Processing and Presentation of Exogenous HLA Class
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病菌学杂志 2005年第5期
Departments of Infectious Diseases and Microbiology
Pathology, Graduate School of Public Health and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
ABSTRACT
Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8+ T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8+ T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.
INTRODUCTION
Combination antiretroviral therapy has resulted in sharp decreases in human immunodeficiency virus type 1 (HIV-1) loads, increases in CD4+ T-cell numbers, and a dramatic clinical improvement in HIV-1-infected persons (34). There is, however, persistence of viral infection and only partial recovery of anti-HIV-1 T-cell immunity after treatment of persons with chronic HIV-1 infection (12, 36). Since the CD8+ T-cell response is important in host control of HIV-1 infection and prevention of AIDS (37), new emphasis has been placed on developing methods that will enhance anti-HIV-1 CD8+ T-cell immune responses during combination antiretroviral therapy (12, 29). One approach for immunotherapy of HIV-1 infection, based on a model that has been used extensively in cancer immunotherapy (4, 8, 35), is activation of CD8+ T cells with dendritic cells (DCs) that have been loaded ex vivo with synthetic 8- to 10-mer peptides representing HIV-1 specific, human leukocyte antigen (HLA) class I cytotyoxic T-lymphocyte (CTL) epitopes. DCs are highly specialized antigen-presenting cells (APCs) that process and present antigens for stimulation of primary and secondary T-cell responses (4, 22). Immature DCs (iDCs) are most efficient at taking up and processing exogenous antigens, a step followed by a maturation process that is required for optimal presentation of the HLA-peptide complexes to T cells. Some reports show that the antigen-presenting capacity of DCs is diminished in persons with progressive HIV-1 infection (16). It is not clear, however, if DCs from HIV-1-infected persons on combination drug therapy are able to process and present peptide antigens as well as DCs from uninfected persons. This is an important consideration for the use of DCs in immunotherapy of HIV-1 infection.
Two distinct pathways for processing and loading of exogenous peptides into major histocompatibility complex (MHC) class I molecules have been demonstrated. Exogenous peptides can be loaded onto MHC class I complexes at the cell surface (41, 51), or they can be internalized and loaded onto these molecules in the endoplasmic reticulum (ER) (14). Moreover, a precise size of peptide (8- to 10-mer) following appropriate N- or C-terminal cleavages during protease processing is required for optimal binding to the MHC class I molecule and successful presentation to CD8+ T cells (41, 42).
The mechanisms and efficiency of processing of peptides by DCs from HIV-1-infected persons are important as a biologic basis for clinical trials that we and others are currently conducting for immunotherapy of HIV-1 infection using DCs matured and loaded with HIV-1 peptides ex vivo. We have therefore characterized exogenous peptide processing and presentation by human blood monocyte-derived DCs from HIV-1-infected subjects for activation of viral peptide-specific CD8+ T-cell activity in vitro. We show that intracellular processing is required for optimal HLA class I presentation of exogenous 9-mer peptides to CD8+ T cells. This peptide processing and presentation function is intact in DCs derived from HIV-1-infected persons on combination antiretroviral therapy. Our study supports the use of peptide-loaded DCs for immunotherapy of HIV-1 infection.
MATERIALS AND METHODS
Study subjects. The study population consisted of 13 HLA A0201-positive, non-HIV-1-infected donors and 12 HLA A0201-positive, HIV-1-infected subjects (median CD4+ T-cell count, 542/μl [range, 226 to 1,169/μl]; median HIV-1 RNA load, <50 copies/ml [range, <50 to 13,446 copies/ml]) who were receiving combination antiretroviral therapy in the Pitt Treatment Evaluation Unit (23) or in the Pittsburgh portion of the Multicenter AIDS Cohort Study, a study of the natural history of HIV-1 infection (28).
DCs. Peripheral blood mononuclear cells (PBMCs) were obtained either from freshly donated, heparinized blood by Ficoll-Hypaque gradient separation or from cryopreserved samples. CD14+ monocytes were positively selected from PBMCs by using anti-CD14 monoclonal antibody (MAb)-coated magnetic microbeads (Miltenyi, Auburn, Calif.) to a purity of >97% and were cultured for 5 to 6 days in AIM V medium (GIBCO, Grand Island, N.Y.) containing 1,000 U of recombinant interleukin 4 (IL-4) (R & D Systems, Minneapolis, Minn.)/ml and 1,000 U of recombinant granulocyte-monocyte colony-stimulating factor (Amgen, Seattle, Wash.)/ml to induce iDCs (17). The iDCs were treated with CD40 ligand (CD40L) (1 μg/ml; Amgen) for 40 h to induce DC maturation. The number of viable DCs was determined by typical DC morphology by exclusion of trypan blue dye. The DCs were stained with a phycoerythrin (PE)-Cy5-conjugated anti-HLA-DR MAb (Immunotech, Marseille, France) and a PE-conjugated MAb specific for lineage markers CD3, CD14, CD16, CD19, and CD56 (BD Immunocytometry Systems, San Jose, Calif.). They were assayed by flow cytometry by gating on large cells with high side scatter and analysis on a two-color histogram (EPICS XL; Coulter, Fullerton, Calif.). Cells that were HLA-DR+ and CD3–, CD14–, CD16–, CD19–, and CD56– were defined as DCs. The DCs were further analyzed after staining with PE-conjugated MAbs specific for HLA ABC (BD) or CD83 (Immunotech) and a fluorescein isothiocyanate-conjugated MAb to CD86 (Ancell, Bayport, Minn.). Appropriate isotype-matched reagents were used as controls. The expression of the cell surface markers was assessed as the mean fluorescence intensity (MFI) and percent positive cells. In some experiments, DCs were treated with metabolic inhibitors prior to stimulation of the T cells in order to assess the effects of metabolic inhibitors on DC surface marker expression (17).
Synthetic peptides. The synthetic peptides used were HLA A0201-restricted influenza A virus (FLU) M158-66 (GILGFVFTL) (6), human cytomegalovirus (CMV) strain AD169 pp65495-503 (NLVPMVATV) (50), Epstein-Barr virus (EBV) BMLF1280-288 (9-mer G-L [GLCTLVAML], N-terminal flanking sequence [AIQNAGLCTLVAML], and C-terminal flanking sequence [GLCTLVAMLEETIFW]) (45), HIV-1 RT476-484 (ILKEPVHGV) (47), and HIV-1 p1777-85 (9-mer S-L [SLYNTVATL], N-terminal flanking sequence [GSEELRSLYNTVATL], and C-terminal flanking sequence [SLYNTVATLYCVHQR]) (46), and HLA B27-restricted HIV-1 p24263-272 (KRWIILGLNK) (10) (prepared by the Protein Research Lab, University of Illinois, Chicago, Ill.).
We determined the relative binding capacities of the synthetic peptides by two methods. First, we examined binding to HLA A0201 in a peptide-induced stabilization assay using TAP-deficient, HLA A0201-expressing T2 cells (American Type Culture Collection, Manassas, Va.) (49). The affinity binding capacity was calculated as the MFI of the test peptide compared to the MFI of T2 cells without added peptide. We also examined the binding of these peptides to soluble HLA A0201 by using the HLA PolyTest kit (Pure Protein, Oklahoma City, Okla.) according to the manufacturer's instructions (24). A fluorescently labeled control peptide and soluble HLA were incubated with each peptide until equilibration of peptide replacement was reached as read on an Analyst AD plate reader (Molecular Devices, Sunnyvale, Calif.). Fifty-percent inhibitory concentration (IC50) was calculated by using a dose-response curve. The log10 IC50 for the peptides used in this study were compared to those of a panel of reference viral peptides with known binding capacity in order to define their relative binding affinities.
Preparation of DCs as stimulators. iDCs and mature DCs (mDCs) were suspended to 0.2 x 106/ml in AIM V medium; 0.1 x 105 cells of this APC suspension were added to each well of a 96-microwell enzyme-linked immunospot assay (Elispot) plate and loaded with peptide for 2 h at 37°C under a 5% CO2 atmosphere (26). The responder cells were added to the plates at responder-to-stimulator (R:S) cell ratios of 10:1, 20:1, and 40:1. In some experiments, brefeldin A, lactacystin, and cycloheximide (Sigma, St. Louis, Mo.) were used as metabolic inhibitors to study peptide processing. APCs were preincubated in the presence of these inhibitors for 30 min at 37°C and then washed with RPMI 1640 medium (Life Technologies, Grand Island, N.Y.) to remove excess inhibitors. Treated and untreated APCs were loaded with peptides for an additional 2 h at 37°C and then mixed with CD8+ T cells at a ratio of 1:10.
Establishment of EBV- and CMV-specific CD8+ T-cell lines and enrichment of CD8+ cells as responder cells for IFN- Elispot assays. PBMCs obtained from freshly donated blood or freeze-thawed samples from HLA A0201-positive, EBV- and CMV-seropositive, non-HIV-1-infected donors were used to generate CD8+ T-cell lines specific for the EBV BMLF1280-288 or CMV pp65495-503 peptide. CD8+ T cells were mixed at a 10-to-1 ratio with EBV BMLF1280-288- or CMV pp65495-503-loaded and gamma-irradiated (3,000 rads) T2-?2M cells as stimulators. The R:S cell mixtures were resuspended in RPMI 1640 medium containing 15% heat-inactivated fetal calf serum, murine anti-CD3 MAb 12F6 (0.1 μg/ml; courtesy of J. Wong, Boston, Mass.), recombinant IL-2 (50 U/ml; Chiron, Emeryville, Calif.), recombinant IL-15 (100 pg/ml; R & D), and gamma-irradiated (3,000 rads) PBMCs from allogeneic normal donors as feeder cells. The stimulator-responder cell mixture was cultured for as long as 12 weeks, with fresh RPMI 1640 medium, IL-2, and IL-15 added every 3 to 4 days. For repetitive stimulation, peptide loaded, gamma-irradiated T2-?2M cells were added to the cultures every 3 to 4 weeks. Cell lines were selected based on peptide-specific gamma interferon (IFN-) responses that were greater than the mean plus 3 standard deviations of the unstimulated responses. CD8+ T cells were positively selected from PBMCs by using anti-CD8 MAb-coated magnetic microbeads (Miltenyi). The purities of CD8+ T cells and T-cell lines were 94 to 99% as determined by staining with a fluorescent dye-conjugated anti-CD8 MAb and flow cytometry.
Elispot assay for IFN- release from single, antigen-specific T cells. We used a modification of our previously described Elispot assay to determine single-cell IFN- production (25). Nitrocellulose membrane-containing 96-well plates (Millipore, Bedford, Mass.) were coated overnight at 4°C with 50 μl of a murine anti-human IFN- MAb (10 μg/ml; Mabtech, Stockolm, Sweden)/well. The antibody-coated plates were washed four times with phosphate-buffered saline (PBS) (BioWhittaker, Walkersville, Md.) and blocked with 180 μl of RPMI 1640 medium containing 10% heat-inactivated human serum (Sigma)/well for 1 h at 37°C. The responder cells (0.05 x 106 to 0.1 x 106) and stimulator cells were added to the wells in AIM V medium and incubated for 18 h at 37°C under 5% CO2. The plates were washed four times with PBS containing 0.05% Tween 20 (Sigma), and the secondary antibody (a biotin-conjugated anti-IFN- MAb) was added at 2 μg/ml in 50 μl/well. Plates were washed four times with PBS containing 0.05% Tween 20 after a 2-h incubation at 37°C under CO2, and avidin-bound biotinylated horseradish peroxidase H (AEC substrate kit; Vector, Burlingame, Calif.) was added to the wells and incubated for 1 h at room temperature. The plates were then washed three times with PBS-Tween and three times with PBS, followed by a 5-min incubation with 100 μl of 3-amino-9-ethylcarbazole (Sigma) per well. The reaction was stopped by addition of running tap water. The spots were counted with an Elispot reader system (Cell Technology, Columbia, Md.). Control wells included cells and medium only or phorbol myristate acetate at 1 ng/ml and ionomycin at 1 μM (Sigma) as negative and positive controls, respectively. IFN- production was expressed either as the number of spots per 106 cells or as net spots per 106 cells (i.e., the number of spots/106 cells in peptide-treated cultures minus the number of spots/106 cells in non-peptide-treated cultures). DCs alone, with and without peptides, produced low levels of IFN- in the Elispot assay (for DCs without peptides, mean IFN- production ± standard error [SE] was 19 ± 8 spots/106 cells for both uninfected [n = 5] and HIV-1-infected [n = 6] persons; for DCs with peptides, IFN- production was 9 ± 7 and 21 ± 7 spots/106 cells for non-HIV-1-infected and infected persons, respectively).
Detection of CTL reactivity. The target cells were cultured iDCs and mDCs. They were treated with or without metabolic inhibitors for 30 min at room temperature, washed, and loaded with synthetic peptide (100 μg/ml) for 2 h at 37°C under a 5% CO2 atmosphere. The target cells were then labeled with Na251CrO4 for 16 h at 37°C under a 5% CO2 atmosphere and subsequently washed three times with cold RPMI 1640 medium containing 5% fetal calf serum. The cell counts and viability of target cells were monitored by trypan blue dye exclusion. The target cells were added to the effector cells in triplicate at two or three effector-to-target cell (E/T) ratios (e.g., 40:1, 20:1, and 10:1) in the 96-well plates. The plates were centrifuged at 50 x g for 3 min and were incubated for 4 h at 37°C under a 5% CO2 atmosphere. Afterward, the radioactivity of the cell-free supernatant was assessed in a gamma counter (Top Count NXT; Perkin-Elmer, Shelton, Conn.). The percentage of lysis was calculated as 100 x (experimental counts per minute – spontaneous counts per minute)/(maximum counts per minute – spontaneous counts per minute). Specific lysis was expressed as the percentage of lysis in peptide-treated targets minus the percentage of lysis in non-peptide-treated targets.
Statistics. Data for the different groups were assessed by analysis of variance with the Scheffe multiple comparison test, and by the one- and two-tailed paired Student t test.
RESULTS
Maturation of monocyte-derived DCs from non-HIV-1-infected and infected persons. The purity of monocyte-derived DCs obtained from HIV-1-infected and uninfected persons was 90% based on the expression of HLA-DR and lack of expression of T-cell, B-cell, NK cell, and monocyte lineage markers by flow cytometry. DC yields, calculated based on the percentage of cells with typical DC morphology per cultured monocyte, were similar for non-HIV-1-infected and infected donors, i.e., 3.6% ± 1.4% (n = 7) and 3.7% ± 11% (n = 11), respectively (P > 0.05). The iDCs derived from 10 non-HIV-1-infected donors and 8 HIV-1-infected subjects displayed similar, characteristic DC morphology (data not shown) and cell surface marker expression (Fig. 1). The iDCs from uninfected and HIV-1-infected persons also responded to stimulation with CD40L with similar increases in MFI of HLA-DR, HLA ABC, CD83, and CD86. The increases in the percentages of DCs positive for these markers after treatment with CD40L were also similar for non-HIV-1-infected and infected subjects (P > 0.05). These data indicate that expression of activation and coreceptor molecules on DCs derived from the blood of HIV-1-infected persons who are receiving combination drug therapy is comparable to that of uninfected persons.
Efficiency of presentation of exogenous peptides to CD8+ T cells by DCs from non-HIV-1-infected and HIV-1-infected persons. We compared iDCs and mDCs loaded with viral peptides for their capacities to stimulate IFN- production in CD8+ T cells from HIV-1-infected and uninfected persons. The HLA binding capacity of peptides is central to their capacity to induce T-cell responses. Therefore, in preliminary experiments, we determined the HLA A0201 binding affinities of HIV-1, EBV, and influenza A virus immunodominant peptides. Using the T2 cell assay and a cutoff based on a mean binding and standard deviation of 0.93 ± 0.19 for the negative control, we found that the binding values were 4.40 for FLU M158-66, 2.60 for HIV-1 p1777-85, 1.15 for HIV-1 RT476-484 and CMV pp65495-503, and 1.10 for EBV BMLF1280-288 (Table 1). Peptide HIV-1 p24263-272, which is HLA B27 restricted (binding value, 0.99), served as an MHC class I mismatched negative control. We also examined binding to soluble HLA (sHLA) A0201 in a peptide replacement assay. The log10 IC50 for binding of these peptides to sHLA A0201 was 3.35 for FLU M158-66, 3.82 for HIV-1 RT476-484, 3.78 for HIV-1 p1777-85, 4.05 for EBV BMLF1280-288, and 4.28 for CMV pp65495-503. There was a strong correlation between these two binding affinity assays (r = –0.88; P < 0.05).
We next showed that loading of these peptides at the 2- or 10-μg/ml concentration into CD40L-treated mDCs for 2 h induced greater peptide-specific IFN- production at all the R:S ratios (10, 20, and 40) than that by iDCs cultured with peptides alone or loaded with peptides and treated with CD40L concurrently for 40 h (data not shown). Higher levels of IFN- production were noted at the 10-μg/ml concentration and the R:S ratio of 10 (data not shown). Consequently, for all further experiments comparing iDCs and mDCs, we loaded the mDCs with 10 μg of peptides/ml for 2 h after their maturation and used an R:S ratio of 10.
We compared iDCs and mDCs derived from a cross-section of three HLA A0201-positive, non-HIV-1-infected subjects and six HIV-1-infected subjects on combination antiretroviral drug therapy for presentation of exogenous peptides to autologous CD8+ T cells. The results show that there was a dose-dependent stimulation of CD8+ T cells with peptide-loaded, autologous iDCs and mDCs from non-HIV-1-infected persons. This was superior to activation of CD8+ T cells by peptide alone for two peptides with differing binding affinities, i.e., FLU M158-66 and EBV BMLF1280-288 (P 0.02 for mDCs compared to no DCs or iDCs at all peptide concentrations) (Fig. 2A and B). A similar, dose-dependent increase in response was demonstrated for iDCs and mDCs from HIV-1-infected persons to increasing concentrations of HIV-1 RT476-484 and HIV-1 p1777-85 (P 0.02 for iDCs or mDCs compared to no DCs at all peptide concentrations) (Fig. 2C and D).
We next limited the possible effect of differences in T-cell function between HIV-1-infected and uninfected persons on peptide presentation by DCs by assessing allogeneic, CD8+ T-cell lines specific for EBV BMLF1280-288 and CMV pp65495-503, which are HLA A0201 peptides with similar HLA binding capacities. Figure 3 shows that IFN- production was relatively high in these CD8+ T-cell lines stimulated by this single concentration of peptide (10 μg/ml) without DCs. This was expected, as the T-cell lines were initially selected by conventional screening for high IFN- responses to these peptides without addition of APCs. More importantly, IFN- production stimulated by allogeneic mDCs derived from HLA A0201-positive non-HIV-1-infected or infected persons and loaded with EBV BMLF1280-288 (Fig. 3A) or CMV pp65495-503 (Fig. 3B), was greater than that stimulated by peptide alone (P < 0.01) or peptide-loaded iDCs (P < 0.05). The same trend was noted with peptide-loaded mDCs from the two autologous non-HIV-1-infected subjects from whom the T-cell lines were derived (data not shown).
The cumulative results from these studies show that iDCs and mDCs derived from persons with chronic HIV-1 infection who were responding to combination antiretroviral drug treatment stimulated more IFN- production in peptide-specific CD8+ T cells than peptide alone.
Effects of metabolic inhibitors on processing of exogenous 9-mer peptides. Binding of exogenous peptides representing optimal T-cell epitopes to MHC class I molecules may occur at the APC surface without the need for intracellular processing (42) or in the ER after intracellular uptake of the peptides (14). A recent study has demonstrated that these mechanisms relate to HLA class I binding capacity, with high-affinity peptides binding to HLA class I on the cell surface and low-affinity peptides binding to HLA class I molecules in the ER (33). We therefore investigated the mechanisms of processing and presentation of exogenous low- and high-affinity peptides in DCs from HIV-1-infected and uninfected persons. For this purpose, we blocked vesicular trafficking of peptides with brefeldin A, blocked protease activity in proteasomes by using lactacystin, and blocked the translocation reaction on ribosomes for protein biosynthesis by using cycloheximide. We first determined the optimal concentrations of these potentially cytotoxic agents by using DC viability and surface expression of DC activation and T-cell receptor and costimulatory molecules. Treatment of iDCs or mDCs for 30 min with various concentrations of brefeldin A (0.05 to 5.0 μg/ml), lactacystin (0.05 to 40 μM), and cycloheximide (0.05 to 5.0 mg/ml) did not alter cell viability (range, 82 to 100% compared to that of untreated cells). Moreover, surface expression of HLA class I, CD86, and CD83 was not altered after treatment of mDCs with these metabolic inhibitors (data not shown). There was, however, decreased expression of HLA-DR on mDCs after treatment with each of the three metabolic inhibitors (data not shown). For further studies, we chose concentrations of brefeldin A (5 μg/ml), lactacystin (10 μM), and cycloheximide (1 mg/ml) that were at an intermediate level within these ranges.
The requirement for intracellular processing for optimal presentation of these peptides to CD8+ T cells was supported by treatment of the APCs with brefeldin A, which inhibited T-cell activation by APCs from both non-HIV-1-infected (representative data shown in Fig. 4A and B) and HIV-1-infected (representative data shown in Fig. 4C and D) persons. Cumulative results for FLU M158-66 and EBV BMLF1280-288 responses showed that brefeldin A decreased CD8+ T-cell reactivity to peptide-loaded iDCs and mDCs by a median of 56% (range, 37 to 91%) (P = 0.08 compared to peptides alone) and 51% (range, 8 to 90%) (P = 0.03), respectively, for four HIV-1-negative subjects (data not shown). Likewise, brefeldin A decreased the T-cell response to HIV-1 p17 and RT peptide-loaded iDCs and mDCs by 59% (range, 35 to 71%) (P = 0.04) and 61% (range, 37 to 89%) (P = 0.04), respectively, for four HIV-1-infected subjects.
Lactacystin did not inhibit IFN- production by CD8+ T cells stimulated by the four peptides expressed by autologous iDCs or mDCs from HIV-1-negative or -positive persons (Fig. 4). Indeed, there was a small increase in stimulation of CD8+ T cells by DCs treated with lactacystin: for iDCs and mDCs of four HIV-1-negative persons, median inhibition was –10% (range for iDCs, –23 to 4% [P = 0.44 compared to peptides alone]; range for mDCs, –37 to 1% [P = 0.04]), while for four HIV-1-positive subjects, median inhibition was –9% (range for iDCs, –40 to 1% [P = 0.07]; range for mDCs, –23 to 21% [P = 0.29]). Thus, as expected, proteasomal processing was not required for these minimal 9-mer epitopes. Finally, cycloheximide inhibited most of the activation of CD8+ T cells by either iDCs or mDCs from both non-HIV-1-infected and infected persons (Fig. 4), with cumulative median results of 95 to 99% (data not shown).
In order to study further the exogenous peptide processing pathway, DCs were loaded with EBV BMLF1280-288 or HIV-1 RT476-484 in the presence or absence of lactacystin (10 μM), brefeldin A (5 μg/ml), or cycloheximide (1 mg/ml) and were used as target cells in a CTL assay. The responder cells were CD8+ T cells from three HLA A0201-positive non-HIV-1-infected donors (Fig. 5A) and three HLA A0201-positive HIV-1-infected donors (Fig. 5B). The recognition of EBV BMLF1280-288- and HIV-1 RT476-484-expressing DCs by CTLs was sensitive to the effect of brefeldin A or cycloheximide but not to that of lactacystin as judged by comparison to non-inhibitor-treated DCs from three non-HIV-1-infected and three HIV-1-infected individuals, respectively.
Taken together, these results indicate that HLA class I processing of exogenous 9-mer peptides by iDCs and mDCs from both non-HIV-1-infected and infected persons occurs in part through intracellular pathways.
Effects of metabolic inhibitors on the HLA class I processing pathway of N and C termini of EBV BMLF1280-288 and HIV-1 p1777-85 peptides. Peptides must be of a precise size (usually 8, 9, or 10 amino acid residues) to bind to an MHC class I molecule. The presence of either the amino- or the carboxyl-terminal flank of dominant peptides is suboptimal for antigen presentation (41). The N-terminal flanks of peptides are trimmed by aminopeptidases in the cytosol, and this process is resistant to proteasome inhibitors such as lactacystin. Trimming of the C-terminal flanks of peptides occurs in the proteasome and consequently is blocked by proteasome inhibitors. Therefore, to investigate further the intracellular peptide-processing capacity of DCs, we assessed the abilities of DCs from HIV-1-infected subjects on combination antiretroviral therapy and uninfected persons to process suboptimal viral peptides into their optimal forms for HLA class I presentation to CD8+ T cells.
We compared a hydrophobic 9-mer EBV BMLF1280-288 peptide (G-L [GLCTLVAML]) (Table 1) to its hydrophobic N-terminal flanking sequence (AIQNAGLCTLVAML) and acidic C-terminal flanking sequence (GLCTLVAMLEETIFW) in non-HIV-1-infected donors, and a hydrophobic 9-mer HIV-1 p17 peptide (S-L [SLYNTVATL]) with its acidic N-terminal flanking sequence (GSEELRSLYNTVATL) and hydrophobic C-terminal flanking sequence (SLYNTVATLYCVHQR) in HIV-1-infected subjects, for presentation by iDCs and mDCs to CD8+ T cells. For these studies, iDCs and mDCs were either left untreated or treated with various concentrations of lactacystin, brefeldin A, or cycloheximide for 30 min, then washed and loaded for 2 h with 10 μg of the 9-mer sequence, N-terminal flanking sequence, or C-terminal flanking sequence/ml. CD8+ T cells were then cultured with these APCs in the 18-h IFN- Elispot assay.
The data show that, as expected, iDCs from HIV-1-negative (Fig. 6A) and HIV-1-infected (Fig. 7A) subjects induced less IFN- production than mDCs (Fig. 6B and 7B, respectively) in response to these peptides at concentrations of 1 μg/ml (data not shown) or 10 μg/ml (Fig. 6 and 7). Lactacystin blocked the presentation of the C-terminal extension but not the N-terminal extension or the 9-mer EBV and HIV-1 peptides by DCs to CD8+ T cells from both HIV-1-negative (Fig. 6) and HIV-1-infected (Fig. 7) persons in a concentration-dependent fashion. In contrast, brefeldin A and cycloheximide blocked the presentation of all three peptides (C-terminal extension, N-terminal extension, and 9-mer peptides). The same effect was evident for the 1 μg/ml (data not shown) and 10 μg/ml (Fig. 6 and 7) concentrations of the peptides. Notably, both the binding affinity and the charge of a peptide are related to the efficiency of attachment and transport into APCs (3, 9). However, these peptides did not differ in their capacity to be blocked by these metabolic inhibitors, based on their HLA class I binding affinities and their neutral, anionic, or cationic charges.
These results indicate that iDCs and mDCs derived from HIV-1-infected persons on combination drug therapy and those from non-HIV-1-infected persons process exogenous peptides for presentation by HLA class I molecules to CD8+ T cells through similar protease-trimming pathways and with comparable efficiencies.
DISCUSSION
Our study demonstrates that DCs from HIV-1-infected persons on combination antiretroviral therapy have a capacity similar to those from non-HIV-1-infected persons to process and present exogenously loaded synthetic peptides to CD8+ T cells. The data show that mDCs derived from the blood of these individuals were more potent APCs than iDCs. This result was associated with an increase in the expression of surface molecules involved in the activation of T cells on the DCs after maturation. Thus, we noted an increase in the level of HLA class I molecules that are required for the presentation of peptides to CD8+ T cells (19), as well as in the level and percentage of cells expressing the T-cell coreceptor CD86 (11) and the T-cell activation factor CD83 (30), after treatment of iDCs with CD40L. These mDCs also expressed higher levels of HLA class II (i.e., HLA-DR). There was no significant difference in the expression of these molecules by DCs between HIV-1-infected and uninfected persons. This finding confirms and extends the results of previous studies from our laboratory showing that marker expression on DCs derived from peripheral blood myeloid precursors of HIV-1-infected persons on combination antiretroviral therapy, and matured with either trimeric CD40L or monocyte-conditioned culture medium, is comparable to that from HIV-1-negative persons (17).
DCs matured with CD40L were efficient APCs for presenting exogenous peptides representing optimal, immunodominant, HLA A0201-restricted viral epitopes for induction of IFN- production in peptide-specific CD8+ T-cell lines. These cell lines controlled for the potential effects of low CD8+ T-cell reactivity known to evolve during long-term combination antiretroviral therapy (27, 40). Even with this lower T-cell activity in such HIV-1-infected subjects, we showed that DCs loaded with peptides could stimulate blood-derived, autologous CD8+ T cells better than the peptides alone. The effects of CD40L, normally provided by CD4+ T cells, on DC stimulation of CD8+ T cells have been related to increases in the surface expression of HLA and costimulatory molecules, as well as to production of cytokines such as IL-12 and IL-15 (48). The iDCs that were loaded with the exogenous 9-mer viral peptides could activate CD8+ T cells to a significant degree, but a lesser degree than mDCs. This result was found for viral peptides with a range of binding affinities for HLA class I and different solubilities. Interestingly, enriched CD8+ T cells and CD8+ T-cell lines were able to respond to these 9-mer peptides in the absence of added APCs, albeit to a lesser degree than T cells stimulated with peptide-loaded DCs. The results suggest that CD8+ T cells were acting as both antigen-presenting and responding cells, a conclusion supported by a recent report that nonprofessional APCs in PBMCs can process peptides for presentation to CD8+ T cells (20).
The increase in T-cell response obtained by using DCs as APCs in our study was statistically significant and approximately 2- to 10-fold higher than that achieved by overnight stimulation of T cells with peptides alone. It may be possible to attain greater levels of response by long-term priming of na?ve T cells (21, 26, 52) in addition to activation of the limited number of memory CD8+ T cells specific for each peptide. Nevertheless, these data support the use of mDCs as APCs for activation of T-cell responses in immunotherapy trials (15).
Approaches to immunotherapy of cancers as well as HIV-1 infection have included the use of DCs loaded either in vivo or ex vivo with single peptides or pools of peptides as antigens (5, 7, 38). Rock and colleagues established that exogenous peptides representing minimal, 8- to 10-mer epitopes for CD8+ T cells do not require intracellular processing by APCs, as they can bind directly to HLA class I molecules at the cell surface (42). Such peptides, however, can be internalized and bind to MHC class I molecules in the ER (14, 33). This processing could relate to the efficacy of DCs loaded with these peptides in immunotherapy of HIV-1 infection. We therefore examined the mechanisms and efficiency of processing of peptides by DCs from non-HIV-1-infected and infected persons. For this purpose, we used metabolic inhibitors to delineate various stages of processing of the extracellular peptides by DCs and other APCs from non-HIV-1-infected and infected subjects. We demonstrated that blocking of intracellular vesicular trafficking in the DCs with brefeldin A reduced the stimulation of CD8+ T cells by 9-mer viral peptides. We extended these findings by assessing the capacities of DCs from HIV-1-infected subjects on combination antiretroviral therapy and uninfected persons to process viral peptides into their optimal forms for HLA class I presentation to CD8+ T cells. Peptides ideally must be a precise size (i.e., 8, 9, or 10 amino acid residues) to bind to an MHC class I molecule. The presence of either the amino- or the carboxyl-terminal flank of dominant peptides is suboptimal for antigen presentation. The N-terminal flanks of peptides are trimmed by aminopeptidases in the cytosol, and this trimming is resistant to the proteasome inhibitor lactacystin. Trimming of the C-terminal flanks of peptides occurs in the proteasome and consequently is blocked by lactacystin. Our results show that DCs from both non-HIV-1-infected and HIV-1-infected subjects were able to process exogenous 9-mer peptides (EBV BMLF1280-288 and HIV-1 p1777-85) with 5 or 6 additional amino acids at their N-terminal or C-terminal flanking end with similar efficiencies. Lactacystin inhibited the processing of the C-terminal flanking peptides, but not of the N-terminal flanking peptides or minimal 9-mer peptides, similarly in both iDCs and mDCs from uninfected and HIV-1-infected subjects. Brefeldin A, however, inhibited processing of all three forms of these exogenously delivered peptides in iDCs and mDCs, as did cycloheximide. The effects of these inhibitors were similar for peptides with different HLA class I binding affinities and solubilities. These data concur with the generation of HIV-1 p1777-85, which is known to be dependent on C-terminal trimming in the proteasome and N-terminal trimming by aminopeptidases in the cytosol (13, 18).
These results confirm that DCs from uninfected and HIV-1-infected persons have similar capacities for intracellular processing of exogenous peptides with a range of binding affinities and charges for presentation to CD8+ T cells. The results also indicate that intracellular uptake and processing are involved in the presentation of these exogenous 9-mer peptides to CD8+ T cells by professional APCs. In this regard, Andrieu and colleagues (1, 2) have recently reported that HIV-1 RT476-484 or its N-terminal extension RT461-484 when bound to a lipid (N-palmytoyl-lysine) is processed intracellularly by iDCs of HIV-1-negative subjects for presentation to CD8+ T-cell lines. We found, however, that the APCs with the greatest capacity for activating peptide-specific CD8+ T cells were mDCs. Moreover, the relatively lower inhibitory effects of brefeldin A on stimulation of T cells by mDCs than on stimulation by the other APCs in our study support the notion that viral peptides are processed in part by direct binding to HLA class I molecules at the cell surface. Factors that could contribute to the relative resistance of mDCs to the effects of these inhibitors are their lack of highly active endocytosis (43) and greater expression of surface HLA class I molecules (31). These factors also may be necessary to counter the limitation on induction of anti-peptide CD8+ T cells due to rapid turnover of MHC class I-complexed peptides by DCs (32).
A major criticism of peptide immunotherapy in HIV-1 infection is the narrow HLA restriction and limited immunodominance of viral peptide vaccines. This limitation, however, may be overcome by use of peptides that bind to multiple HLA class I molecules ("HLA supertype peptides"), a strategy that effectively broadens the targeted population (44). Moreover, peptide-based cancer vaccines can induce T-cell reactivity specific for other cancer antigens that are distinct from those used in the vaccine (a process termed determinant spreading, or epitope spreading) (39). It is postulated that lysis of cancer cells by CD8+ T cells specific for the vaccinating peptides releases other cancer antigens that are cross-presented to CD8+ T cells by HLA class I molecules on DCs. Such mechanisms could increase the potential efficacy of immunotherapy of HIV-1 infection with HIV-1 peptides loaded into DCs ex vivo or targeted to DCs in vivo. Our study supports the use of DC-based peptide immunotherapy to elicit broad, prolonged, and elevated T-cell responses to HIV-1 in persons receiving combination antiretroviral drug treatment.
ACKNOWLEDGMENTS
This work was supported by National Institute of Allergy and Infectious Diseases grants R01 AI41870, U01 AI37984, and U01 AI35041 and by a grant from Merck Research Laboratories.
We thank S. D. Lyman, E. Thomas, K. Phalmer, and K. Picha (Amgen/Immunex, Seattle, Wash.) for providing the CD40L; A. Hoji, C. Kalinyak, L. Borowski, W. Jiang, L. Zheng, X. Zhao, and P. Zhang for technical assistance; and W. Buchanan and C. Tripoli for clinical assistance.
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Pathology, Graduate School of Public Health and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
ABSTRACT
Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8+ T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8+ T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.
INTRODUCTION
Combination antiretroviral therapy has resulted in sharp decreases in human immunodeficiency virus type 1 (HIV-1) loads, increases in CD4+ T-cell numbers, and a dramatic clinical improvement in HIV-1-infected persons (34). There is, however, persistence of viral infection and only partial recovery of anti-HIV-1 T-cell immunity after treatment of persons with chronic HIV-1 infection (12, 36). Since the CD8+ T-cell response is important in host control of HIV-1 infection and prevention of AIDS (37), new emphasis has been placed on developing methods that will enhance anti-HIV-1 CD8+ T-cell immune responses during combination antiretroviral therapy (12, 29). One approach for immunotherapy of HIV-1 infection, based on a model that has been used extensively in cancer immunotherapy (4, 8, 35), is activation of CD8+ T cells with dendritic cells (DCs) that have been loaded ex vivo with synthetic 8- to 10-mer peptides representing HIV-1 specific, human leukocyte antigen (HLA) class I cytotyoxic T-lymphocyte (CTL) epitopes. DCs are highly specialized antigen-presenting cells (APCs) that process and present antigens for stimulation of primary and secondary T-cell responses (4, 22). Immature DCs (iDCs) are most efficient at taking up and processing exogenous antigens, a step followed by a maturation process that is required for optimal presentation of the HLA-peptide complexes to T cells. Some reports show that the antigen-presenting capacity of DCs is diminished in persons with progressive HIV-1 infection (16). It is not clear, however, if DCs from HIV-1-infected persons on combination drug therapy are able to process and present peptide antigens as well as DCs from uninfected persons. This is an important consideration for the use of DCs in immunotherapy of HIV-1 infection.
Two distinct pathways for processing and loading of exogenous peptides into major histocompatibility complex (MHC) class I molecules have been demonstrated. Exogenous peptides can be loaded onto MHC class I complexes at the cell surface (41, 51), or they can be internalized and loaded onto these molecules in the endoplasmic reticulum (ER) (14). Moreover, a precise size of peptide (8- to 10-mer) following appropriate N- or C-terminal cleavages during protease processing is required for optimal binding to the MHC class I molecule and successful presentation to CD8+ T cells (41, 42).
The mechanisms and efficiency of processing of peptides by DCs from HIV-1-infected persons are important as a biologic basis for clinical trials that we and others are currently conducting for immunotherapy of HIV-1 infection using DCs matured and loaded with HIV-1 peptides ex vivo. We have therefore characterized exogenous peptide processing and presentation by human blood monocyte-derived DCs from HIV-1-infected subjects for activation of viral peptide-specific CD8+ T-cell activity in vitro. We show that intracellular processing is required for optimal HLA class I presentation of exogenous 9-mer peptides to CD8+ T cells. This peptide processing and presentation function is intact in DCs derived from HIV-1-infected persons on combination antiretroviral therapy. Our study supports the use of peptide-loaded DCs for immunotherapy of HIV-1 infection.
MATERIALS AND METHODS
Study subjects. The study population consisted of 13 HLA A0201-positive, non-HIV-1-infected donors and 12 HLA A0201-positive, HIV-1-infected subjects (median CD4+ T-cell count, 542/μl [range, 226 to 1,169/μl]; median HIV-1 RNA load, <50 copies/ml [range, <50 to 13,446 copies/ml]) who were receiving combination antiretroviral therapy in the Pitt Treatment Evaluation Unit (23) or in the Pittsburgh portion of the Multicenter AIDS Cohort Study, a study of the natural history of HIV-1 infection (28).
DCs. Peripheral blood mononuclear cells (PBMCs) were obtained either from freshly donated, heparinized blood by Ficoll-Hypaque gradient separation or from cryopreserved samples. CD14+ monocytes were positively selected from PBMCs by using anti-CD14 monoclonal antibody (MAb)-coated magnetic microbeads (Miltenyi, Auburn, Calif.) to a purity of >97% and were cultured for 5 to 6 days in AIM V medium (GIBCO, Grand Island, N.Y.) containing 1,000 U of recombinant interleukin 4 (IL-4) (R & D Systems, Minneapolis, Minn.)/ml and 1,000 U of recombinant granulocyte-monocyte colony-stimulating factor (Amgen, Seattle, Wash.)/ml to induce iDCs (17). The iDCs were treated with CD40 ligand (CD40L) (1 μg/ml; Amgen) for 40 h to induce DC maturation. The number of viable DCs was determined by typical DC morphology by exclusion of trypan blue dye. The DCs were stained with a phycoerythrin (PE)-Cy5-conjugated anti-HLA-DR MAb (Immunotech, Marseille, France) and a PE-conjugated MAb specific for lineage markers CD3, CD14, CD16, CD19, and CD56 (BD Immunocytometry Systems, San Jose, Calif.). They were assayed by flow cytometry by gating on large cells with high side scatter and analysis on a two-color histogram (EPICS XL; Coulter, Fullerton, Calif.). Cells that were HLA-DR+ and CD3–, CD14–, CD16–, CD19–, and CD56– were defined as DCs. The DCs were further analyzed after staining with PE-conjugated MAbs specific for HLA ABC (BD) or CD83 (Immunotech) and a fluorescein isothiocyanate-conjugated MAb to CD86 (Ancell, Bayport, Minn.). Appropriate isotype-matched reagents were used as controls. The expression of the cell surface markers was assessed as the mean fluorescence intensity (MFI) and percent positive cells. In some experiments, DCs were treated with metabolic inhibitors prior to stimulation of the T cells in order to assess the effects of metabolic inhibitors on DC surface marker expression (17).
Synthetic peptides. The synthetic peptides used were HLA A0201-restricted influenza A virus (FLU) M158-66 (GILGFVFTL) (6), human cytomegalovirus (CMV) strain AD169 pp65495-503 (NLVPMVATV) (50), Epstein-Barr virus (EBV) BMLF1280-288 (9-mer G-L [GLCTLVAML], N-terminal flanking sequence [AIQNAGLCTLVAML], and C-terminal flanking sequence [GLCTLVAMLEETIFW]) (45), HIV-1 RT476-484 (ILKEPVHGV) (47), and HIV-1 p1777-85 (9-mer S-L [SLYNTVATL], N-terminal flanking sequence [GSEELRSLYNTVATL], and C-terminal flanking sequence [SLYNTVATLYCVHQR]) (46), and HLA B27-restricted HIV-1 p24263-272 (KRWIILGLNK) (10) (prepared by the Protein Research Lab, University of Illinois, Chicago, Ill.).
We determined the relative binding capacities of the synthetic peptides by two methods. First, we examined binding to HLA A0201 in a peptide-induced stabilization assay using TAP-deficient, HLA A0201-expressing T2 cells (American Type Culture Collection, Manassas, Va.) (49). The affinity binding capacity was calculated as the MFI of the test peptide compared to the MFI of T2 cells without added peptide. We also examined the binding of these peptides to soluble HLA A0201 by using the HLA PolyTest kit (Pure Protein, Oklahoma City, Okla.) according to the manufacturer's instructions (24). A fluorescently labeled control peptide and soluble HLA were incubated with each peptide until equilibration of peptide replacement was reached as read on an Analyst AD plate reader (Molecular Devices, Sunnyvale, Calif.). Fifty-percent inhibitory concentration (IC50) was calculated by using a dose-response curve. The log10 IC50 for the peptides used in this study were compared to those of a panel of reference viral peptides with known binding capacity in order to define their relative binding affinities.
Preparation of DCs as stimulators. iDCs and mature DCs (mDCs) were suspended to 0.2 x 106/ml in AIM V medium; 0.1 x 105 cells of this APC suspension were added to each well of a 96-microwell enzyme-linked immunospot assay (Elispot) plate and loaded with peptide for 2 h at 37°C under a 5% CO2 atmosphere (26). The responder cells were added to the plates at responder-to-stimulator (R:S) cell ratios of 10:1, 20:1, and 40:1. In some experiments, brefeldin A, lactacystin, and cycloheximide (Sigma, St. Louis, Mo.) were used as metabolic inhibitors to study peptide processing. APCs were preincubated in the presence of these inhibitors for 30 min at 37°C and then washed with RPMI 1640 medium (Life Technologies, Grand Island, N.Y.) to remove excess inhibitors. Treated and untreated APCs were loaded with peptides for an additional 2 h at 37°C and then mixed with CD8+ T cells at a ratio of 1:10.
Establishment of EBV- and CMV-specific CD8+ T-cell lines and enrichment of CD8+ cells as responder cells for IFN- Elispot assays. PBMCs obtained from freshly donated blood or freeze-thawed samples from HLA A0201-positive, EBV- and CMV-seropositive, non-HIV-1-infected donors were used to generate CD8+ T-cell lines specific for the EBV BMLF1280-288 or CMV pp65495-503 peptide. CD8+ T cells were mixed at a 10-to-1 ratio with EBV BMLF1280-288- or CMV pp65495-503-loaded and gamma-irradiated (3,000 rads) T2-?2M cells as stimulators. The R:S cell mixtures were resuspended in RPMI 1640 medium containing 15% heat-inactivated fetal calf serum, murine anti-CD3 MAb 12F6 (0.1 μg/ml; courtesy of J. Wong, Boston, Mass.), recombinant IL-2 (50 U/ml; Chiron, Emeryville, Calif.), recombinant IL-15 (100 pg/ml; R & D), and gamma-irradiated (3,000 rads) PBMCs from allogeneic normal donors as feeder cells. The stimulator-responder cell mixture was cultured for as long as 12 weeks, with fresh RPMI 1640 medium, IL-2, and IL-15 added every 3 to 4 days. For repetitive stimulation, peptide loaded, gamma-irradiated T2-?2M cells were added to the cultures every 3 to 4 weeks. Cell lines were selected based on peptide-specific gamma interferon (IFN-) responses that were greater than the mean plus 3 standard deviations of the unstimulated responses. CD8+ T cells were positively selected from PBMCs by using anti-CD8 MAb-coated magnetic microbeads (Miltenyi). The purities of CD8+ T cells and T-cell lines were 94 to 99% as determined by staining with a fluorescent dye-conjugated anti-CD8 MAb and flow cytometry.
Elispot assay for IFN- release from single, antigen-specific T cells. We used a modification of our previously described Elispot assay to determine single-cell IFN- production (25). Nitrocellulose membrane-containing 96-well plates (Millipore, Bedford, Mass.) were coated overnight at 4°C with 50 μl of a murine anti-human IFN- MAb (10 μg/ml; Mabtech, Stockolm, Sweden)/well. The antibody-coated plates were washed four times with phosphate-buffered saline (PBS) (BioWhittaker, Walkersville, Md.) and blocked with 180 μl of RPMI 1640 medium containing 10% heat-inactivated human serum (Sigma)/well for 1 h at 37°C. The responder cells (0.05 x 106 to 0.1 x 106) and stimulator cells were added to the wells in AIM V medium and incubated for 18 h at 37°C under 5% CO2. The plates were washed four times with PBS containing 0.05% Tween 20 (Sigma), and the secondary antibody (a biotin-conjugated anti-IFN- MAb) was added at 2 μg/ml in 50 μl/well. Plates were washed four times with PBS containing 0.05% Tween 20 after a 2-h incubation at 37°C under CO2, and avidin-bound biotinylated horseradish peroxidase H (AEC substrate kit; Vector, Burlingame, Calif.) was added to the wells and incubated for 1 h at room temperature. The plates were then washed three times with PBS-Tween and three times with PBS, followed by a 5-min incubation with 100 μl of 3-amino-9-ethylcarbazole (Sigma) per well. The reaction was stopped by addition of running tap water. The spots were counted with an Elispot reader system (Cell Technology, Columbia, Md.). Control wells included cells and medium only or phorbol myristate acetate at 1 ng/ml and ionomycin at 1 μM (Sigma) as negative and positive controls, respectively. IFN- production was expressed either as the number of spots per 106 cells or as net spots per 106 cells (i.e., the number of spots/106 cells in peptide-treated cultures minus the number of spots/106 cells in non-peptide-treated cultures). DCs alone, with and without peptides, produced low levels of IFN- in the Elispot assay (for DCs without peptides, mean IFN- production ± standard error [SE] was 19 ± 8 spots/106 cells for both uninfected [n = 5] and HIV-1-infected [n = 6] persons; for DCs with peptides, IFN- production was 9 ± 7 and 21 ± 7 spots/106 cells for non-HIV-1-infected and infected persons, respectively).
Detection of CTL reactivity. The target cells were cultured iDCs and mDCs. They were treated with or without metabolic inhibitors for 30 min at room temperature, washed, and loaded with synthetic peptide (100 μg/ml) for 2 h at 37°C under a 5% CO2 atmosphere. The target cells were then labeled with Na251CrO4 for 16 h at 37°C under a 5% CO2 atmosphere and subsequently washed three times with cold RPMI 1640 medium containing 5% fetal calf serum. The cell counts and viability of target cells were monitored by trypan blue dye exclusion. The target cells were added to the effector cells in triplicate at two or three effector-to-target cell (E/T) ratios (e.g., 40:1, 20:1, and 10:1) in the 96-well plates. The plates were centrifuged at 50 x g for 3 min and were incubated for 4 h at 37°C under a 5% CO2 atmosphere. Afterward, the radioactivity of the cell-free supernatant was assessed in a gamma counter (Top Count NXT; Perkin-Elmer, Shelton, Conn.). The percentage of lysis was calculated as 100 x (experimental counts per minute – spontaneous counts per minute)/(maximum counts per minute – spontaneous counts per minute). Specific lysis was expressed as the percentage of lysis in peptide-treated targets minus the percentage of lysis in non-peptide-treated targets.
Statistics. Data for the different groups were assessed by analysis of variance with the Scheffe multiple comparison test, and by the one- and two-tailed paired Student t test.
RESULTS
Maturation of monocyte-derived DCs from non-HIV-1-infected and infected persons. The purity of monocyte-derived DCs obtained from HIV-1-infected and uninfected persons was 90% based on the expression of HLA-DR and lack of expression of T-cell, B-cell, NK cell, and monocyte lineage markers by flow cytometry. DC yields, calculated based on the percentage of cells with typical DC morphology per cultured monocyte, were similar for non-HIV-1-infected and infected donors, i.e., 3.6% ± 1.4% (n = 7) and 3.7% ± 11% (n = 11), respectively (P > 0.05). The iDCs derived from 10 non-HIV-1-infected donors and 8 HIV-1-infected subjects displayed similar, characteristic DC morphology (data not shown) and cell surface marker expression (Fig. 1). The iDCs from uninfected and HIV-1-infected persons also responded to stimulation with CD40L with similar increases in MFI of HLA-DR, HLA ABC, CD83, and CD86. The increases in the percentages of DCs positive for these markers after treatment with CD40L were also similar for non-HIV-1-infected and infected subjects (P > 0.05). These data indicate that expression of activation and coreceptor molecules on DCs derived from the blood of HIV-1-infected persons who are receiving combination drug therapy is comparable to that of uninfected persons.
Efficiency of presentation of exogenous peptides to CD8+ T cells by DCs from non-HIV-1-infected and HIV-1-infected persons. We compared iDCs and mDCs loaded with viral peptides for their capacities to stimulate IFN- production in CD8+ T cells from HIV-1-infected and uninfected persons. The HLA binding capacity of peptides is central to their capacity to induce T-cell responses. Therefore, in preliminary experiments, we determined the HLA A0201 binding affinities of HIV-1, EBV, and influenza A virus immunodominant peptides. Using the T2 cell assay and a cutoff based on a mean binding and standard deviation of 0.93 ± 0.19 for the negative control, we found that the binding values were 4.40 for FLU M158-66, 2.60 for HIV-1 p1777-85, 1.15 for HIV-1 RT476-484 and CMV pp65495-503, and 1.10 for EBV BMLF1280-288 (Table 1). Peptide HIV-1 p24263-272, which is HLA B27 restricted (binding value, 0.99), served as an MHC class I mismatched negative control. We also examined binding to soluble HLA (sHLA) A0201 in a peptide replacement assay. The log10 IC50 for binding of these peptides to sHLA A0201 was 3.35 for FLU M158-66, 3.82 for HIV-1 RT476-484, 3.78 for HIV-1 p1777-85, 4.05 for EBV BMLF1280-288, and 4.28 for CMV pp65495-503. There was a strong correlation between these two binding affinity assays (r = –0.88; P < 0.05).
We next showed that loading of these peptides at the 2- or 10-μg/ml concentration into CD40L-treated mDCs for 2 h induced greater peptide-specific IFN- production at all the R:S ratios (10, 20, and 40) than that by iDCs cultured with peptides alone or loaded with peptides and treated with CD40L concurrently for 40 h (data not shown). Higher levels of IFN- production were noted at the 10-μg/ml concentration and the R:S ratio of 10 (data not shown). Consequently, for all further experiments comparing iDCs and mDCs, we loaded the mDCs with 10 μg of peptides/ml for 2 h after their maturation and used an R:S ratio of 10.
We compared iDCs and mDCs derived from a cross-section of three HLA A0201-positive, non-HIV-1-infected subjects and six HIV-1-infected subjects on combination antiretroviral drug therapy for presentation of exogenous peptides to autologous CD8+ T cells. The results show that there was a dose-dependent stimulation of CD8+ T cells with peptide-loaded, autologous iDCs and mDCs from non-HIV-1-infected persons. This was superior to activation of CD8+ T cells by peptide alone for two peptides with differing binding affinities, i.e., FLU M158-66 and EBV BMLF1280-288 (P 0.02 for mDCs compared to no DCs or iDCs at all peptide concentrations) (Fig. 2A and B). A similar, dose-dependent increase in response was demonstrated for iDCs and mDCs from HIV-1-infected persons to increasing concentrations of HIV-1 RT476-484 and HIV-1 p1777-85 (P 0.02 for iDCs or mDCs compared to no DCs at all peptide concentrations) (Fig. 2C and D).
We next limited the possible effect of differences in T-cell function between HIV-1-infected and uninfected persons on peptide presentation by DCs by assessing allogeneic, CD8+ T-cell lines specific for EBV BMLF1280-288 and CMV pp65495-503, which are HLA A0201 peptides with similar HLA binding capacities. Figure 3 shows that IFN- production was relatively high in these CD8+ T-cell lines stimulated by this single concentration of peptide (10 μg/ml) without DCs. This was expected, as the T-cell lines were initially selected by conventional screening for high IFN- responses to these peptides without addition of APCs. More importantly, IFN- production stimulated by allogeneic mDCs derived from HLA A0201-positive non-HIV-1-infected or infected persons and loaded with EBV BMLF1280-288 (Fig. 3A) or CMV pp65495-503 (Fig. 3B), was greater than that stimulated by peptide alone (P < 0.01) or peptide-loaded iDCs (P < 0.05). The same trend was noted with peptide-loaded mDCs from the two autologous non-HIV-1-infected subjects from whom the T-cell lines were derived (data not shown).
The cumulative results from these studies show that iDCs and mDCs derived from persons with chronic HIV-1 infection who were responding to combination antiretroviral drug treatment stimulated more IFN- production in peptide-specific CD8+ T cells than peptide alone.
Effects of metabolic inhibitors on processing of exogenous 9-mer peptides. Binding of exogenous peptides representing optimal T-cell epitopes to MHC class I molecules may occur at the APC surface without the need for intracellular processing (42) or in the ER after intracellular uptake of the peptides (14). A recent study has demonstrated that these mechanisms relate to HLA class I binding capacity, with high-affinity peptides binding to HLA class I on the cell surface and low-affinity peptides binding to HLA class I molecules in the ER (33). We therefore investigated the mechanisms of processing and presentation of exogenous low- and high-affinity peptides in DCs from HIV-1-infected and uninfected persons. For this purpose, we blocked vesicular trafficking of peptides with brefeldin A, blocked protease activity in proteasomes by using lactacystin, and blocked the translocation reaction on ribosomes for protein biosynthesis by using cycloheximide. We first determined the optimal concentrations of these potentially cytotoxic agents by using DC viability and surface expression of DC activation and T-cell receptor and costimulatory molecules. Treatment of iDCs or mDCs for 30 min with various concentrations of brefeldin A (0.05 to 5.0 μg/ml), lactacystin (0.05 to 40 μM), and cycloheximide (0.05 to 5.0 mg/ml) did not alter cell viability (range, 82 to 100% compared to that of untreated cells). Moreover, surface expression of HLA class I, CD86, and CD83 was not altered after treatment of mDCs with these metabolic inhibitors (data not shown). There was, however, decreased expression of HLA-DR on mDCs after treatment with each of the three metabolic inhibitors (data not shown). For further studies, we chose concentrations of brefeldin A (5 μg/ml), lactacystin (10 μM), and cycloheximide (1 mg/ml) that were at an intermediate level within these ranges.
The requirement for intracellular processing for optimal presentation of these peptides to CD8+ T cells was supported by treatment of the APCs with brefeldin A, which inhibited T-cell activation by APCs from both non-HIV-1-infected (representative data shown in Fig. 4A and B) and HIV-1-infected (representative data shown in Fig. 4C and D) persons. Cumulative results for FLU M158-66 and EBV BMLF1280-288 responses showed that brefeldin A decreased CD8+ T-cell reactivity to peptide-loaded iDCs and mDCs by a median of 56% (range, 37 to 91%) (P = 0.08 compared to peptides alone) and 51% (range, 8 to 90%) (P = 0.03), respectively, for four HIV-1-negative subjects (data not shown). Likewise, brefeldin A decreased the T-cell response to HIV-1 p17 and RT peptide-loaded iDCs and mDCs by 59% (range, 35 to 71%) (P = 0.04) and 61% (range, 37 to 89%) (P = 0.04), respectively, for four HIV-1-infected subjects.
Lactacystin did not inhibit IFN- production by CD8+ T cells stimulated by the four peptides expressed by autologous iDCs or mDCs from HIV-1-negative or -positive persons (Fig. 4). Indeed, there was a small increase in stimulation of CD8+ T cells by DCs treated with lactacystin: for iDCs and mDCs of four HIV-1-negative persons, median inhibition was –10% (range for iDCs, –23 to 4% [P = 0.44 compared to peptides alone]; range for mDCs, –37 to 1% [P = 0.04]), while for four HIV-1-positive subjects, median inhibition was –9% (range for iDCs, –40 to 1% [P = 0.07]; range for mDCs, –23 to 21% [P = 0.29]). Thus, as expected, proteasomal processing was not required for these minimal 9-mer epitopes. Finally, cycloheximide inhibited most of the activation of CD8+ T cells by either iDCs or mDCs from both non-HIV-1-infected and infected persons (Fig. 4), with cumulative median results of 95 to 99% (data not shown).
In order to study further the exogenous peptide processing pathway, DCs were loaded with EBV BMLF1280-288 or HIV-1 RT476-484 in the presence or absence of lactacystin (10 μM), brefeldin A (5 μg/ml), or cycloheximide (1 mg/ml) and were used as target cells in a CTL assay. The responder cells were CD8+ T cells from three HLA A0201-positive non-HIV-1-infected donors (Fig. 5A) and three HLA A0201-positive HIV-1-infected donors (Fig. 5B). The recognition of EBV BMLF1280-288- and HIV-1 RT476-484-expressing DCs by CTLs was sensitive to the effect of brefeldin A or cycloheximide but not to that of lactacystin as judged by comparison to non-inhibitor-treated DCs from three non-HIV-1-infected and three HIV-1-infected individuals, respectively.
Taken together, these results indicate that HLA class I processing of exogenous 9-mer peptides by iDCs and mDCs from both non-HIV-1-infected and infected persons occurs in part through intracellular pathways.
Effects of metabolic inhibitors on the HLA class I processing pathway of N and C termini of EBV BMLF1280-288 and HIV-1 p1777-85 peptides. Peptides must be of a precise size (usually 8, 9, or 10 amino acid residues) to bind to an MHC class I molecule. The presence of either the amino- or the carboxyl-terminal flank of dominant peptides is suboptimal for antigen presentation (41). The N-terminal flanks of peptides are trimmed by aminopeptidases in the cytosol, and this process is resistant to proteasome inhibitors such as lactacystin. Trimming of the C-terminal flanks of peptides occurs in the proteasome and consequently is blocked by proteasome inhibitors. Therefore, to investigate further the intracellular peptide-processing capacity of DCs, we assessed the abilities of DCs from HIV-1-infected subjects on combination antiretroviral therapy and uninfected persons to process suboptimal viral peptides into their optimal forms for HLA class I presentation to CD8+ T cells.
We compared a hydrophobic 9-mer EBV BMLF1280-288 peptide (G-L [GLCTLVAML]) (Table 1) to its hydrophobic N-terminal flanking sequence (AIQNAGLCTLVAML) and acidic C-terminal flanking sequence (GLCTLVAMLEETIFW) in non-HIV-1-infected donors, and a hydrophobic 9-mer HIV-1 p17 peptide (S-L [SLYNTVATL]) with its acidic N-terminal flanking sequence (GSEELRSLYNTVATL) and hydrophobic C-terminal flanking sequence (SLYNTVATLYCVHQR) in HIV-1-infected subjects, for presentation by iDCs and mDCs to CD8+ T cells. For these studies, iDCs and mDCs were either left untreated or treated with various concentrations of lactacystin, brefeldin A, or cycloheximide for 30 min, then washed and loaded for 2 h with 10 μg of the 9-mer sequence, N-terminal flanking sequence, or C-terminal flanking sequence/ml. CD8+ T cells were then cultured with these APCs in the 18-h IFN- Elispot assay.
The data show that, as expected, iDCs from HIV-1-negative (Fig. 6A) and HIV-1-infected (Fig. 7A) subjects induced less IFN- production than mDCs (Fig. 6B and 7B, respectively) in response to these peptides at concentrations of 1 μg/ml (data not shown) or 10 μg/ml (Fig. 6 and 7). Lactacystin blocked the presentation of the C-terminal extension but not the N-terminal extension or the 9-mer EBV and HIV-1 peptides by DCs to CD8+ T cells from both HIV-1-negative (Fig. 6) and HIV-1-infected (Fig. 7) persons in a concentration-dependent fashion. In contrast, brefeldin A and cycloheximide blocked the presentation of all three peptides (C-terminal extension, N-terminal extension, and 9-mer peptides). The same effect was evident for the 1 μg/ml (data not shown) and 10 μg/ml (Fig. 6 and 7) concentrations of the peptides. Notably, both the binding affinity and the charge of a peptide are related to the efficiency of attachment and transport into APCs (3, 9). However, these peptides did not differ in their capacity to be blocked by these metabolic inhibitors, based on their HLA class I binding affinities and their neutral, anionic, or cationic charges.
These results indicate that iDCs and mDCs derived from HIV-1-infected persons on combination drug therapy and those from non-HIV-1-infected persons process exogenous peptides for presentation by HLA class I molecules to CD8+ T cells through similar protease-trimming pathways and with comparable efficiencies.
DISCUSSION
Our study demonstrates that DCs from HIV-1-infected persons on combination antiretroviral therapy have a capacity similar to those from non-HIV-1-infected persons to process and present exogenously loaded synthetic peptides to CD8+ T cells. The data show that mDCs derived from the blood of these individuals were more potent APCs than iDCs. This result was associated with an increase in the expression of surface molecules involved in the activation of T cells on the DCs after maturation. Thus, we noted an increase in the level of HLA class I molecules that are required for the presentation of peptides to CD8+ T cells (19), as well as in the level and percentage of cells expressing the T-cell coreceptor CD86 (11) and the T-cell activation factor CD83 (30), after treatment of iDCs with CD40L. These mDCs also expressed higher levels of HLA class II (i.e., HLA-DR). There was no significant difference in the expression of these molecules by DCs between HIV-1-infected and uninfected persons. This finding confirms and extends the results of previous studies from our laboratory showing that marker expression on DCs derived from peripheral blood myeloid precursors of HIV-1-infected persons on combination antiretroviral therapy, and matured with either trimeric CD40L or monocyte-conditioned culture medium, is comparable to that from HIV-1-negative persons (17).
DCs matured with CD40L were efficient APCs for presenting exogenous peptides representing optimal, immunodominant, HLA A0201-restricted viral epitopes for induction of IFN- production in peptide-specific CD8+ T-cell lines. These cell lines controlled for the potential effects of low CD8+ T-cell reactivity known to evolve during long-term combination antiretroviral therapy (27, 40). Even with this lower T-cell activity in such HIV-1-infected subjects, we showed that DCs loaded with peptides could stimulate blood-derived, autologous CD8+ T cells better than the peptides alone. The effects of CD40L, normally provided by CD4+ T cells, on DC stimulation of CD8+ T cells have been related to increases in the surface expression of HLA and costimulatory molecules, as well as to production of cytokines such as IL-12 and IL-15 (48). The iDCs that were loaded with the exogenous 9-mer viral peptides could activate CD8+ T cells to a significant degree, but a lesser degree than mDCs. This result was found for viral peptides with a range of binding affinities for HLA class I and different solubilities. Interestingly, enriched CD8+ T cells and CD8+ T-cell lines were able to respond to these 9-mer peptides in the absence of added APCs, albeit to a lesser degree than T cells stimulated with peptide-loaded DCs. The results suggest that CD8+ T cells were acting as both antigen-presenting and responding cells, a conclusion supported by a recent report that nonprofessional APCs in PBMCs can process peptides for presentation to CD8+ T cells (20).
The increase in T-cell response obtained by using DCs as APCs in our study was statistically significant and approximately 2- to 10-fold higher than that achieved by overnight stimulation of T cells with peptides alone. It may be possible to attain greater levels of response by long-term priming of na?ve T cells (21, 26, 52) in addition to activation of the limited number of memory CD8+ T cells specific for each peptide. Nevertheless, these data support the use of mDCs as APCs for activation of T-cell responses in immunotherapy trials (15).
Approaches to immunotherapy of cancers as well as HIV-1 infection have included the use of DCs loaded either in vivo or ex vivo with single peptides or pools of peptides as antigens (5, 7, 38). Rock and colleagues established that exogenous peptides representing minimal, 8- to 10-mer epitopes for CD8+ T cells do not require intracellular processing by APCs, as they can bind directly to HLA class I molecules at the cell surface (42). Such peptides, however, can be internalized and bind to MHC class I molecules in the ER (14, 33). This processing could relate to the efficacy of DCs loaded with these peptides in immunotherapy of HIV-1 infection. We therefore examined the mechanisms and efficiency of processing of peptides by DCs from non-HIV-1-infected and infected persons. For this purpose, we used metabolic inhibitors to delineate various stages of processing of the extracellular peptides by DCs and other APCs from non-HIV-1-infected and infected subjects. We demonstrated that blocking of intracellular vesicular trafficking in the DCs with brefeldin A reduced the stimulation of CD8+ T cells by 9-mer viral peptides. We extended these findings by assessing the capacities of DCs from HIV-1-infected subjects on combination antiretroviral therapy and uninfected persons to process viral peptides into their optimal forms for HLA class I presentation to CD8+ T cells. Peptides ideally must be a precise size (i.e., 8, 9, or 10 amino acid residues) to bind to an MHC class I molecule. The presence of either the amino- or the carboxyl-terminal flank of dominant peptides is suboptimal for antigen presentation. The N-terminal flanks of peptides are trimmed by aminopeptidases in the cytosol, and this trimming is resistant to the proteasome inhibitor lactacystin. Trimming of the C-terminal flanks of peptides occurs in the proteasome and consequently is blocked by lactacystin. Our results show that DCs from both non-HIV-1-infected and HIV-1-infected subjects were able to process exogenous 9-mer peptides (EBV BMLF1280-288 and HIV-1 p1777-85) with 5 or 6 additional amino acids at their N-terminal or C-terminal flanking end with similar efficiencies. Lactacystin inhibited the processing of the C-terminal flanking peptides, but not of the N-terminal flanking peptides or minimal 9-mer peptides, similarly in both iDCs and mDCs from uninfected and HIV-1-infected subjects. Brefeldin A, however, inhibited processing of all three forms of these exogenously delivered peptides in iDCs and mDCs, as did cycloheximide. The effects of these inhibitors were similar for peptides with different HLA class I binding affinities and solubilities. These data concur with the generation of HIV-1 p1777-85, which is known to be dependent on C-terminal trimming in the proteasome and N-terminal trimming by aminopeptidases in the cytosol (13, 18).
These results confirm that DCs from uninfected and HIV-1-infected persons have similar capacities for intracellular processing of exogenous peptides with a range of binding affinities and charges for presentation to CD8+ T cells. The results also indicate that intracellular uptake and processing are involved in the presentation of these exogenous 9-mer peptides to CD8+ T cells by professional APCs. In this regard, Andrieu and colleagues (1, 2) have recently reported that HIV-1 RT476-484 or its N-terminal extension RT461-484 when bound to a lipid (N-palmytoyl-lysine) is processed intracellularly by iDCs of HIV-1-negative subjects for presentation to CD8+ T-cell lines. We found, however, that the APCs with the greatest capacity for activating peptide-specific CD8+ T cells were mDCs. Moreover, the relatively lower inhibitory effects of brefeldin A on stimulation of T cells by mDCs than on stimulation by the other APCs in our study support the notion that viral peptides are processed in part by direct binding to HLA class I molecules at the cell surface. Factors that could contribute to the relative resistance of mDCs to the effects of these inhibitors are their lack of highly active endocytosis (43) and greater expression of surface HLA class I molecules (31). These factors also may be necessary to counter the limitation on induction of anti-peptide CD8+ T cells due to rapid turnover of MHC class I-complexed peptides by DCs (32).
A major criticism of peptide immunotherapy in HIV-1 infection is the narrow HLA restriction and limited immunodominance of viral peptide vaccines. This limitation, however, may be overcome by use of peptides that bind to multiple HLA class I molecules ("HLA supertype peptides"), a strategy that effectively broadens the targeted population (44). Moreover, peptide-based cancer vaccines can induce T-cell reactivity specific for other cancer antigens that are distinct from those used in the vaccine (a process termed determinant spreading, or epitope spreading) (39). It is postulated that lysis of cancer cells by CD8+ T cells specific for the vaccinating peptides releases other cancer antigens that are cross-presented to CD8+ T cells by HLA class I molecules on DCs. Such mechanisms could increase the potential efficacy of immunotherapy of HIV-1 infection with HIV-1 peptides loaded into DCs ex vivo or targeted to DCs in vivo. Our study supports the use of DC-based peptide immunotherapy to elicit broad, prolonged, and elevated T-cell responses to HIV-1 in persons receiving combination antiretroviral drug treatment.
ACKNOWLEDGMENTS
This work was supported by National Institute of Allergy and Infectious Diseases grants R01 AI41870, U01 AI37984, and U01 AI35041 and by a grant from Merck Research Laboratories.
We thank S. D. Lyman, E. Thomas, K. Phalmer, and K. Picha (Amgen/Immunex, Seattle, Wash.) for providing the CD40L; A. Hoji, C. Kalinyak, L. Borowski, W. Jiang, L. Zheng, X. Zhao, and P. Zhang for technical assistance; and W. Buchanan and C. Tripoli for clinical assistance.
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