Identification of a Conserved Universal Th Epitope in HIV-1 Reverse Transcriptase That Is Processed and Presented to HIV-Specific CD4⁺ T Cells by at Least Four Unrelated HLA-DR Molecules

Antigens

Peptides were synthesized by solid-phase strategies on an automated multiple peptide synthesizer (Abimed AMS 422, Langenfeld, Germany). Peptides were analyzed by reverse phase HPLC, dissolved in 10–20 μl DMSO, diluted in PBS to a final concentration of 2.5 mg/ml, and stored at −20°C. The peptides that were used in the lymphocyte stimulation assay were of the following purity: RT_171–190, 80%; RT_271–290, 70%; RT_251–270, 50%; RT_301–320, 80%; and RT_431–450, 80%.

RT protein was obtained by subcloning the gene coding for HIV-1 RT_HXB2 from its original vector pHRTRX2 (32) into pET-19b. Correct insertion of the RT sequence was confirmed using the T7 sequencing kit (Pharmacia, Roosendaal, The Netherlands). RT protein was expressed as a fusion protein containing 10 histidine residues plus a 13-amino acid linker attached to its amino terminus. For overproduction, the Escherichia coli strain BL21 (DE3) was used, in which the T7 RNA polymerase is under the control of the Lac promoter (33). At OD₆₀₀ 0.6 overproduction was induced with 1 mM isopropyl β-d-thiogalactoside. After 5 h bacteria were collected by centrifugation, and the pellet was washed with 50 mM sodium phosphate, pH 8, and 300 mM NaCl. Pellets were subsequently stored at −20°C until purification. Proteins were purified by nickel-chelate affinity chromatography according to the recommendations of the supplier (Qiagen, Chatsworth, CA).

HPV16-E7 protein, produced from bacteria transformed with Pet-19b-HPV16-E7, was purified under the same conditions as RT. HPV16-E7 protein served as a control in the proliferation assays (34).

A stock preparation of Psoralen-Plus UV-inactivated HIV-1 HXB2 virions containing 10¹⁰ particles/ml was a gift from Dr. F. Manca (San Martino Hospital, Genoa, Italy) and was used as previously described (31).

Generation of purified DR molecules

As a source of DR molecules, B lymphoblastoid cell lines homozygous for DR were used: LG2.1 (DRB*0101, DR1), IWB (DRB1*0201, DR2), HAR (DRB*0301, DR3), BSM (DRB*0401, DR4), ATH (DRB*1101, DR5), and Pitout (DRB1*0701, DR7). Cells were cultured in RPMI 1640 (Life Technologies, Paisley, U.K.), supplemented with 2 mM l-glutamine, 100 U/100 μg/ml penicillin/streptomycin solution (Life Technologies), and 10% heat-inactivated FCS (Life Technologies). DR molecules were purified by affinity chromatography (21, 35). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 2% Renex (Accurate Chemicals, Westbury, NY), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were cleared of nuclear and other debris by centrifugation at 10,000 × g for 20 min. The lysates were passed through the following columns using a flow rate of 30 ml/h: Sepharose CL-4B (10 ml), protein A-Sepharose (5 ml), W6/32-protein A-Sepharose (10 ml), and B8.11.2 (anti-DR)-protein A-Sepharose (10 ml). The columns were washed with 10 column volumes of 10 mM Tris-HCl (pH 8.0) with 0.1% Renex, 2 column volumes of PBS, and 2 column volumes of PBS-1% octyl-glucoside. Bound DR was eluted from the B8.11.2 column with 50 mM diethylamine in 150 mM NaCl containing 1% octylglucoside and 0.02% NaN₃ (pH 11.5). The eluate was immediately neutralized with 2 M glycine (pH 2.5) and concentrated through an Amicon 8050 YM30 membrane (Amicon, Beverly, MA) under N₂ pressure. Protein content was evaluated by a bicinchoninic acid assay (Pierce, Rockford, IL), and purity was confirmed by SDS-PAGE.

HLA-DR-peptide binding assay

The analysis of peptide binding to purified DR molecules was performed as described previously (21, 35), using N-terminally fluorescence-labeled standard peptides. As standard fluorescent peptides in the binding assays, HA_308–319 (PKYVKQNTLKLAT, DR1 and DR2), heat shock protein 65 (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) (KTIAYDEEARR, DR3), HA_308–319 Y→F (PKFVKQNTLKLAT, DR4), tetanus toxin (830–843) (QYIKANSKFIGITE, DR5), and Ii_80–103 (LPKPPKPVSKMRMATPLLMQALPM, DR7) were used.

Lymphocyte stimulation assay

PBMC isolated from blood obtained from HIV-1-seronegative donors were seeded at a density of 1.5 × 10⁵ cells/well of a 96-well U-bottom plate (Costar, Cambridge, MA) in 200 μl of Iscove’s medium (Life Technologies) enriched with 10% autologous serum in the presence or the absence of 10 μg/ml of the indicated peptide. As a positive control, PBMC were cultured in the presence of 4 μg/ml of PHA (Murex Diagnostics, Dartford, U.K.) or 10 μg/ml of the influenza virus HA peptide (PKYVQNTLKLAT) (36). Peptide-specific proliferation was measured on day 6. Results are given as counts per minute for each separate well. Wells are scored positive when the stimulation index of all test wells was ≥3, and 50% of the test wells exceeded the mean proliferation + 3 times the SD of medium control wells.

The stimulation index (SI) is calculated as SI = mean cpm of 10 wells stimulated with peptide ÷ mean cpm + 3 times the SD of medium control. MHC class II typing of the healthy donors is shown in Table I⇓.

Generation of primary peptide-specific CD4⁺ T cell lines and T cell clones

Peptide-specific CD4⁺ T cell bulks were generated as described previously (37). Briefly, 15 × 10⁶ PBMC, obtained from HIV-1-seronegative volunteers were cultured in T25 flasks (Becton Dickinson, Lincoln Park, NJ) in Iscove’s medium containing 10% autologous serum and 5 μg/ml peptide. On day 7, cultures were supplemented with 15 × 10⁶ PBMC as well as 5 μg/ml peptide. Thereafter, responders were counted and stimulated twice with an equal amount of irradiated (30 Gy) autologous PBMC and 5 μg/ml peptide. Two days after each restimulation T cell growth factor (Biotest, Dreieich, Germany) was added to a final concentration of 10%. Specific proliferation was measured by incubation of 50,000 responders with an equal amount of irradiated (30 Gy) APC and peptide, protein, or HIV-1 virions as indicated.

RT peptide and protein-specific bulk T cells were cloned by limiting dilution. Responder cells were plated in a total volume of 100 μl of Iscove’s medium at 1 or 0.3 cell/well in 96-well U-bottom plates in the presence of 5% human pooled sera, 10% T cell growth factor, and a stimulator mix. This stimulator mix contained 10⁴ irradiated (100 Gy) B lymphoblastoid cell lines, 8 × 10⁴ irradiated (30 Gy) PBMC derived from a pool of six blood bank donors, and 10⁴ irradiated (30 Gy) autologous PBMC incubated with 1 μg/ml peptide for 1 h at 37°C. Cultures were restimulated with 100 μl of stimulator mix on day 7. On day 14, cultures were split into eight wells, of which six were used to measure peptide- and protein-specific proliferation. Positive clones were restimulated each week with the stimulator mix described above.

Proliferation assay

Cultures were pulsed with 0.5 μCi of [³H]thymidine (5 Ci/mM; Amersham, Aylesbury, U.K.)/well for 18 h. Plates were harvested with a Microcell Harvester (Skatron, Lier, Norway). Filters were packed in plastic bags containing 10 ml of scintillation fluid and were subsequently counted in a 1205 Betaplate counter (Wallac, Turku, Finland).

Cytokine assays

To determine specific excretion of cytokines, T cell clones were stimulated by incubation of 50,000 T cells with an equal amount of APC (30 Gy) together with 10 μg/ml peptide, control peptide, RT protein, or E7 protein as indicated. After 24 h of incubation supernatant was harvested, and replicate wells were pooled.

Cytokine production was measured by ELISA as described previously (38). Briefly, culture supernatant was tested for IFN-γ, IL-4, or IL-10 content by sandwich ELISA. Primary mAbs were mAb 45B3 (American Type Culture Collection, Manassas, VA; 10 μg/ml), mAb 7A3-3 (Department of Nephrology, Leiden University Medical Center, The Netherlands; 10 μg/ml), and mAb 9D7 (provided by Dr. J. Bancherau, Schering Plough, Dardilly, France; 1 μg/ml), respectively. Maxisorb 96-well ELISA plates (Nunc, Copenhagen, Denmark) were coated overnight at room temperature with 100 μl/well of the capturing Ab, appropriately diluted in PBS. After three washings with PBS containing 0.05% Tween-20 (PBST), nonspecific binding sites were blocked with PBST containing 1% BSA (IFN-γ) or 10% FCS (Life Technologies, Breda, The Netherlands) for IL-4 and IL-10. Cell-free culture supernatants were diluted 1/2 and 1/10, added, and incubated at 37°C. Each assay was standardized using serial twofold dilutions of human IFN-γ (range, 50,000 to 390 pg/ml), IL-4 (10,000 to 80 pg/ml), or IL-10 (1000 to 8 pg/ml). The standard sera yielded OD values in a dose-dependent linear fashion. Bound IFN-γ was detected by mAb MD1 (a gift from Dr. P. van der Meide, TNO (Netherlands Central Organization for Applied Scientific Research), Rijswijk, The Netherlands), IL-4 by mAb 1A6-10 (Department of Nephrology, Leiden University Medical Center), and IL-10 by mAb 12G8 (provided by Dr. J. Bancherau, Schering Plough), all conjugated to digoxigenin (Boehringer Mannheim, Mannheim, Germany) and consecutively incubated with sheep anti-digoxigenin-horseradish peroxidase (Boehringer Mannheim). Between each step the wells were washed three times with PBST. Finally, enzyme substrate (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonate]; Sigma) containing 0.0075% H₂O₂ was added. The reaction was stopped by adding 2% oxalic acid, and OD was measured at 415 nm using a microplate reader (Bio-Kinetics EL 312e, Biotek Instruments, Winooski, VT). Concentrations were obtained by interpolation on the standard curves using Kineticalc (EIA Application Software, Biotek Instruments).

MHC blocking

MHC class II blocking experiments were conducted as described previously (37), using murine mAbs anti-DQ SPV.L3, anti-DR B8.11.2, and anti-DP B7/21 (Becton Dickinson, Mountain View, CA). Abs were added to APC 1 h before peptide-APC incubation.

Identification of RT peptides that bind to multiple HLA-DR molecules

We set out to identify HIV-derived peptides that are restricted by multiple HLA-DR molecules and that consequently should be able to elicit HIV-1-specific T cell help in a large fraction of the human population. To maximize the chance that this T cell help is indeed relevant with respect to the challenging HIV-1 virus, we focused our search on peptides derived from the highly conserved RT protein. Several interesting candidate peptide epitopes have been described by Manca et al., who used RT-specific T cell lines obtained by in vitro stimulation of healthy donor-derived lymphocytes with protein to identify RT-derived peptides (31). Although the MHC class II restriction of these peptides was not investigated, their data indicated that these peptides represented naturally processed epitopes. The capacity of these peptides for binding to various class II MHC molecules was tested in a quantitative peptide/MHC binding assay (21).The HLA-DR1, -2, -3, -4, -5, and -7 molecules for which peptide binding was analyzed together encompass 70–80% of the Caucasian, Oriental, and Negroid populations (39).

We found three peptides to bind with high affinity to four different HLA-DR molecules. Peptide RT_251–270 bound to DR1, -2, -3, and -5, while peptides RT_301–320 and RT_431–450 bound to DR1, -2, -3, and -4. In addition, we identified two peptides that bound to five different HLA-DR molecules. Peptide RT_171–190 displayed high affinity binding to DR1, -2, -3, -4, and -7, whereas peptide RT_271–290 bound well to DR1, -2, -3, -5, and -7. Importantly, these latter peptides failed to bind to, respectively, DR5 and DR4, indicating that their binding, although rather ubiquitous in character, was specific (Table II⇓).

Immunogenicity of peptides that display promiscuous HLA-DR-binding

The five peptides that were able to bind to four or more different HLA-DR molecules, and which therefore could represent universal epitopes, were subsequently tested in lymphocyte stimulation assays. PBMC obtained from four different healthy individuals, together covering HLA-DR1, -2, -3, -4, and -5, were pulsed with 10 μg/ml peptide, and proliferation was measured on day 6. Peptide RT_171–190 consistently elicited proliferative responses in the PBMC cultures of donors that displayed one of the HLA molecules DR1, DR2, DR3, or DR4, but not of an HLA-DR5-positive donor. Peptide RT_271–290 stimulated PBMC of donors that carried HLA-DR1, -DR2, and -DR3, but not HLA-DR5. From the other three peptides only peptide RT_431–450 was able to strongly stimulate PBMC of donor 1, whereas PBMC from donor 3 was weakly stimulated. Peptide RT_301–320 showed a weak capacity to stimulate PBMC of donor 3 but not of others. These latter three peptides were apparently not immunogenic in the context of most DR molecules to which they exhibited binding, and therefore were less likely to represent the universal epitopes we were looking for (Table III⇓ and Fig. 1⇓).

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FIGURE 1.

Immunogenicity of peptides binding to multiple HLA-DR molecules. PBMC derived from healthy blood bank donors were incubated with 10 μg/ml of the indicated peptides for 6 days. Influenza HA-derived peptide served as positive control, and no peptide served as background proliferation (medium). PBMC (10 wells/peptide) were subsequently pulsed with [³H]thymidine and harvested following overnight incubation. This graph shows a representative example of the proliferative response of PBMC (donor 2) stimulated with the indicated peptides. The [³H]thymidine incorporation (counts per minute) of each individual well is given, grouped per peptide. Only peptides 171–190 and 271–290 were capable of consequently stimulating naive PBMC obtained from different donors.

To investigate whether the peptides RT_171–190 and RT_271–290 indeed represent Th epitopes that are naturally processed into multiple HLA-DR molecules, the T cell responses to these peptides were analyzed in more detail. Peptide-specific bulk T cell cultures were initiated with PBMC from the first three donors, which all responded to both peptides (Tables I and III). FACS analysis showed that the resulting responder populations consisted of CD4⁺ T cells (not shown). These T cells were subsequently tested for recognition of stimulator cells that were pulsed with whole RT protein instead of RT-derived peptides. In this setting presentation of the relevant peptide-epitopes requires Ag uptake and processing, rather than exogenous loading of class II MHC molecules at the cell surface. Importantly, T cell cultures generated against peptide RT_171–190 showed proliferation against RT protein-pulsed stimulator cells (Fig. 2⇓A). This indicates that peptide RT_171–190 is naturally processed into several different HLA-DR molecules. On the other hand, T cell cultures generated with peptide RT_271–290 do not react against RT-pulsed stimulator cells (Fig. 2⇓B). Peptide RT_271–290 appears not to be naturally processed in the context of DR types 1–4 and therefore cannot serve as a universal Th epitope. Furthermore, these data indicate that the RT protein used does not contain contaminants that cause nonspecific T cell proliferation. Peptide RT_171–190 was further studied in two other donors (no. 5 and 6) expressing HLA-DR1, -7 and HLA-DR3, -7. Donor 4 was not used since peptide RT_171–190 displayed a lack of capacity to bind HLA-DR5 and, accordingly, did not elicit a proliferative response in this donor (Tables II and III). Both donors 5 and 6 mounted peptide RT_171–190-specific CD4⁺ T cells capable of recognizing RT protein-pulsed stimulator cells (Fig. 2⇓C). Interestingly, donor 5 appeared to use more than one MHC class II molecule as a restriction element (Fig. 3⇓A). The T cell culture of donor 5 proliferated specifically against DR7- and DQ2-matched stimulators as well as against DR1- and DQ5-matched stimulators. The donor 6-derived T cell bulk showed specific proliferation when stimulated with DR7- and DQ2-matched stimulator cells, but not when DR3- and DQ2-matched stimulator cells were used (Fig. 3⇓B), indicating that this T cell bulk was restricted by DR7.

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FIGURE 2.

Peptide 171–190 induced T cell responses recognize naturally processed Ag. PBMC from three different donors were primed and restimulated with peptide 171–190 (A) or peptide 271–290 (B). Following two restimulations, the responding T cells were tested in a 3-day proliferation assay. Responder cells (R) and autologous PBMC (stimulators (S)) were incubated with the indicated Ags: recombinant RT, recombinant HPV16-derived E7 protein (E7), peptide 171–190, or an irrelevant peptide. Bars indicate the mean of triplicate wells and the SEM in counts per minute. Note that peptide 171–190 induces T cells capable of recognizing protein-pulsed stimulator cells, whereas peptide 271–290 failed to induce such T cells. C, Reactivity of two additional donors, displaying different sets of HLA-DR molecules at the cell surface, against peptide 171–190.

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FIGURE 3.

MHC class II restrictions of T cells in a responding bulk T cell culture. Responding T cells of donors 5 and 6 were tested in a proliferation assay. The following stimulatory cells were used: autologous PBMC (stimulators (S)) or partially MHC class II-matched PBMC (S (class II match)) incubated with peptide 171–190 or an irrelevant peptide.

HLA-DR restriction and cytokine profile of RT-reactive Th clones

From the different T cell populations found to react against RT-pulsed stimulator cells, clones were derived through limiting dilution. T cell clones recognizing peptide only as well as T cell clones capable of recognizing both peptide and protein-pulsed stimulator cells were obtained. Since the latter category of clones is representative of a physiologically relevant anti-HIV response, their DR restriction was determined. Stimulation of the clones with different APCs matched for selected HLA-DR molecules showed that clones obtained from donor 1 were restricted by HLA-DR4 (Fig. 4⇓). A panel of matched stimulator cells used to test two donor 5-derived Th clones showed restriction by DR1 (Fig. 4⇓). A donor 6-derived T cell line as well as two T cell clones recognized RT-pulsed stimulator cells in the context of DR7 (Fig. 4⇓). Cytokine production upon specific stimulation with peptide was tested by sandwich ELISA for four T cell clones of donors 1 and 6. The T cell clones were stimulated as in a normal proliferation assay with RT_171–190 or irrelevant peptide. All four T cell clones secreted IFN-γ at levels ranging from 4,000–40,000 pg/ml. Clones 16 and 74, which were also tested against RT protein or HPV16 E7-derived control protein, displayed a somewhat lower, but very specific, IFN-γ response against RT protein, probably due to the fact that protein first needs to be processed before stimulation of the T cell clone occurs. Donor 1-derived T cell clone 36 secreted a low amount of IL-10 (280 pg/ml) and no IL-4 (detection level, 80 pg/ml), whereas T cell clone 44 secreted no IL-10 (detection level, 8 pg/ml) but a low amount of IL-4 (460 pg/ml). The T cell clones of donor 6 did not produce detectable amounts of IL-4 (Fig. 5⇓). Thus, these T cell clones displayed a typical Th type 1 profile.

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FIGURE 4.

MHC class II restriction of T cell clones. Top, An HLA-DR4-restricted CD4⁺ Th cell clone obtained from donor 1 was stimulated with autologous PBMC (stimulators (S)) or single DR only-matched PBMC (S/DR2 or S/DR4) pulsed with peptide 171–190. Bottom, Th cell clones 68 (donor 5) and 16 (donor 6) were stimulated with the indicated partially matched PBMC obtained from healthy blood donors (S (class II match)). Clone 68 responded when DR1-matched PBMC pulsed with peptide or protein was used. Clone 16 responded when DR7-matched PBMC were used as stimulators. Bars indicate the mean of two wells.

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FIGURE 5.

Peptide 171–190 induced CD4⁺ Th clones display a Th type 1 cytokine profile. T cell clones 36 and 44 derived from donor 1 and T cell clones 16 and 74 from donor 6 were stimulated with APC pulsed with peptide RT_171–190 (open bars), control peptide (hatched bars), RT protein (black bars), or control HPV16 E7 protein (gray bars). Bars indicate the amount of cytokine produced in 24 h. T cell clones 36 and 44 were tested for IFN-γ, IL-4, and IL-10 upon stimulation with peptide. T cell clones 16 and 74 were tested for IFN-γ and IL-4 upon stimulation with peptide or protein. Stars indicates that cytokine production was below the detection level of the sandwich ELISA used.

Th clones raised against RT peptide react against HIV-1

For a number of clones we explored the capacity of in vitro peptide-induced RT-specific CD4⁺ T cells to recognize APC that had been pulsed with HIV-1 virions. As shown in Fig. 6⇓, Th clones obtained from three different donors were indeed capable of responding to stimulator cells loaded with peptide, RT protein, or HIV-1 virions. We therefore conclude that peptide RT_171–190 can be employed for the generation of functional anti-HIV-1 Th responses in the context of several different HLA-DR molecules.

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FIGURE 6.

Peptide 171–190-induced CD4⁺ Th clones recognize HIV-1. Peptide 171–190-induced, peptide- and protein-specific T cell clones 36 (donor 1), 68 (donor 5), and 16 (donor 6) specifically proliferated when stimulated with APC pulsed with peptide 171–190, RT protein, or inactivated HIV-1 virions. Bars indicate the proliferation, in counts per minute, of the mean of triplicate wells and the SEM.