of the DNA binding). In the 43 cells, W/Z/S, X-1, X-2, and Y box DNA binding was present and could be displaced by the unlabeled W/Z/S, X-1, X-2, and Y probes (Fig. 1⇓A). However, in the 43_HIV cells, there was no binding to either the W/Z/S, X-1, X-2, or Y boxes, respectively (Fig. 1⇓A). Because of the conflicting reports regarding the loss of HLA-DR expression in primary monocytes after HIV-1 infection (18, 19, 22, 25), we wanted to validate our findings in the 43_HIV cells using primary HIV-1_BaL-infected monocytes. In freshly isolated monocytes from normal blood donors, we detected decreased surface expression of HLA-DR in 3 of 10 preparations 8 days after infection with HIV-1_BaL (Table I⇓). Similar results were obtained when we infected the primary monocytes with HIV-1_ADA, HIV-1_87.9, or primary isolates from HIV-1-infected donors (data not shown). We separately pooled the nuclear extracts from the HLA-DR⁺ and HLA-DR⁻ HIV-1_BaL-infected monocytes and performed W/Z/S, X-1, X-2, and Y box-binding studies. Consistent with the findings in the 43_HIV cells, there was reduced or absent W/Z/S, X-1, X-2, and Y box DNA binding in the HLA-DR⁻ HIV-1_BaL-infected monocytes compared with the HLA-DR⁺ HIV-1_BaL-infected monocytes (Fig. 1⇓A). CIITA is a non-DNA-binding protein and serves as a coactivation factor for class II expression (41). Lack of CIITA expression has been reported in class II-deficient patients with the bare lymphocyte syndrome (BLS) (40, 41). Competition between Tat and CIITA for binding to the P-TEBFb transcription factor has also been reported in HIV-1-infected THP-1 cells that lost class II expression (26). Although DNA binding to W/Z/S, X-1, X-2, and Y boxes can be demonstrated in CIITA-deficient patients (44), it is still possible that CIITA production may be affected by HIV-1 infection in our system and may contribute to the loss of HLA-DR expression. CIITA production was assessed by PCR in 43 and 43_HIV cells and in the HLA-DR⁺ and HLA-DR⁻ HIV-1_BaL-infected monocytes. A 350-bp band corresponding to CIITA was detected in both the HIV-1-infected and uninfected cells (Fig. 1⇓B).

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FIGURE 1.

DNA-binding studies in 43 and 43_HIV cells and in HIV-1_BaL-infected monocytes. A, Nuclear protein was extracted from HIV-1-infected (43_HIV) and uninfected 43 cells, probed with ³²P-labeled oligonucleotides corresponding to the W/Z/S, X-1, X-2, and Y box bindings, and run on a 10% polyacrylamide gel. Unlabeled probe was added to assess the specificity of the DNA binding (43+competitor). The gels were dried and exposed to x-ray film overnight. The arrows represent DNA binding. This is a representative experiment repeated five times. B, PCR for CIITA. RNA was extracted from 43, 43_HIV, and HIV_BaL-infected monocytes (HLA-DR⁺ and HLA-DR⁻). The RNA was reverse transcribed, probed with a CIITA or an actin-specific probe, and then run on a 5% agarose gel. Negative controls were performed omitting the RNA from the cDNA synthesis and amplification step. This is representative of an experiment repeated five times.

Production of X and Y box-binding proteins

Because there was no DNA binding to the W/Z/S, X-1, X-2, and Y boxes, we investigated whether there was loss of the W/Z/S, X-1, X-2, or Y box-binding proteins in 43_HIV, and in the HLA-DR⁻ HIV_BaL-infected monocytes. Because many DNA-binding proteins have been found to associate with class II regulatory sequences (45, 46, 47, 48, 49, 50, 51, 52, 53), we only assayed for those binding proteins that have been associated with the loss of class II expression in BLS or that cooperatively interact with other binding proteins (54). To this end, we determined the presence or absence of four DNA-binding proteins: RFX-5 (W/Z/S, X-1), hX-2BP (X-2), NF-YA, and NF-YB (Y box) by PCR in the 43_HIV cells. Amplified fragments corresponding to RFX-5 (504 bp)-, hX-2BP (738 bp)-, and NF-YB (490 bp)-binding proteins could be demonstrated in the uninfected 43 and 43_HIV cells (Fig. 2⇓). However, a 926-bp fragment, corresponding to NF-YA, was absent in the 43_HIV cells. We again wanted to validate these findings in the HLA-DR⁻ HIV_BaL-infected monocyte preparations. Fragments corresponding to RFX-5 (504 bp), hX-2BP (738 bp), and NF-YB (490 bp) DNA-binding proteins were present, but there was no detectable NF-YA (926 bp) (Fig. 2⇓). Because the loss of NF-YA could potentially explain the loss of HLA-DR after HIV-1 infection in our system, we transfected the 43_HIV cells with NF-YA in an attempt to restore expression.

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FIGURE 2.

PCR for X and Y box-binding proteins. RNA was isolated from 43 and 43_HIV cells and HIV_BaL-infected monocytes (HLA-DR⁺ and HLA-DR⁻), reverse transcribed, and probed with RFX-5, hX-2BP, NF-YA-A, NF-YB, and actin-specific probes, and then run on a 5% agarose gel. This is representative of an experiment repeated five times.

Transfection of 43_HIV with the NF-YA DNA box-binding protein

We transfected full-length cDNA for the NF-YA DNA-binding protein driven by a CMV IE promoter into 43_HIV cells using DEAE-dextran. The surface expression of HLA-DR was lost in the 43_HIV cells 2 wk after infection, but was transiently restored after transfection with NF-YA (Fig. 3⇓). Because the NF-YA-transfected 43_HIV cells expressed HLA-DR, we investigated whether these were functional by assessing whether they could present TT to HLA-matched (HLA-DR2⁺) responder T cells. APC-depleted T cells and TT (40 μg/ml) were cocultured in the presence of varying concentrations (10³-10⁵) of either 43 and 43_HIV cells or NF-YA box-transfected 43_HIV cells. In the absence of accessory cells, there was no T cell proliferation in response to TT (Fig. 4⇓). Uninfected 43 cells induced a TT-specific proliferative response in the HLA-DR2⁺ T cells, inducing increases in thymidine incorporation, whereas 43_HIV failed to induce any T cell proliferation. After transfection with NF-YA, the 43_HIV cells were still unable to induce T cell proliferation in response to TT (Fig. 4⇓). One possible explanation is loss of CD80 and CD86 expression in the 43_HIV cells. However, the surface expression of CD80 and CD86 was unchanged in the 43_HIV cells compared with the uninfected 43 cells (Fig. 5⇓). We have reported that after prolonged HIV-1 infection (35 days), 43_HIV can induce apoptosis in cocultured T cells through gp120, Fas ligand, and the production of a soluble proapoptotic factor (22, 36). Even though the 43_HIV cells were infected for only 2 wk, we performed annexin V staining to ensure that apoptosis of the cocultured T cells didn’t account for the lack of a T cell response. There was no difference in annexin V staining in the T cells cocultured with 43_HIV or 43_HIV cells transfected with NF-YA compared with T cells cultured with the uninfected cells (data not shown). These findings are in line with our previous studies, which demonstrated that HLA-DR-transfected 43_HIV cells that expressed surface HLA-DR were still incapable of inducing an Ag-specific T cell response (25). Furthermore, in colocalization experiments, we demonstrated a diminished capacity to form HLA-DR-Ag complexes (25). The HLA-DR molecules assemble in the endocytoplasmic reticulum associated with Ii, a nonpolymorphic class II gene product that is coordinately expressed with HLA-DR and targets the molecule to either the late endosome or lysosome (55). Decreased production of Ii in the 43_HIV cells may explain the inability of the cells to induce an Ag-specific T cell response in our system. We performed PCR for Ii production in 43_HIV cells and in the HLA-DR⁺ and HLA-DR⁻ HIV-1_BaL-infected monocytes to determine whether HIV-1 infection also blocked Ii mRNA production.

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FIGURE 3.

Transfection of NF-YA-binding protein. The 43_HIV cells were transfected with NF-YA cDNA using DEAE-dextran and were then stained with FITC-labeled anti-HLA-DR mAbs. The cells were analyzed by flow cytometry, gating on the live cells. The shaded areas represent staining with the isotype control Abs. The percentage of cells stained with anti-HLA-DR Abs is indicated in the upper right corner. This is representative of an experiment repeated five times.

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FIGURE 4.

The NF-YA-transfected 43_HIV cells fail to present Ag to TT-specific T cells. HLA-DR2⁺ TT-specific, monocyte-depleted T cells were incubated with TT (40 μg/ml) (T+TT), 43 (T+43), 43 plus TT (T+TT+43), 43_HIV plus TT (T+TT+43HIV), and NF-YA-transfected 43_HIV cells plus TT (T+TT+43HIV+T). The cells were maintained in CM at 37°C for 5 days, and 1 μCi [³H]thymidine was added to the cultures during the last 16 h. The y-axis represents incorporated thymidine. These data represent experiments that were repeated three times.

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FIGURE 5.

Expression of CD80 and CD86 on 43_HIV cells. The 43 cells and 43_HIV cells transfected with NF-YA were stained with FITC-labeled anti-CD80 and CD86 mAbs and examined by flow cytometry with gating on live cells. The shaded areas represent staining with isotype control Abs, and the percentage of cells stained with FITC-labeled anti-CD80 and CD86 Abs is indicated in the upper right corner. This is representative of an experiment repeated three times.

Expression of Ii, HLA-DM-α, and HLA-DM-β in 43_HIV and 63_HIV

We extracted RNA from 43 and 43_HIV cells and from the HLA-DR⁺ and HLA-DR⁻ HIV-1_BaL-infected monocytes to amplify Ii-specific base pair fragments (410 bp) (Fig. 6⇓) by PCR. Bands (410 bp) corresponding to Ii were detected in 43 and 43_HIV as well as in the HIV_BaL HLA-DR⁺- and HLA-DR⁻-infected monocytes (Fig. 6⇓). Other HLA gene products (HLA-DM-α and HLA-DM-β) are also coordinately expressed with Ii and are essential in Ag processing (55). HLA-DM-α and HLA-DM-β further cleave antigenic peptides in subcellular processing compartments, allowing for HLA-DR binding permitting Ag recognition by T cells (56). In the processing compartments, Ii is degraded into several peptides, including CLIP and leupeptin-induced protein (LIP). CLIP binds to the Ag-binding groove of the class II molecules and is removed first before endocytically generated antigenic peptides can bind. The removal of CLIP is catalyzed by HLA-DM. LIP is a 14-kDa NH₂-terminal fragment of the Ii that accumulates in the presence of the protease inhibitor leupeptin (37). Impaired production of HLA-DM-α and HLA-DM-β could possibly explain the failure to induce T cell proliferation in response to TT in the 43_HIV cells. We determined by PCR whether HLA-DM-α and HLA-DM-β were present in 43, 43_HIV, and in HLA-DR⁺ and HLA-DR⁻ HIV_BaL-infected monocytes. Similar to the results obtained with Ii, there was no difference in expression of either HLA-DM-α (341 bp) or HLA-DM-β (321 bp) in 43, 43_HIV, or in the HLA-DR⁺ and HLA-DR⁻ HIV-1_BaL-infected monocytes (Fig. 5⇑). However, the Ii breakdown products CLIP and LIP were present in the uninfected 43 cells, but absent in the 43_HIV cells (Fig. 7⇓, A and B). The PIN.1 mAb identifies not only LIP, but also the α-chain (molecular mass 30 kDa) and β-chain (molecular mass 28 kDa) of HLA-DR, which are present in both the 43 and 43_HIV cells. The presence of Ii and HLA-DM-α and HLA-DM-β in the 43_HIV cells in the absence of Ii breakdown products suggested a sorting defect.

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FIGURE 6.

PCR for Ii, HLA-DM-α, and HLA-DM-β. RNA was isolated from the 43, 43_HIV, and HIV-1_BaL HLA-DR⁺ and HLA-DR⁻ monocytes. The RNA was reverse transcribed, probed with HLA-DM-α, HLA-DM-β, Ii, and actin-specific probes, and then run on a 5% agarose gel. This is representative of an experiment repeated five times.

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FIGURE 7.

Ii breakdown products in the 43 and 43_HIV cells. A, The presence of CLIP in the 43 and the 43_HIV cells was demonstrated by intracytoplasmic staining with FITC-labeled anti-CLIP mAbs with gating on live cells. The shaded areas represent staining with isotype control Abs. The percentage of cells stained with CLIP is indicated in the upper right corner. This is representative of an experiment repeated three times. B, Presence of LIP in the 43 and 43_HIV cells. Lysates from the 43 and 43_HIV cells were run on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane, blotted with the anti-PIN.1 mAb, and then developed. This is representative of an experiment repeated three times.

Impaired class II sorting in 43_HIV cells

To follow the sorting of HLA-DR into subcellular trafficking compartments, we permeabilized uninfected 43 cells along with the NF-YA-transfected 43_HIV cells and stained with FITC-labeled anti-HLA-DR Abs and Texas Red-labeled anti-early endosome (cathepsin D), anti-late endosome (M6PR), anti-lysosome (CD63), and anti-acidic compartment Abs (DAMP). In the uninfected 43 cells, HLA-DR was present (yellow staining) in the late endosomes, acidic compartments, and lysosomes (Fig. 8⇓). However, in the NF-YA-transfected 43_HIV cells, HLA-DR did not localize into any subcellular compartment (Fig. 8⇓). It is possible that there was competition between HIV-1 peptides and normal endosomal and lysosomal targeting that may have prevented HLA-DR sorting. To address this possibility, we introduced env, gag, pol, and nef proteins into uninfected 43 cells using vaccinia virus that contained vectors for different HIV-1 proteins, and then attempted to colocalize HLA-DR in the early and late endosomes, lysosomes, and acidic compartments.

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FIGURE 8.

Colocalization of HLA-DR in different trafficking compartments in 43 and 43_HIV cells. The NF-YA-transfected HIV-1-infected and uninfected 43 cells were stained intracytoplasmically with FITC-labeled anti-HLA-DR and Texas Red-labeled anti-cathepsin D (early events), anti-DAMP (acidic compartments), anti-M6PR (late endosomes), and anti-CD63 (lysosomes) Abs. The cells were then examined by laser confocal microscopy. Yellow staining (red plus green equals yellow) represents colocalization. Two observers routinely examined 10 separate fields. This is representative of an experiment repeated five times.

Effect of HIV-1 proteins on class II sorting in 43_HIV cells

To ensure that there were comparable amounts of the env, gag, pol, and nef proteins in the 43 cells before performing the colocalization experiments, we stained the cells intracytoplasmically with FITC-labeled human polyclonal anti-HIV-1 Ab (35). Using an empty vaccinia virus vector as a negative control, peak channel shifts corresponding to the presence of intracytoplasmic env, gag, pol, and nef proteins were detected to a comparable degree in the 43 cells after staining (Fig. 9⇓). We then repeated the colocalization studies with FITC-labeled anti-HLA-DR Abs and the Texas Red-labeled anti-cathepsin D, anti-M6PR, anti-lysosome, and anti-DAMP Abs. HLA-DR localized (yellow staining) in the late endosomes, lysosomes, and acidic compartments in the 43 cells infected with the empty vaccinia virus (Fig. 10⇓). HLA-DR also localized in the same compartments in the 43 and 63 cells that were infected with vaccinia virus containing the pol, gag, and nef proteins (Fig. 8⇑). However, in the 43 cells infected with vaccinia virus that contained the env proteins, HLA-DR did not localize in any of the trafficking compartments (Fig. 8⇑).

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FIGURE 9.

Presence of HIV-1 proteins in vaccinia virus-infected 43 cells. Vaccinia virus containing HIV-1 pol, gag, nef, and env was cultured with uninfected 43 and 63 cells at a virus to cell ratio of 10⁸:1. The presence of different HIV-1 proteins in the cells was determined by intracytoplasmic staining with polyclonal human anti-HIV-1 Abs, followed by flow cytometry with gating on live cells. The shaded areas represent staining with control isotype Abs. The percentage of cells stained with the anti-HIV-1 Abs is indicated in the upper right corner. This is representative of an experiment repeated three times.

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FIGURE 10.

Colocalization of HLA-DR in subcellular compartments. The 43 cells containing an empty vector and the pol, gag, nef, and env HIV-1 proteins (vaccinia virus infected) were stained intracytoplasmically with Texas Red-labeled anti-cathepsin D (early endosomes), anti-DAMP (acidic compartments), anti-M6PR (late endosomes), anti-CD63 (lysosomes) Abs, and FITC-labeled anti-DR Abs. The cells were examined by laser confocal microscopy. Yellow staining (red plus green equals yellow) represents colocalization. Two observers routinely examined 10 separate fields. This is representative of an experiment repeated five times.