Frank, Ines; Santos, John J.; Mehlhop, Erin; Villamide-Herrera, Loreley; Santisteban, Christine; Gettie, Agegnehu; Ignatius, Ralf; Lifson, Jeffrey D.; Pope, Melissa
It is becoming clear that a successful vaccine strategy against HIV-1 will likely have to induce broad specific immune responses comprising the CD4+ and CD8+ T-cell arms as well as neutralizing antibodies (Abs). 1–6 For evaluation of many vaccine approaches, experimental simian immunodeficiency virus (SIV) infection of rhesus macaques 7 is arguably the best animal model system used for immunization and virus challenge studies. Attempts to induce immunity in macaques against vigorous SIV challenge have used combinations of subunit proteins, live-attenuated or recombinant viral vectors, and/or DNA immunization approaches. 6 Despite some modest successes, 1–6,8–10 most approaches have not provided solid protection against robust challenges with pathogenic viruses, stressing the need for improved strategies. Given their status as the most potent antigen-presenting cells (APCs) in the body and their central role in inducing immune responses, many investigators believe that delivery of appropriate antigens (Ags) to dendritic cells (DCs) will be an important step in achieving an effective vaccine. Except for 1 report, this has proved problematic. 11–13
Myeloid-derived DCs (MDCs) are potent professional APCs that induce strong Ag-specific immune responses. DCs have been shown to specifically stimulate CD4+ TH1 cells and cytotoxic T lymphocytes (CTLs), to activate B cells, 14,15 and to elicit T-cell memory. 11 DCs show specific characteristics based on their differentiation stage. Typically, as immature DCs, they are preferentially located at the body surfaces and in blood and are well equipped to nonspecifically ingest large amounts of Ag via fluid phase uptake, which, along with locally secreted inflammatory stimuli, leads to their activation. Triggered DCs migrate to lymph nodes (LNs) using multidrug resistance proteins and the homing receptor CCR7, 16 undergoing further maturation, which subsequently leads to enhanced capacity to induce Ag-specific activation of naive and memory T- and B-cell responses on interaction with lymphocytes in the LNs. 17 Mature DCs at this stage characteristically possess limited phagocytic capacities but retain receptor-mediated uptake mechanisms. 18,19
MDCs from healthy and SIV-infected macaques are comparable to their human counterparts in phenotype, function, and how they interact with immunodeficiency viruses. 20–26 Similar to the results for human cells, 27 macaque mature monocyte-derived DCs (moDCs) are most reliably obtained by exposing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 cultured monocytes to a cocktail of inflammatory factors, including prostaglandin E2 (PGE2), tumor necrosis factor-α (TNFα), IL-1β, and IL-6. 24 In particular, cocktail-matured moDCs exhibit a reproducibly uniform phenotype, enhanced viability, and potent immunostimulatory capacities while maintaining their receptor-mediated uptake mechanisms. 24
DCs play a central yet dichotomous role in the biology of primate lentivirus infection. On one hand, DCs are critically involved as key APCs in inducing virus-specific immune responses. 11,13 On the other hand, DCs efficiently transmit HIV-1 and SIV to T cells, resulting in potent amplification of infection (S. Turville et al, submitted). 28,28a To clarify this critical aspect of AIDS pathogenesis, we have developed methods for studying virus-DC interactions in vitro. 18,29 This work has been facilitated by the availability of a novel form of chemically inactivated virus that retains conformationally and functionally intact envelope glycoproteins. 30,31 Treatment of retroviruses with 2,2′-dithiodipyridine (aldrithiol-2 [AT-2]; Aldrich, Milwaukee, WI) results in covalent modification of the free sulfhydryl groups of cysteine residues on internal structural proteins without appreciably affecting the disulfide-bonded cysteines of the proteins on the virion surface. 30,32 In practice, this results in noninfectious virions with functionally intact envelope glycoproteins. Such virions are being evaluated as a candidate vaccine immunogen (J. Lifson et al, in preparation) 33,34 and are also proving to be useful reagents for studies of virion interactions with various cellular populations. 18,24,29,35–39 For such studies, AT-2–inactivated virions offer the advantage of being able to monitor authentic envelope glycoprotein–dependent interactions of native virions with host cells and cell surface receptors in the absence of potentially confounding effects due to concomitant infection.
In this study, we primarily assessed the comparative ability of AT-2 SIV to stimulate lymphocyte responses in mononuclear cells isolated from blood (peripheral blood mononuclear cells [PBMCs]) and LNs as well as the capacity of immature and mature moDCs to induce virus-specific T-cell responses after being loaded with the inactivated virus. AT-2 SIV activated dose-dependent SIV-specific interferon-γ (IFNγ) and proliferative responses by CD4+ and CD8+ T cells in contrast to the predominant CD8+ T-cell responses induced by SIV-recombinant canarypox or vaccinia vectors. When mature DCs were loaded with AT-2 SIV, they reproducibly stimulated strong CD4+ and CD8+ T-cell responses, whereas the immature DCs preferentially led to CD4+ T-cell activation. In both cases, the response was independent of the amount of gp120 carried per virion. Hopefully, improved understanding of these attributes of DC-virus interactions will help to advance applications for in vivo priming against SIV and facilitate the development of effective vaccine approaches for the prevention of HIV-1 infection and AIDS.
MATERIALS AND METHODS
Adult male and female rhesus macaques (Macaca mulatta) used in this study were housed at the Tulane National Primate Research Center (TNPRC) and tested negative by polymerase chain reaction (PCR) for simian type D retroviruses (SRV) and simian T-cell leukemia virus (STLV-1). The weight and age of the animals at the time of study initiation ranged from 13 to 18 kg and 10 to 12 years, respectively. Both naive and SIV-infected macaques were used for these studies. All animals were healthy at the time of study unless otherwise indicated. Animals were anesthetized with ketamine-HCl (10 mg/kg) prior to all procedures. Based on a previously published protocol known to prime against SIV, 40 animals were inoculated via intravenous injection of 2.35 × 104 50% tissue culture infectious doses (TCID50) of SIVmac239 delta nef (delta nef) 15 weeks prior to being challenged intravenously with 102 TCID50 of SIVmac239 wild type (AR 76, AR 77, AR 81, AR 83, AR 84, AT 62, M284, L507, P075). Such delta nef–vaccinated and wild-type–challenged animals show strong SIV-specific immune responses and have not shown evidence of SIV-associated disease over at least 2 years of postchallenge follow-up. For the present studies, these macaques have been used as a source of SIV-primed T cells to monitor the presentation of AT-2 SIV in vitro. Peripheral blood or superficial LN samples were collected to monitor SIV-specific T-cell responses. To measure the presentation of AT-2 SIV by different APC populations, DCs were generated from CD14+ cells isolated from blood (see below), and the animals were bled again approximately 1 week later to obtain autologous T cells (and fresh CD14+ cells where necessary). The blood and LNs from 1 monkey infected with wild-type SIVmac239 (M806) that had to be euthanized for health reasons were also studied. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the TNPRC. Animal care procedures were in compliance with the regulations detailed under the Animal Welfare Act and in the “Guide for the Care and Use of Laboratory Animals.”
Culture Medium and Dendritic Cell Maturation Reagents
The medium used throughout these studies was RPMI 1640 (Cellgro; Fisher Scientific, Springfield, NJ) supplemented with 2 mM of l-glutamine (GIBCO-BRL Life Technologies, Grand Island, NY), 50 μM of 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM of HEPES (GIBCO-BRL Life Technologies), penicillin (100 U/mL)-streptomycin (100 μg/mL) (GIBCO-BRL Life Technologies), and 1% heparinized human plasma (complete RPMI, “cRPMI”). Recombinant human (rh) IL-4, IL-1β, IL-6, and rh TNFα were purchased from R&D Systems (Minneapolis, MN), rh GM-CSF from Berlex Laboratories (Montville, NJ), and PGE2 (P6532) from Sigma Chemical Company.
Infectious and chemically inactivated noninfectious SIVmneE11S (E11S) was provided by the AIDS Vaccine Program (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD). E11S was obtained from supernatants of the cloned E11S cell line. 41 Wild-type SIVmac239 (239) was generously provided by R. Desrosiers, Harvard Medical School, and the mutant variants of SIVmac239 (SIVmac239/251 tail and SIVmac239/C5) were provided by J. Hoxie, University of Pennsylvania. SIVmac239 and SIVmac239/251 tail were grown in the 174 × CEM cell line (obtained from the NIH AIDS Research and Reference Reagent Program). SIVmac239/251 tail virions contain approximately 10- to 20-fold more surface gp120 than wild-type SIVmac239, whereas the gp120 intermediate variant SIVmac239/C5 was grown in the SuP T1 cell line, and virions contained approximately 5- to 10-fold less gp120 than the SIVmac239/251 tail isolate. SIV CP-mac and SIV NC-mac 42 (both derived from SIVmacBK28) were propagated in SuP T1 cells transduced to express CCR5. 43 Although SIV CP-mac virions have full-length TM and contain high levels of gp120SU with an SU/TM ratio of 1:1 comparable to SIVmac239/251 tail, SIV NC-mac has approximately the same amount of TM as SIV CP-mac but approximately 10-fold less gp120, with an SU/TM ratio of approximately 1:10 instead of 1:1. 43 Preparations of infectious viruses were inactivated by treatment with AT-2 as described. 30,31 Virus content of purified concentrated preparations was determined with an Ag capture immunoassay for the SIV gag p27 Ag (AIDS Vaccine Program) and/or by high-performance liquid chromatography (HPLC) analysis as described. 43 Virus stocks were diluted in 1% bovine serum albumin (BSA; Intergen, New York, NY) in phosphate-buffered saline (PBS) and stored as aliquots (3 μg of p27 Ag equivalents per milliliter) at −80°C until use. SIV-recombinant canarypox virus vCP180 (encoding the SIVmac142 gag, pol, and env genes) and its parental strain (ALVAC) as well as vaccinia virus vP1071 (encoding the SIVmac142 gag, pol, and env genes) and its parental vector (NYVAC) were kindly provided by Aventis Pasteur (Aventis Pasteur, Swiftwater, PA) and stored at 2 × 109 PFU/μL at −80°C.
Generation of Cell Subsets
Peripheral Blood Mononuclear Cells and T Cells
PBMCs were isolated from heparinized blood from either SIV-infected or naive macaques using a Ficoll-Hypaque density gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden). For the depletion of specific cell populations from whole PBMCs, the Magnetic Cell Sorting System (MACS; Miltenyi Biotech, Auburn, CA) was employed.
To analyze whether SIV-specific responses are mediated by CD4+ or CD8+ cell subsets, PBMC suspensions were divided, and fractions were either depleted of CD8+ or CD4+ cell subsets by directly adding anti-CD8 (clone SK1) or anti-CD4 MACS beads before the cell suspensions were loaded onto separation columns (LS columns; Miltenyi Biotech). To remove distinct APC subsets, PBMCs were incubated with phycoerythrin (PE)-labeled anti-CD14, anti-CD20, anti-human leukocyte antigen (HLA)-DR Abs, or isotype Ab control (fluorescein isothiocyanate [FITC]-IgG1 and PE-IgG2a), all from Becton Dickinson (Mountainview, CA), versus medium alone for 20 minutes on ice. The cells were then washed and incubated with anti-PE-labeled MACS beads before cells were loaded onto depletion columns (LD columns; Miltenyi Biotech).
Bulk purified T-cell populations were obtained by adding anti-CD14, anti-CD11b, and anti-HLA-DR MACS beads to the PBMC suspension to remove APCs and natural killer (NK) cells. For the analysis of CD4+- or CD8+-mediated SIV-specific T-cell responses, suspensions were again divided, and anti-CD8 or anti-CD4 MACS beads (as above) were included with the anti-CD11b/CD14/HLA-DR beads for the depletion. CD11b−CD14−HLA-DR−CD8− fractions provided the CD4+ T cells, and CD11b−CD14−HLA-DR−CD4− fractions provided the CD8+ T cells. When CD14+ cells were used as APCs, PBMCs were first depleted of the CD14+ cells (keeping that fraction as an APC population), and the CD14− cells were subsequently depleted of CD11b+HLA-DR+ (and CD4+ or CD8+) cells.
To characterize macaque T cells and analyze the purity of cell preparations, cells were monitored by 2-color flow cytometry using an FITC-conjugated mouse monoclonal antibody (mAb) combined with a panel of PE-conjugated mouse mAbs. T-cell preparations (bulk vs. CD8- or CD4-depleted cell fractions) were stained with a FITC-conjugated anti-human CD3 (V 5T-CD30.05; PharMingen) and PE-conjugated anti-CD4 (Leu 3; Becton Dickinson), anti-CD8 (Leu 2; Becton Dickinson), anti-CD11b (F6.2; Exalpha Biologic, MA), anti-CD14 (Becton Dickinson), anti-CD20 (Becton Dickinson), or anti-HLA-DR (Becton Dickinson). For the analysis of purity in the populations depleted of the various APCs (CD14+, CD20+, or HLA-DR+ cell subsets), selected cell fractions were stained with FITC-conjugated anti-HLA-DR (Becton Dickinson) combined with the PE-conjugated anti-CD4, anti-CD8, anti-CD11b, anti-CD14, and anti-CD20. Isotype-matched controls (FITC-IgG1 and PE-IgG2a; Becton Dickinson) were included for all Ab staining experiments. The staining was performed as previously described. 18
Lymph Node Cells
Superficial inguinal LNs were biopsied from the indicated delta nef–infected wild-type–challenged animals using standard surgical procedures or a wild-type–infected animal at necropsy. Single cell suspensions of total leukocytes were obtained as previously described. 21,22 LN cells (LNCs) were kept as a bulk population or depleted of either CD8+ or CD4+ cell subsets (see above), and the T-cell purity was verified by 2-color flow cytometry as described previously.
Macaque moDCs were generated from a highly enriched population of CD14+ cells as previously described. 18,24 After 6 to 7 days of culture in GM-CSF (1000 U/mL) and IL-4 (100 U/mL), immature DCs were used. When comparing immature and mature DCs, immature DCs (day 6) were cultured for an additional 2 days in medium containing GM-CSF and IL-4 (maintaining immature DCs) or were matured with a cocktail of IL-1β (10 ng/mL), IL-6 (10 ng/mL), TNFα (10 ng/mL), and PGE2 (10−6 M). 24 The phenotype of immature and mature DCs as well as the purity of DC populations was routinely monitored by 2-color flow cytometry as described previously. 18 For phenotypic comparison of DCs and CD14+ monocytes, FITC-conjugated anti-HLA-DR was combined with PE-conjugated anti-CD14, anti-CD25, anti-CD80, and anti-CD86 (all from Becton Dickinson) as well as anti-CD83 (PN IM2218; Immunotech-Beckman-Coulter, Marseille, France). Phenotypically, immature DCs express negligible levels of CD14 unlike monocytes; both express little if any CD25, CD80, or CD83 but show intermediate levels of CD86. In contrast, mature DCs significantly upregulate all the activation markers (CD25, CD80, CD83, and CD86) while remaining negative/weak for CD14.
Loading of Antigen-Presenting Cells with Aldrithiol-2 Simian Immunodeficiency Virus
For the immune assays, aliquots of 1 to 2 × 105 DCs or CD14+ monocytes isolated from infected animals were used, and 3 to 5 × 105 cells from naive and infected macaques were used for immunostaining. Cells were placed into 1.5-mL Eppendorf tubes, pelleted, resuspended with AT-2 SIV (30 ng of p27 per 105 cells) in 50 μL of cRPMI, and incubated for 1 hour at 37°C. Control cells were incubated with the equivalent amount of buffer (1% BSA in PBS). The cells were then washed 5 times to remove cell-free virus, 18 and viable large cells were recounted by Trypan blue exclusion and used in immune assays or monitored for the presence of viral protein by immunostaining (see below). 18
Interferon-γ Enzyme-Linked Immunospot Assay
The secretion of IFNγ by SIV-specific T cells was measured by IFNγ enzyme-linked immunospot (ELISPOT) assays (U-Cytech, Utrecht, The Netherlands). The IFNγ ELISPOT assays were performed according to the manufacturer's instructions and as previously described. 24 PBMCs/LNCs or the variously depleted fractions were plated 2 × 105 per well in 100 μL of medium. Cells were incubated with AT-2 SIV (0.003–30 ng of p27 per 2 × 105 cells) versus medium as the control, with vaccinia virus vP1071 and its parental vector (NYVAC), or with canarypox virus vCP180 and its parental vector (ALVAC) at a multiplicity of infection (MOI) of 1 or 3, respectively. For the APC-T-cell cultures, T cells were plated (2 × 105 per well per 50 μL) and cocultured with 50 μL of autologous DCs or CD14+ cells pulsed with virus versus buffer at APC/T-cell ratios ranging from 1:20 to 1:180. To verify the capacity of the T cells to respond to stimulation, 5 ng/mL of the superantigen staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL) was added to 5 × 104 cells. For the APC-T-cell assay, SEB was added to cocultures of T cells (5 × 104 per well) with SIV-pulsed or untreated APCs added at the indicated DC/T-cell ratios. After an 18- to 24-hour incubation at 37°C, the assay was developed according to the manufacturer's instructions. IFNγ spot-forming cells (SFCs) were enumerated under an inverted light microscope. Results are represented as means of triplicate cultures of single experiments.
T-Cell Proliferation Assay
To measure T-cell expansion, AT-2 SIV (0.003–30 ng of p27) was added to 1 × 105 PBMCs, LNCs, or cells depleted of CD8+ or CD4+ subsets. Cells were cultured in a final volume of 100 μL per well in 96-well round-bottomed plates (Microtest U-Bottom; Becton Dickinson, NJ). Alternatively, DCs or CD14+ cells were pulsed with AT-2 SIV or buffer (see above) and added to 1 × 105 T cells at a ratio of 1:20 to 1:180 per well of a 96-well flat-bottomed plate (Microtest 96; Becton Dickinson). After 5 days of culture at 37°C, 1 μCi of (3H)-thymidine (3H-TdR) was added to each well, and the amount of 3H-TdR incorporated during the last 8 hours of culture was measured to assess T-cell division. To verify the responsiveness of the T cells in each of the cultures, control cultures were set up where cells were incubated with SEB (5 ng/mL) for 3 days. T-cell proliferation was measured using a semiautomated harvesting system (Brandel MWXR-96TI; Gaithersburg, MD) and liquid scintillation counter (1450 Wallac MicroBeta JET; Perkin Elmer Life Sciences, Turku, Finland). Results are represented as mean counts per minute (cpm) of 3H-TdR uptake of triplicate cultures of single experiments.
Viral Envelope Staining with Biotinylated CD4-IgG2
The presence of AT-2 SIV associated with immature and mature moDCs or with CD14+ monocytes was examined by using the biotinylated human CD4-IgG2, 44 recognizing the CD4-binding epitope of conformationally intact gp120. 18 In brief, virus- or buffer-treated cells were adhered to Alcian blue precoated slides and fixed with 4% paraformaldehyde before nonspecific binding was blocked (100 μg/mL of human IgG; Jackson ImmunoResearch Laboratories) and were thereafter incubated with 25 μg/mL of biotinylated CD4-IgG2 or biotinylated human IgG (Jackson ImmunoResearch Laboratories), followed by horseradish peroxidase (HRP)-conjugated streptavidin (1:100) and the amplification reagent (fluorescein-labeled TSA kit). Before slides were mounted, the cell nuclei were stained with DAPI (1.75 ng/mL of D-1306; Molecular Probes). Mounted slides were then examined using a Nikon Eclipse E800 epifluorescence microscope equipped with the Image-Pro Plus deconvolution module (Media Cybernetics, Silver Spring, MD). Composite figures were made using Photoshop (Adobe Systems).
Dose-Dependent Activation of Simian Immunodeficiency Virus–Specific T Cells in the Blood and Lymph Nodes of Simian Immunodeficiency Virus–Infected Macaques by Aldrithiol-2 Simian Immunodeficiency Virus
To evaluate the antigenicity of AT-2 SIV, the activation of SIV-primed T cells in the PBMCs or LNCs of SIV-infected versus naive macaques was analyzed by assessing IFNγ-secreting T cells in ELISPOT and T-cell expansion in proliferation assays following in vitro stimulation with inactivated virus. Rhesus macaques chronically infected with SIVmac239 delta nef that had controlled subsequent challenge with wild-type SIVmac239 were used extensively in these studies as a reliable source of SIV-primed T cells with intact immune function. 24 Cultures of PBMCs from these animals (Figs. 1A, B) showed comparable SIV-specific IFNγ responses to AT-2–inactivated preparations of 2 different SIV isolates: SIVmneE11S (E11S) and SIVmac239 (239). In contrast to infected animals, the PBMCs from naive animals did not respond to even the highest dose of virus tested (see Fig. 1A).
To evaluate whether the amount of viral surface envelope glycoprotein on virions affects the level of T-cell activation, titrated doses of several AT-2–inactivated virus isolates differing in virion envelope glycoprotein content were added to PBMC cultures and tested for their ability to stimulate IFNγ responses (see Fig. 1C). Virus isolates compared included AT-2–treated SIVmac239/251 tail, SIV CP-mac, and SIVmneE11S as examples of high envelope–expressing viruses, SIVmac239/C5 as an example of an intermediate envelope–expressing virus, and SIVmac239 and SIV NC-mac as examples of viruses expressing low gp120 levels. IFNγ responses were dependent on the amount of virus added to the cultures (normalized by p27 content) but were not influenced by the virus isolate or the gp120SU content of the virions (see Fig. 1C). In parallel testing of PBMCs and LNCs from a chronically delta nef–infected wild-type–challenged animal, comparable dose-dependent SIV-specific IFNγ release and T-cell expansion in response to AT-2 E11S were observed (see Fig. 1D). These results confirm that AT-2–inactivated viruses are able to stimulate potent SIV-specific T-cell responses in whole PBMC and LNC assays in vitro.
Aldrithiol-2 Simian Immunodeficiency Virus Induces CD4+ and CD8+ T-Cell Responses in Blood and Lymph Nodes
To identify the T cells activated by AT-2 SIV, PBMCs or LNCs of SIV-primed or naive animals were depleted of either CD8+ or CD4+ cell subsets and tested in parallel with unfractionated PBMCs and LNCs. SIV recombinant canarypox and vaccinia viruses were compared with AT-2 SIV for their ability to stimulate CD4+ and CD8+ T-cell subsets. As shown in Figure 2, both AT-2 SIV and the viral vectors stimulated responses in the PBMCs and LNCs of SIV-infected animals. Notably, AT-2 SIV induced SIV-specific IFNγ secretion (see Figs. 2A, C, D) and proliferation (see Fig. 2B) of both CD4+ and CD8+ T-cell subsets in a dose-dependent manner. This is in contrast to the preferential CD8+ T-cell responses induced by SIV-recombinant vaccinia and canarypox viruses (see Figs. 2A, C, D). Thus, although variable in intensity, CD4+ and CD8+ T-cell responses against AT-2 SIV were reproducibly observed in the blood and LNs of several healthy delta-nef–infected and wild-type–challenged animals (see Figs. 2A–C) as well as in those of a wild-type–infected animal examined at necropsy (see Fig. 2D).
Simian Immunodeficiency Virus–Specific Interferon-γ Release in Peripheral Blood Mononuclear Cell Cultures by Aldrithiol-2 Simian Immunodeficiency Virus Requires Human Leukocyte Antigen-DR+ Accessory Cells
The fact that the AT-2–treated virions are able to activate both CD4+ T cells and CD8+ T cells indicated the requirement for APCs to capture and process whole virus and properly present viral Ags on major histocompatibility complex (MHC) class I and II molecules. Therefore, we investigated which APC subset(s) in PBMC cultures are responsible for the induction of T-cell responses against AT-2 SIV. APCs such as monocytes (CD14+), B cells (CD20+), and HLA-DR+ cells (comprising all APCs, including blood DCs) were selectively removed from PBMCs before the remaining cells were stimulated with AT-2 SIV in an IFNγ ELISPOT assay. The data from 3 separate experiments are summarized in Table 1. The efficiency of the removal of each subset was verified by FACS analyses in each experiment, and the depletion of the CD14+, CD20+, or HLA-DR+ cell fraction was routinely >95% (data not shown). Although neither the depletion of CD20+ cells nor the treatment with the matched isotype Ab (or medium) had a demonstrable impact on the stimulation of IFNγ production, the removal of CD14+ cells had a variable influence on the magnitude of the IFNγ responses measured (reducing responses from ∼15%–85%) (see Table 1). In contrast, SIV-specific T-cell responses were consistently abrogated after depletion of HLA-DR+ cells. These results indicated the requirement for HLA-DR+ APCs in the activation of CD4+ and CD8+ T cells rather than virus directly activating the T-cell subsets.
Aldrithiol-2 Simian Immunodeficiency Virus–Loaded Mature Dendritic Cells Efficiently Activate Simian Immunodeficiency Virus–Primed T Cells
The fact that B cells appeared not to take part and CD14+ cells only contributed to variable extents in the SIV-specific T-cell activation suggested the involvement of DCs in the T-cell activation of whole PBMCs or LNCs. DCs show differentiation stage-dependent functions. The use of in vitro generated moDCs enabled us to compare classic immature and mature DCs for their capacity to present AT-2 SIV and to induce virus-specific T-cell responses.
To compare the capacity of immature versus mature DCs to process and present captured AT-2 SIV, immature and mature DCs were pulsed with AT-2 SIV and washed to remove unbound virus before being recultured with autologous SIV-primed T cells at varying DC/T-cell ratios. Because CD14+ may contribute to some of the presentation of AT-2 SIV as indicated in Table 1, CD14+ monocytes were initially assayed in parallel. As summarized in Figure 3A, although SIV-specific T-cell responses such as proliferation and IFNγ were observed with the AT-2 SIV-loaded monocytes as APCs, greater responses were stimulated by DCs. Remarkably, the mature DCs tended to induce slightly higher IFNγ responses, and this was most apparent at the higher DC/T-cell ratios. Even at cell ratios of 1 APC to 180 T cells, immature and mature DCs provoked strong T-cell responses, whereas Ag presentation by monocytes did not. Although the magnitudes of each response varied for different donors, the patterns remained consistent between donors. This is in contrast to T cells isolated from naive animals, which did not show measurable responses, even when stimulated with AT-2 SIV–pulsed autologous mature DCs (see Fig. 3B). T-cell responses to SEB were strong and comparable in infected (data not shown) and naive animals (see Fig. 3B), verifying the functionality of the APCs and T cells in each culture.
We were interested to ascertain whether the lower responses induced by the monocytes may relate to the amount of virus Ags captured by these cells. Although both immature and mature DCs capture AT-2 SIV, mature DCs sequester virus in large vacuoles localized deeper within the cells, whereas immature DCs retain virus in smaller vacuoles that are typically more dispersed around the cell closer to the periphery. 18,29 To qualitatively compare the uptake of AT-2 SIV by DCs and CD14+ monocytes at the single cell level, we compared monocytes with immature and mature DCs from the same donor and incubated the cells for 1 hour at 37°C with AT-2 SIV before immunostaining for viral proteins. As shown in Figure 4, all 3 populations captured AT-2 SIV. Although the level of protein staining in CD14+ cells was qualitatively consistently lower than that seen in immature and mature DCs, the localization of the viral proteins was comparable to that of the immature DCs. Here, viral envelope proteins were detected in small vesicles that were mostly found closer to the cell periphery. This is true for naive (data not shown) and infected animals (see Fig. 4). Similar results have been observed with human cells, where lower levels of viral uptake by CD14+ cells compared with DCs were also confirmed by reverse transcriptase (RT) PCR analysis (data not shown).
CD4+ and CD8+ Simian Immunodeficiency Virus–Specific T-Cell Responses Are Predominantly Induced by Mature Dendritic Cells
The observation that both immature and mature DCs capture AT-2 SIV, hold it in different intracellular locations (see Fig. 4), 18 and possess the ability to process and present captured AT-2 SIV (see Fig. 3A) leads to the question of whether distinct DC populations might activate different T-cell subpopulations. Additionally, previous results strongly suggested that the uptake of AT-2 virus by DCs is dependent on the presence of functional viral envelope glycoproteins. 18,35 Therefore, we also addressed the question of whether uptake of more AT-2 SIV by DCs results in enhanced activation of T-cell subsets. To do so, DCs were loaded with AT-2 inactivated wild-type SIVmac239 virus (Low env) or SIVmac239/251 tail virus (High env), which varies in gp120SU content by approximately 10-fold, and then recultured with autologous CD4+ or CD8+ T cells. As shown in Figure 5, the gp120SU content of the virions tested did not appear to affect the type or magnitude of the T-cell response induced at the input virus dose tested. Thus, although less virus was captured by the DCs after pulsing with the low env–expressing SIV, 18 this was sufficient for the DCs to activate SIV-specific T cells. Strikingly, although immature DCs preferentially activated CD4+ T cells, mature DCs induced IFNγ release from both CD4+ and CD8+ T cells (see Fig. 5). These data suggest that mature DCs are also equipped to process cell-associated whole AT-2 SIV as well as to then present virion-derived peptides on MHC class I and II molecules to activate CD8+ and CD4+ T-cell responses.
To appreciate better how DCs can be used to boost vaccine efficacy, these studies evaluated the presentation of a chemically modified whole inactivated virion immunogen, which retains functional envelope glycoproteins, thereby mimicking authentic interactions with DCs and T cells. 18,29,30 By virtue of their particulate nature and functional envelope glycoproteins (even when expressed at low levels), these virions are taken up and presented by mature DCs to stimulate MHC class I– and II–restricted responses, activating CD8+ and CD4+ T cells. Preliminary data from in vivo studies in macaques indicate that inactivated SIV with conformationally and functionally intact envelope glycoproteins induces neutralizing Ab responses as well as CD4+ and CD8+ T-cell responses (J. Lifson et al, in preparation). Herein, we have also carefully studied some of the variables affecting the efficiency of Ag-specific stimulation mediated by such inactivated virions, including dose response relationships, the comparison of the responses stimulated by inactivated virions to those stimulated by the presentation of similar Ags via avipox vectors, the envelope content of the virions, and the ability of different APCs to present inactivated virion-derived Ags.
When added to in vivo primed SIV-specific T cells, whole inactivated virions successfully activated dose-dependent CD4+ and CD8+ T-cell responses. A qualitative shift in the T-cell response toward the sole activation of CD8+ T cells was noted when similar Ags were presented via classic vectors such as vaccinia or canarypox viruses, as previously described for HIV-1–specific responses. 45,46 The magnitudes of the (CD8+) responses against the SIV-recombinant avipox constructs were comparable to, if not stronger than, those induced in PBMCs by HIV-1–recombinant avipox vectors, 36,45 comparable to the levels seen when immature DCs were directly infected with HIV-1–recombinant vaccinia or canarypox virus and used to stimulate autologous HIV-1–specific T cells. 39,46 The observation of the significant CD8+ responses resulting from avipox vector stimulation compared with the more balanced CD4+ and CD8+ responses induced by AT-2 SIV reveals an important difference with possible major implications for vaccine potential.
The most prominent difference accounting for the qualitative distinctions between the activation of T cells through the presentation of inactivated virion-derived Ags versus similar Ags presented via the avipox vectors most likely lies in the nature of the Ag and how it is handled by the APC. In contrast to whole replication incompetent inactivated SIV, viral vectors infect APCs 46–48 and thereby lead to de novo biosynthesis of the encoded Ags within the APCs, representing the classic requirement for endogenously derived Ags to trigger CD8+ T cells, whereas Ag has to be captured by APCs from the environment (exogenously) to access the MHC class II processing pathway, likely accounting for the activation of CD4+ T cells by inactivated virus. Simultaneously, the stimulation of CD8+ T cells by AT-2 SIV is indicative of the involvement of a potent APC capable of introducing exogenous virus to the MHC class I processing pathway, probably occurring as a result of the virus fusing with the cell membranes of the APCs, allowing Ags to enter the cytosol and gain access to the MHC class I processing machinery. 35 Besides the capacity of the inactivated virus to activate CD4+ and CD8+ T cells, the fact that different virus clones were equally well recognized by the T cells indicated the potency of these inactivated whole virions as a promising Ag source in successfully providing a wide spectra of immunologically relevant Ags for the activation of T cells. Furthermore, these responses were comparable to the level of T-cell responses induced by live virus.
AT-2 SIV is internalized by both immature and mature DCs, with the mature DCs retaining larger numbers of intact particles even though viral RNA levels were the same or higher in immature DCs. 18 Remarkably, although the uptake of AT-2 SIV by DCs is influenced by the amount of gp120 expressed on the virus surface (higher levels of virus-associated envelope favor more virus uptake), 18 it did not have a significant impact on the magnitude or the type of T-cell response induced (in the DC/T-cell and PBMC assays). These data imply that the uptake of even small amounts of AT-2 SIV by DCs (and possibly other APCs) is enough for efficient processing of Ag to activate CD4+ or CD8+ T cells. Notably, in PBMCs using heated virus, in which envelope is stripped from the virion and the native conformation is lost, the magnitude of T-cell responses can only be reduced by as much as one third at the higher Ag doses (3–300 ng/mL) and two thirds at the lower doses (0.3 and 0.03 ng/mL) (data not shown). Removal of the bulk of the stripped envelope (by centrifugation) does not further influence the response to heated virus, suggesting that at least some responses are specific for determinants found in conformationally intact envelope. The possibility that some virus uptake by APCs occurs independent of envelope (which would allow other determinants to be presented) is likely. Definitive identification of the T-cell specificity (and frequency) induced by AT-2 viruses would require AT-2 virus–activated T cells to be restimulated with recombinant vectors expressing select viral genes or specific peptide pools.
A direct comparison of DCs at different activation stages showed that both immature and mature DCs activate autologous SIV-primed T cells in an Ag-specific manner, although the magnitude of the T-cell response was often higher with the mature DCs. These results are in agreement with the literature of previous in vitro and in vivo observations, which show that immature DCs loaded with the inactivated virus can lead to T-cell activation 35,36,39,49 and that the more activated IFNα–treated DCs lead to stronger responses than DCs generated with IL-4 and GM-CSF only. 49 Thus, these data suggest that immature DCs, although specialized for the uptake of Ag and less well equipped for the activation of T cells, are able to induce immune responses, whereas mature DCs, which have a diminished capacity of macropinocytic uptake but are well suited for Ag presentation, also have the capacity to internalize and process whole structurally intact virions and present the virion-derived peptides on MHC molecules. The finding that the Ag processing machinery is most efficient in mature DCs further supports this. 50 Our recent studies also revealed that although considerable amounts of AT-2 (or live) virus captured by immature and mature DCs is degraded, the virus proteins are localized in intracellular compartments that do not stain with classic endosomal/lysosomal markers (S. Turville et al, submitted). This contrasts where monomeric gp120 is located and suggests that whole virus diverts the normal processing pathways in DCs to favor virus spread over immune activation. Immature DCs tend to degrade virus more rapidly than mature DCs and may limit the access of virus to the Ag processing pathways. Studies to identify the unique compartment(s) in which immature versus mature DCs sequester and process virus are underway.
Closer analysis uncovered that immature DCs preferentially evoked CD4+ T-cell responses with only low levels of CD8+ T-cell activation, whereas matured DCs efficiently stimulated both CD4+ and CD8+ T cells. Thus, using the in vitro–generated immature and mature moDCs (phenotype was confirmed by FACS) revealed that capture of the inactivated virus by immature DCs 18 targets at least some of the virus to compartments allowing the association of processed antigenic peptides with MHC class II molecules, 51 facilitating the activation of CD4+ T cells. Interestingly, a recent report described how tubular endosomes extend within the DC and polarize toward the interacting T cell but only when Ag-laden DCs encounter T cells of the appropriate specificity, 52,53 explaining how maturing DCs increase the levels of MHC class II peptide complexes for more efficient T-cell activation. In this scenario, an SIV-specific DC/T-cell encounter could favor the direct transport of AT-2 SIV–derived peptide-loaded MHC class II molecules to the interaction site, thereby favoring the activation of SIV-specific CD4+ T cells. On the other hand, virus captured by DCs moves rapidly to the point of contact with a CD4+ T cell, inferring a mode of virus transmission to the T cell (S. Turville et al, submitted). 54 Although we have demonstrated that immature DCs can stimulate CD4+ T cells, it is possible that the virus hijacks this MHC class II peptide–transporting tubule machinery to facilitate DC-to-T-cell transmission of virus. If true, this would provide an effective way in which the virus could more efficiently target virus-specific CD4+ T cells as suggested by Douek et al. 55 This further highlights how the virus may specifically exploit immature DCs at the body surface to facilitate infection and cell-to-cell spread while inducing suboptimal immune responses.
In contrast, mature DCs stimulated CD4+ and CD8+ T-cell responses. Larsson et al 39 made similar observations, where mature DCs exposed to inactivated HIV-1 also activated both CD4+ and CD8+ T cells from seropositive donors. Noteworthy, the use of X4-versus R5-tropic strains or different DC activation stimuli (monocyte conditioned media [MCM] vs. a cocktail of inflammatory factors) had no significant impact on the induced immune response, indicating that the processing of exogenously captured AT-2 virus by mature DCs is independent of virus tropism. The ability of DCs to capture whole SIV particles and to process and present at least some of this to CD8+ T cells also agrees with earlier work that highlighted how this is largely mediated by virus fusing with the cell membrane (probably surface as well as compartment membranes) to access an exogenous MHC class I pathway. 35
The nature of DCs being able to direct qualitative aspects of the T-cell response may account for the CD4+ and CD8+ responses observed in PBMC cultures. The fact that the depletion of HLA-DR+ cells from PBMCs comprising all APCs, including MDCs and plasmacytoid-derived DCs (PDCs), completely abolished the T-cell responses strongly implicated the involvement of APCs in the activation of T cells. A further attempt to define the HLA-DR+ cell subset based on the surface expression of the MDC marker CD11c was not possible due to the heterogeneous expression pattern of CD11c on monkey leukocytes (I. Frank and M. Pope, unpublished observations). Therefore, the contribution of MDCs (which get activated during overnight culture to favor CD4+ and CD8+ responses; N. Teleshova and M. Pope, unpublished observations) to the presentation of AT-2 SIV in PBMC cultures could not be addressed in this way. Furthermore, depletion of the CD123+ PDCs, which represent less than 0.5% of the leukocytes in freshly isolated macaque blood (N. Teleshova and M. Pope, unpublished observation), did not significantly alter the T-cell response (data not shown). Direct analysis of the ability of PDCs and MDCs to present AT-2 SIV is being performed in separate ongoing studies.
In contrast to the depletion of HLA-DR+ cells, the response in PBMC cultures was unaffected by the depletion of B cells and was only partially diminished when CD14+ cell subsets were removed. Although CD14+ monocytes can capture and become infected by HIV-1/SIV, 56,57 their capacity to capture whole AT-2 SIV was marginal; more significantly, they were much less efficient in inducing strong T-cell responses compared with the DCs. Although the possibility that some monocytes carrying a few of the inactivated virions convert into macrophages cannot be excluded, macrophages are less likely responsible for the overall response due to their much weaker MHC class I–restricted Ag-presenting capacity compared with DCs. 35 Monocytes/macrophages also show only poor or no expression of CD80 and CD86, 2 costimulatory molecules critical for the activation of T cells (confirmed here for macaque CD14+ cells, data not shown). A subset of CD14+CD16+ cells representing a precursor for DCs in blood was recently characterized by the expression of high levels of HLA-DR and a robust capacity to induce T-cell proliferation. 58 In addition to resident MDCs, this newly defined subset of blood DC precursors could also be involved in the responses observed in the PBMC cultures.
This work demonstrates that in contrast to traditional viral vectors expressing HIV-1/SIV Ags, whole inactivated virions represent a reliable way to stimulate antigenically broad and strong dose-dependent CD4+ and CD8+ T-cell responses in the periphery as well as in LNs. Loaded onto DCs, exogenous AT-2 SIV is captured by immature and mature DCs and can access the MHC class II processing pathway in both cases, but only the virus associated with the mature DCs could also be presented on MHC class I molecules. Although a recent report suggested that AT-2 SIV–loaded maturing DCs can boost immunity and antiviral responses in vivo, 12 our study suggests that targeting fully mature DCs with AT-2 SIV might provide the most efficient stimulus to prime and/or boost SIV-specific immunity. AT-2 SIV represents a promising immunogen in the development and further improvement of antiviral therapeutic vaccine strategies against HIV-1.
Animals used for these studies were housed at the TNPRC. The authors thank James Blanchard, Richard Rockar, and Marion Ratterree for providing care and support for the animals; Ronald Desrosiers (Harvard Medical School/New England National Primate Research Center) and James Hoxie (University of Pennsylvania Medical School) for providing viruses used in these studies; Larry Arthur and Julian Bess, Jr (AIDS Vaccine Program, SAIC Frederick) for the AT-2–inac-tivated virus preparations; Elena Chertova (AIDS Vaccine Program) for characterization of the envelope glycoprotein content of these preparations; and William C. Olson (Progenics Pharmaceuticals, Tarrytown, NY) for providing the biotinylated CD4-IgG2. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 174 × CEM from Peter Cresswell.
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