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Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF-B Ligand, and TNF- in the Activation of Dendritic Cells and the Expansion of Viral Specific CD8⁺ T Cell Memory Responses in HIV-1-Infected and HIV-1-Uninfected Individuals ¹

Qigui Yu^*, Jenny X. Gu^*, Colin Kovacs^*, John Freedman, Elaine K. Thomas and Mario A. Ostrowski^2,*,

^* Clinical Sciences Division, University of Toronto, Toronto, Canada; St. Michael’s Hospital, University of Toronto, Toronto, Canada; and Immunex Corporation, Seattle, WA 98101

	Abstract

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Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-B ligand (RANKL), and TNF-, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF- alone to mature DCs and to expand CTL. In healthy volunteers, TNF- or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF- or RANKL in combination with CD40L were variable. TNF- and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF- enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4⁺ T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4⁺ T cells can provide help to CD8⁺ T cells independently of CD40L, RANKL, or TNF-.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A vigorous and sustained CD8⁺ T cell cytotoxic (CTL) response in the host is required to control infections caused by persistent viruses such as lymphocytic choriomeningitis virus, CMV, EBV, and possibly HIV-1 and SIV (1, 2, 3, 4, 5, 6, 7). In this regard, CD4⁺ T cell help has been shown to be instrumental in maintaining effective CTL memory responses, through at least two ways. In murine models, activated Ag-specific CD4⁺ T cells have been shown to help CTL by local IL-2 production and more importantly through the expression of CD40 ligand (CD40L)³ (8, 9, 10, 11). CD40L is a type II membrane protein of the TNF superfamily predominantly expressed on activated CD4⁺ T cells. CD40 is constitutively expressed on immature dendritic cells (DCs), and CD40 ligation of immature DCs induces their maturation and activation (12, 13), as manifested by the increased surface expression of MHC class I and II molecules, the costimulatory molecules CD80 (B7-1) and CD86 (B7-2), and the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-30). In addition, CD40 ligation of mature DCs stimulates the production of proinflammatory cytokines IL-1, IL-6, and TNF-, and T cell growth and differentiation factors, IL-12 and IL-15, as well as increasing DC survival (12, 14, 15, 16, 17, 18, 19, 20). These phenotypic changes confer an enhanced capacity of DCs to induce CD8⁺ T cell proliferation and cytotoxicity. Thus, CD4⁺ T cells help CTL responses via their effects on DC activation and maturation.

Other members of the TNF receptor/ligand superfamily have also been proposed to play a role in either enhancing or bypassing CD4⁺ T cell help in eliciting potent antiviral CTL responses. The receptor activator of NF-B ligand (RANKL)-RANK (also called TRANCE (TNF-related activation-induced cytokine)-TRANCE receptor) pathway has recently been shown in murine models to have similar functions to that of the CD40L-CD40 pathway in the generation of antiviral CD4⁺ T-specific immune responses (21, 22, 23). RANKL is a member of the TNF superfamily sharing greatest amino acid similarity (28%) with CD40L (CD154). RANKL is expressed on both activated CD4⁺ T and CD8⁺ T cells (24). RANK, the receptor for RANKL, is constitutively expressed on DCs (22); however, RANK expression is greater on mature DCs or on CD40-ligated DCs (24). Similar to CD40L, conditioning of murine DCs with RANKL has been shown to elicit production of proinflammatory cytokines, IL-1 and IL-6, and T cell growth and differentiation factors, IL-12 and IL-15. RANKL also dramatically inhibits DC apoptosis via increased Bcl-x_L expression (22, 23, 24, 25). RANKL, however, does not markedly up-regulate DC surface expression of MHC molecules, costimulatory molecules (CD80 and CD86), CD40, or adhesion molecules (CD54), which is observed with CD40L conditioning. It has been postulated that the primary effect of RANKL on DCs is by enhancing DC survival in tissues, thus allowing greater opportunity for DCs to prime T cells (26). The role of the RANKL-RANK pathway in induction of murine or human CTL responses, however, has not yet been evaluated.

TNF- is produced and secreted early in the inflammatory response by activated monocytes, NK cells, and Th1 T cells. TNF- has been shown to differentiate CD34⁺ progenitor cells into DCs. In addition, TNF- has been shown to up-regulate adhesion and costimulatory molecules of immature monocyte-derived DCs (iMDDCs), but not to the extent as that seen with CD40L (14).

Thus, DC activation and maturational signals can originate from multiple sources. The relative importance of each of these signals, either alone or in combination, has not been systematically evaluated, although knowledge of this would be important for vaccine design. Lapointe et al. (27) recently showed that human DCs most optimally stimulated antitumor immune responses if they received combined signals from LPS and CD40L, rather than alone. Similarly, Morel et al. (28) showed that the TNF superfamily member LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor) could enhance the ability of CD40L to mature DCs.

The current study evaluates the ability of three TNF superfamily molecules, CD40L, RANKL, and TNF-, either alone or in combination, to mature human MDDCs, and to elicit antiviral CTL responses. We wanted to determine in a systematic fashion, the optimal signals required to maximally expand virus-specific memory CTL responses both in the presence or absence of CD4⁺ T cell help. We also compared the effects of these TNF family members on DC maturation and CTL induction in cells taken from healthy HIV-1-uninfected and HIV-1-infected individuals.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study subjects

Two HIV-1-uninfected individuals, who were HLA-A*0201 positive and had detectable EBV-specific CD8⁺ T cell IFN- responses by ELISPOT assay (data not shown), were recruited for leukopheresis to obtain large amounts of PBMCs. Three HIV-1-seropositive individuals at different stages of disease were also studied; this included a long-term nonprogressor (patient 1, who was HIV-1 infected for 10 years, had stable CD4⁺ T cell counts >500 µl, and a viral load <50 copies/ml by bDNA), a chronic progressor (patient 2, who was HIV-1 infected for 5 years, CD4⁺ T cell count 410, viral load 45,000 copies/ml, asymptomatic), and a recent seroconverter (patient 3, HIV-1 infected for 4 mo, CD4⁺ T cell count 400, viral load 1000 copies/ml). All HIV-1-infected individuals were not taking antiretroviral drugs during the study. Before the study, individuals were class I HLA typed and screened for EBV- or HIV-1-specific CTL by culturing PBMC with HLA-restricted EBV or HIV-1 peptides and detecting IFN--producing cells by ELISPOT assay, as previously described (data not shown) (29). Informed consent was obtained from participants in accordance with the guideline for conduction of clinical research at the University of Toronto and St. Michael’s Hospital. All investigational protocols were approved by the University of Toronto and St. Michael’s Hospital institutional review boards.

Peptide synthesis

Peptides were synthesized by F-moc chemistry using a Zinnser Analytical Synthesizer (Research Genetics, Huntsville, AL), and purity was established by HPLC. Peptides were dissolved in RPMI medium, and concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The following peptides were used for CD8⁺ T cell expansion: 1) the HLA-A *0201-restricted BMLF1 region of EBV (GLCLVAML); and 2) the HIV-1-specific peptides HLA-B 27-restricted P24 (KRWIIGLNK) for patient 1, HLA-A 24-restricted P17 (KYKLKHIVW) for patient 2, and HLA-A *0201-restricted P17 (SLYNTVATL) for patient 3.

Generation of MDDCs, cell cultures, and stimulation

MDDCs were generated by a modification of a method previously described (19, 30). Briefly, PBMCs obtained by the Ficoll-Paque gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) were separated on multistep Percoll gradients (Sigma-Aldrich, St. Louis, MO). The recovered monocytes were depleted of contaminating B cells, T cells, NK cells, and granulocytes using Ab-conjugated magnetic beads from the Monocyte Negative Isolation Kit (Dynal, Oslo, Norway). Purified monocytes were cultured at 1 x 10⁶ cells/ml in medium consisting of RPMI 1640 plus 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics in the presence of 50 ng/ml human rGM-CSF and 100 ng/ml human rIL-4 (PeproTech, Rocky Hill, NJ). GM-CSF and IL-4 were added again on days 3 and 5 with the fresh complete RPMI 1640 medium. After 7 days of culture, more than 50% of the cells were CD1a^high, MHC class II⁺, CD80^low, and CD14^-, which represents an immature DC phenotype. The immature DCs were then aliquoted and cultured for 72 h in the following conditions: 1) medium alone; 2) soluble recombinant human trimeric CD40L (CD40LT) at 1 µg/ml; 3) soluble recombinant human trimeric RANKL (RANKLT) at a final concentration of 1 µg/ml (CD40LT and RANKLT were obtained as a gift from Immunex, Seattle, WA); 4) soluble human rTNF- at a final concentration of 10 ng/ml (PeproTech); 5) CD40LT + RANKLT; 6) CD40LT + TNF-; 7) RANKLT + TNF-; or 8) CD40LT + RANKLT + TNF-. After 72-h stimulation, cells were harvested and supernatants were saved for cytokine analysis.

Induction of peptide-specific CTL

The protocol for expanding circulating memory CTL ex vivo was described previously (19). MDDCs stimulated for 3 days with CD40LT, RANKLT, or TNF-, alone or in combination, were pulsed with the specific HLA class I-restricted peptide at 40 µg/ml for 1 h at 37°C, then plated in 24-well plates (5 x 10⁵ pulsed or nonpulsed MDDCs/well) in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics. Freshly isolated or thawed autologous PBMCs were prepared in both unfractionated (CD4⁺ T cell-containing) and CD4⁺ T cell-depleted conditions and then added to MDDCs at a 1:10 ratio (5 x 10⁶ cells/well in 2 ml medium). CD4⁺ T cells were depleted from PBMCs using two rounds of magnetic bead depletion (Dynal); the purity of depletion as tested by FACS analysis was always <0.1% CD4⁺ T cell contamination. The percentage of CD8⁺ T cells within total unfractionated PBMCs and CD4⁺ T cell-depleted PBMCs was determined by FACS analysis so that equal input of CD8⁺ T cells could be plated in both unfractionated (total PBMCs) and CD4⁺ T cell-depleted conditions. Thus, the following conditions were included in all experiments using both unfractionated PBMCs or CD4⁺ T cell-depleted PBMCs: 1) MDDCs not pulsed with peptides; 2) MDDCs pulsed with peptides; and 3) MDDCs stimulated with CD40LT, RANKLT, and TNF-, alone or in combination, then pulsed with peptides. On days 3, 5, and 7, the medium was changed. On day 10, duplicate wells were pooled and cells were harvested and tested for CTL activity by intracellular IFN- staining and, in some experiments, also by standard ⁵¹Cr release assay. The percentages of CD8⁺ T cells in both unfractionated and CD4⁺-depleted conditions were again determined by FACS analysis before CTL assays to assure for equal inputs of CD8⁺ T cells for all conditions. Experiments were repeated in HIV-1-negative subject 2 and HIV-1-positive patient 1 with similar results.

Intracellular staining

Intracellular staining was performed to enumerate the number of IFN-- or IL-12-producing cells, as previously described (31). Briefly, for peptide-specific IFN- staining, 0.25 x 10⁶ cells were cultured in U-bottom 96-well plates in the presence of peptide-pulsed (1–10 µM) autologous B-lymphoblastoid cell lines (B-LCL) or autologous T cell-depleted PBMCs as stimulator cells; nonpeptide-pulsed stimulator cells were used as background controls. Positive control cells were stimulated with the bacterial superantigen staphylococcal enterotoxin B (1 µg/ml) (Sigma-Aldrich). Cells were incubated with peptide-pulsed or nonpeptide-pulsed stimulator cells for 6 h at 37°C in 6% CO₂. Monensin was added for the duration of the culture period to facilitate intracellular cytokine accumulation. Cell surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD PharMingen, San Diego, CA) in accordance with the manufacturer’s recommendations. The following Abs were used for the intracellular staining: anti-IFN- (clone 4S.B3) and anti-IL-12 (p40/p70, clone C11.5). All Abs were obtained from BD PharMingen.

Flow cytometry

PBMCs or MDDCs were stained in PBS/1% FCS/0.02% NaN₃ using fluorochrome-conjugated Abs obtained from BD PharMingen. The Abs used were anti-CD80, anti-CD83, anti-CD1a, anti-CD14, anti-HLA-DR, anti-CD4, anti-CD8, anti-CD3, anti-IL-12, and anti-IFN-. Events were acquired using FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). Dead cells were excluded on the basis of forward and side light scatter. For intracellular IFN- assay, a total of 50,000–100,000 events were collected for each sample, and CD8⁺ T cells were enumerated after gating on CD3-positive cells. Data were analyzed using CellQuest (BD Biosciences) or FlowJo (San Carlos, CA) software.

Cytotoxicity assay

Autologous B-LCL were labeled by incubating in 100 µCi sodium ⁵¹Cr chromate and pulsed with the specific peptide at 10 µM for 1 h at 37°C. Control B-LCL were either pulsed with an irrelevant peptide or cultured in complete RPMI 1640 alone. Labeled target cells and serial dilutions of effector cells in triplicate were incubated in complete RPMI 1640 medium for 4 h. Supernatants were then collected and analyzed in a microplate scintillation counter (TopCount; Packard Instrument, Meriden, CT). Background chromium release was always <10%. Percentage of lysis was calculated from the formula: 100% x (E - M)/(T - M), in which E is experimental release, M is the release in the presence of complete RPMI 1640 medium, and T is the release in the 5% Triton X-100 detergent. Specific lysis was determined by subtracting the lysis of control targets from the lysis of peptide-pulsed targets.

RNA isolation and RT-PCR

For RT-PCR analysis, total RNA was extracted from MDDCs cultured in complete RPMI 1640 medium treated for 16 h with CD40LT, RANKLT, TNF-, or the combinations thereof, using TRIzol reagent (Life Technologies, Grand Island, NY). In some experiments, to prime MDDCs to enhance IL-12 production after stimulation, MDDCs were also cultured in the presence of 100 ng/ml IFN-. A total of 500 ng of total RNA extracted from conditioned MDDCs was used for cDNA synthesis using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) in a total volume of 20 µl, and 4 µl of cDNA was then subjected to RT-PCR with the PCR SuperMix High Fidelity kit (Life Technologies). The final RT-PCR volume was 50 µl, and the conditions were: 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. PCR was performed in a 4800 DNA thermal cycler (PerkinElmer/Cetus, Wellesley, MA). The following IL-12 primers were used, resulting in RT-PCR products of 637 bp for IL-12 p35 and 818 bp for IL-12 p40 (Coronel, 2001, 680): IL-12 p35, sense, 5'-CCCTGCAGTGCCGGCTCAGCATGTG-3', and antisense, 5'-GCCCGAATTCTGAAAGCATGAAG-3'; IL-12 p40, sense, 5'-TCTCTGCAGAGAGTCAGAGGG-3', and antisense, 5'-ACGGATCCTGATGGATCAGGTCATAAGAG-3'. GAPDH primers were sense, 5'-TCACCATCTTCCAGGAGGG-3', and antisense, 5'-CTGCTTCACCACCTTCTTGA-3'. A total of 6 µl of PCR product from each sample was size fractionated using ethidium-stained 1.5% agarose gel electrophoresis. Band intensity on gels was quantitated by an Image Analyzer (Bio-Rad).

MDDC survival analysis

Duplicate wells containing 0.51 x 10⁵ MDDCs were cultured in flat-bottom 96-well plates in complete RPMI 1640 medium for 24 h in the presence or absence of CD40LT (1 µg/ml), RANKLT (1 µg/ml), TNF- (10 ng/ml), or the combinations thereof. Cell viability was assessed by trypan blue exclusion. Cell apoptosis was determined by detecting the phosphatidylserine on the outer leaflet of apoptotic cell membranes using annexin V-fluorescein staining (Boehringer Mannheim, Indianapolis, IN), according to the manufacturer’s protocol. Apoptotic MDDCs were analyzed by a FACSCalibur using CellQuest, and the data were analyzed using FlowJo software, as previously described (19).

Cytokine measurements

MDDCs were cultured at a concentration of 10⁶/ml in 24-well plates and stimulated for 72 h with CD40LT (1 µg/ml), RANKLT (1 µg/ml), and TNF- (10 ng/ml), alone or in combinations. Supernatants were harvested and stored at -80°C until analysis. IL-10 and IL-15 production was measured using ChemiKine human IL-10 and IL-15 sandwich ELISA kits (Chemicon International, Temecula, CA), according to the manufacturer’s specifications. The detection limits of these ELISA tests were 1.6 and 2.7 pg/ml for IL-10 and IL-15, respectively.

Statistical analysis

Data were compared using the Wilcoxon signed rank test for paired samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of phenotypical maturation of MDDCs by TNF superfamily members CD40L, RANKL, and TNF-, or with combinations thereof

iMDDCs generated from healthy volunteers or HIV-1-infected individuals were conditioned for 72 h with soluble trimeric CD40L (CD40LT, 1 µg/ml), trimeric RANKL (RANKLT, 1 µg/ml), TNF- (10 ng/ml), or the following combinations: CD40LT with RANKLT, CD40LT with TNF-, RANKLT with TNF-, or all three. In comparison with medium alone, conditioning with either TNF- or RANKLT alone induced small cell clusters; TNF- plus RANKLT induced greater clustering, whereas CD40LT alone or in combination with either TNF- or RANKLT or both induced the greatest degree of clustering (data not shown). Similar morphologic effects were seen in MDDCs derived from both HIV-1-uninfected and HIV-1-infected individuals (data not shown). The cell surface phenotypes of these differentially matured DCs were compared by flow cytometry (Fig. 1). As shown in Fig. 1, the nontreated DCs were HLA-DR^low and expressed high levels of CD1a and low levels of CD80 and CD83. CD40LT was the most potent inducer of DC maturation, as demonstrated by decreased CD1a expression and increased CD83 expression (12). Although TNF- and RANKLT were mild inducers of DC maturation, as shown by CD1a down-regulation, additive effects were not seen when used in combination. Addition of either TNF- or RANKLT to CD40LT minimally altered CD1a and CD83 expression. CD40LT induced the greatest expression of HLA-DR and of the costimulatory molecule CD80. TNF- and RANKLT also induced moderate levels of CD80 and HLA-DR, which were additive in combination. The addition of TNF- or RANKLT to CD40LT increased the levels of CD80 or HLA-DR when compared with only CD40LT-treated DCs. The combination of all three molecules, CD40LT, RANKLT, and TNF-, induced the most extensive expression of HLA-DR and CD80. Thus, although CD40LT is the most potent inducer of DC maturation, the addition of either TNF- or RANKLT further enhances the activation state of DCs.

View larger version (45K): [in this window] [in a new window]   FIGURE 1. TNF superfamily members induce the maturation and activation of MDDCs. iMDDCs were treated for 72 h with medium (negative control), CD40LT (1 µg/ml), RANKLT (1 µg/ml), TNF- (10 ng/ml), or combinations thereof. Cells were harvested, and expression of surface molecules was analyzed by flow cytometry using PE-conjugated mAbs against CD1a, CD80, CD83, and HLA-DR (BD PharMingen). Values represent the mean fluorescence intensity subtracted from the value of matched isotype control mouse mAbs (dotted histogram). Data are analyzed using FlowJo software. Data from HIV-1-infected patient 1 are shown and are representative of five experiments performed with MDDC obtained from two HIV-1-uninfected blood donors and three HIV-1-infected individuals.

Role of conditioned MDDC by CD40L, RANKL, and TNF-, or their combinations in the expansion of EBV-specific memory CTL in vitro in both CD4⁺-containing and -depleted conditions

To determine the role that the TNF superfamily molecules CD40L, RANKL, and TNF- play in the expansion of virus-specific memory CTL responses, we used an in vitro coculture method in which peptide-pulsed conditioned MDDCs stimulate CD8⁺ T cells in the absence of exogenous cytokines, as previously described (19). We wanted to determine the optimal signals provided by TNF superfamily molecules that would induce the most effective Ag-specific CTL response. To determine whether these molecules could completely substitute for CD4⁺ T cell help of CTL responses, we also performed experiments in both CD4⁺ T cell-containing and -depleted conditions. Two healthy, HIV-1-uninfected individuals who had previously demonstrated an HLA-A*0201-restricted response to the EBV epitope of BMLF1 (lytic cycle Ag) were studied. MDDCs from these two HIV-1-uninfected individuals were conditioned for 3 days by the TNF superfamily members CD40L, RANKL, and TNF-, alone or in combinations, then pulsed with EBV-specific peptide, and cocultured with autologous CD8⁺ T cells in CD4⁺ T cell-containing or -depleted conditions. After 10 days of coculture, CTL effector activity was assessed by two assays: direct cytolysis of peptide-pulsed targets and intracellular IFN- production after exposure to peptide-pulsed autologous B-LCL or T cell-depleted PBMC. A representative experiment measuring CTL by intracellular IFN- flow cytometry from subject 1 and chromium release assay from subject 2 is illustrated in Fig. 2, a and b, respectively. Summary IFN- flow cytometric data from subject 2 are illustrated in Fig. 2c. iMDDC treated with TNF-, RANKLT, or CD40LT significantly enhanced EBV-specific memory CTL responses when compared with medium-treated iMDDCs (p < 0.05) (Fig. 2). Of the three, DC treated with CD40LT generally gave the most potent induction of CTL (see Fig. 2, a and c). When TNF superfamily molecules were combined, MDDCs conditioned with CD40LT combined with either TNF- or RANKLT induced greater CTL than with CD40LT alone or TNF- plus RANKLT (p < 0.05) (Fig. 2). Treatment with all three molecules provided even greater enhancement of CTL in subject 1, but not in subject 2. Thus, CD40LT was a stronger inducer of CTL than RANKLT or TNF-. However, the addition of either TNF- or RANKLT could further enhance CTL responses induced by CD40LT. CTL responses were always higher in CD4⁺ T cell-containing conditions even in those containing CD40LT (p < 0.05) or a combination of TNF superfamily molecules (Fig. 2). Thus, although addition of TNF family molecules can dramatically enhance CTL responses in CD4⁺ T cell-depleted conditions, the addition of CD4⁺ T cells can still help CTL responses further. This suggests that CD4⁺ T cells may also provide help to CD8⁺ T cells independently of CD40L, RANKL, or TNF- in our culture system.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Effects of TNF superfamily members CD40L, RANKL, and TNF-, alone or in combination, on the induction of EBV-specific antiviral CTL activity. CD4+ T cell-containing or -depleted PBMC from an EBV-infected healthy donor were cocultured with HLA-A*0201-restricted peptide-pulsed or nonpulsed autologous MDDCs that were previously conditioned for 72 h with CD40LT (1 µg/ml), RANKLT (1 µg/ml), and TNF- (10 ng/ml), alone or in combinations thereof. On day 10, EBV-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN--producing cells, and specific CTL lysis directed against EBV epitope-pulsed autologous target cells was measured. a, Representative intracellular IFN- flow cytometric data obtained from subject 1 are shown. For intracellular cytokine flow cytometric analysis, cells were gated for CD3 and CD8 to enumerate IFN--producing CD8+ T cells only. b, CTL as measured by chromium release assay from subject 2. Corresponding cocultures were tested at E:T ratios ranging from 40:1 to 2.5:1 in a standard 4-h 51Cr release assay on autologous B-LCL in the presence or absence of HLA-A *0201-restricted epitope of the EBV. Results were expressed as percentage of specific lysis determined by subtracting lysis percentage of control from the lysis percentage of peptide-pulsed targets. c, Summary data from intracellular IFN- flow cytometry of subjects 2 are graphically depicted. The experiment from subject 2 was repeated with similar results. Statistical comparisons of data pooled from both subjects were performed between the following culture conditions: DCP vs CD40LT, p < 0.05; DCP vs TNF-, p < 0.05; DCP vs RANKLT, p < 0.05; CD40LT vs CD40LT + RANKLT or CD40LT + TNF-, p < 0.05.; CD40LT vs CD40LT + RANKLT + TNF, p = NS; CD4+ T cell containing vs CD4+ T cell depleted, p < 0.05. DC = nonpeptide-pulsed DC; DCP = DC pulsed with peptides; 40LT = CD40 ligand trimer.

Role of conditioned MDDC by CD40L, RANKL, and TNF-, or their combinations in the expansion of HIV-1-specific memory CTL in vitro in both CD4⁺-containing and -depleted PBMCs

Three HIV-1-seropositive individuals with different rates of disease progression were examined in this study: a long-term nonprogressor (patient 1), a chronic progressor (patient 2), and a recent seroconverter (patient 3) (see Materials and Methods). iMDDCs derived from these individuals were treated with TNF family molecules for 3 days, then pulsed with an HLA class I-restricted HIV-1-specific epitope (determined by standard IFN- ELISPOT assays obtained from that individual; see Materials and Methods), and cocultured with autologous PBMC in CD4⁺ T cell-containing or CD4⁺ T cell-depleted conditions. After 10-day culture, CTL responses were measured by intracellular IFN- production after exposure to peptide-pulsed autologous B-LCL or T cell-depleted PBMC. Summary data from all three individuals are illustrated in Fig. 3.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Effects of TNF superfamily members CD40L, RANKL, and TNF-, alone or in combination, on the induction of HIV-1-specific antiviral CTL activity. CD4+ T cell-containing or -depleted PBMC from three HIV-1-infected individuals were cocultured with autologous MDDCs that were either not pulsed or pulsed with HLA-resticted epitopes of HIV-1 proteins (see Materials and Methods). MDDCs were previously conditioned for 72 h with CD40LT (1 µg/ml), RANKLT (1 µg/ml), and TNF- (10 ng/ml), alone or in combinations thereof. On day 10, HIV-1-specific CTL activitiy was assessed by intracellular flow cytometric analysis of IFN--producing cells. Summary data from intracellular IFN- flow cytometry of patients 1–3 are graphically depicted. The experiment from patient 1 was repeated with similar results. Statistical comparisons on data pooled from all patients were performed between the following culture conditions: DCP vs CD40LT, p < 0.05; DCP vs TNF-, p < 0.05; DCP vs RANKLT, p < 0.05; CD40LT vs CD40LT + RANKLT or TNF-, p = NS; CD40LT vs CD40LT + RANKLT + TNF-, p = NS; CD4+ T cell containing vs CD4+ T cell depleted, p < 0.05. DC = nonpeptide-pulsed DC; DCP = DC pulsed with peptides; 40LT = CD40 ligand trimer.

For most culture conditions, CTL responses were higher in CD4⁺ T cell-containing conditions, including those containing CD40LT (p < 0.05). (Fig. 3), suggesting that CD4⁺ T cells can provide help to CD8⁺ T cells independently of CD40L, RANKL, or TNF- in our culture system.

The role of TNF family members CD40L, RANKL, and TNF- in the regulation of IL-10, IL-12, and IL-15 production from MDDCs

IL-12 is a central regulator of Th1 responses (32). IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8⁺ T cells, and has been shown to be induced by DCs after CD40 ligation (19). IL-10, an immunoregulatory cytokine, is also induced after DC activation (19, 27). Possible explanations for differences in CTL responses from various TNF family combinations may be related to differential cytokine production. We thus compared the ability of TNF superfamily molecules to regulate the production of IL-12, IL-15, and IL-10 in MDDCs. For IL-12,iMDDCs were stimulated for 16 h with CD40L, RANKL, and TNF-, and combinations thereof, and IL-12 induction was measured both by RNA and protein expression. Analysis of IL-12 p35 and p40 transcripts by RT-PCR revealed that CD40LT-containing conditions induced the greatest amounts of p35 and p40 in MDDCs, followed by TNF-, and then by RANKLT (Fig. 4a). This finding correlated with intracellular IL-12 expression (Fig. 4b), as assessed by intracellular flow cytometry. For example, in subject 2, CD40LT stimulation induced 98-fold intracellular IL-12 p40/p70 production, compared with unstimulated MDDCs (Fig. 4b). RANKLT alone failed to induce stainable IL-12 secretion, whereas TNF- alone induced an 11-fold increase of IL-12 secretion. TNF- cooperated with CD40L to increase IL-12 p40/p70 production, whereas RANKL failed to enhance the effect of CD40L stimulation on the IL-12 p40/p70 production. The combination of all three TNF family members, however, gave the strongest induction of IL-12 p40/p70 production. Thus, CD40LT is the strongest inducer of IL-12 in iMDDCs, which can be further enhanced by TNF-, or with TNF- in combination with RANKLT. Similar findings were observed in iMDDCs obtained from both HIV-1-uninfected and HIV-1-infected individuals (data not shown). Detection of IL-15 and IL-10 in the supernatants of MDDCs cultured for 72 h with CD40L, RANKL, and TNF-, and combinations thereof showed that CD40L-containing conditions induced high levels of both IL-10 and IL-15 in MDDCs derived from HIV-1-uninfected and HIV-1-infected individuals (see Table I). IL-10 and IL-15 production from CD40L-stimulated cells was not significantly enhanced by addition of either RANKL or TNF- or both (Table I).

View larger version (64K): [in this window] [in a new window]   FIGURE 4. Effects of TNF family members CD40L, RANKL, and TNF- on IL-12 expression by MDDCs. MDDCs were stimulated for 16 h with CD40LT (1 µg/ml), RANKLT (1 µg/ml), and TNF- (10 ng/ml), alone or in the combinations. a, Expression of mRNA for IL-12 p35 and p40 subunits and for human housekeeping gene GAPDH was determined on reverse-transcribed mRNA from the conditioned MDDCs described, followed by PCR amplification using specific primer sets. The resulting cDNA fragments were visualized after electrophoresis on an ethidium-stained 1.5% agarose gel. The amplified cDNA fragments with lengths corresponding to transcripts of p35 and p40 and GAPDH are shown. Lane M, A 100-bp DNA size marker (Life Technologies); signal intensities of p35 and p40 bands were quantitated by image analysis and compared with medium control. Ratios of respective p35 and p40 band intensity over medium control are indicated in the parentheses: Lanes 1–8, medium (1, 1), CD40LT (98, 12), RANKLT (1, 6), CD40LT + RANKLT (99, 13), TNF- (6, 6), TNF- + CD40LT (121, 13), TNF- + RANKLT (8, 6), TNF- + CD40LT + RANKLT (155, 13). b, Protein expression was determined by IL-12 intracellular staining using anti-IL-12 (p40/p70) Ab (clone C11.5) (BD PharMingen) after overnight incubation with Monensin. The numbers in upper right corner represent percentage of IL-12 p40/p70-positive MDDCs per total MDDCs. Data are taken from HIV-1-uninfected subject 2 and are representative of experiments with MDDCs derived from HIV-1-infected and HIV-1-uninfected individuals. Experiments were performed with or without priming MDDCs with 100 ng/ml IFN- to further enhance IL-12 production, with similar trends observed.

View this table: [in this window] [in a new window]   Table I. IL-10 and IL-15 production in the supernatant of cultured MDDCs stimulated with TNF family members CD40L, RANKL, and TNF-, alone or in combinationsa

The role of TNF family members CD40L, RANKL, and TNF- in MDDC survival

Inhibition of DC apoptosis with resultant DC survival has been postulated to be one mechanism whereby memory CTL responses can be maintained (12). The effects of TNF family members CD40L, RANKL, and TNF- on MDDC survival were studied (Fig. 5). MDDCs treated with RANKLT were remarkably prevented from apoptosis, as determined by annexin V expression compared with untreated cells (4-fold inhibition), whereas CD40LT and TNF- were mild inhibitors of MDDC apoptosis (Fig. 5). Addition of RANKLT also inhibited apoptosis in CD40LT- or TNF--stimulated conditions, but not to the level seen with RANKLT alone. These findings also correlated with viable cell counts, as determined by trypan blue exclusion (data not shown). Similar results were obtained when MDDC from HIV-1-infected individuals were studied, regardless of their viral load (data not shown). Thus, the TNF family members have effects on DC apoptosis. RANKLT alone is the greatest inhibitor of DC apoptosis, and the addition of RANKLT can inhibit apoptosis in MDDCs treated with CD40LT or TNF- from both HIV-1-uninfected and HIV-1-infected individuals.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Regulation of MDDC survival by TNF family members CD40L, RANKL, and TNF-, alone or in combination. MDDCs were incubated with CD40LT (1 µg/ml), RANKLT (1 µg/ml), and TNF- (10 ng/ml), or the combinations thereof. MDDCs were then assessed for the phosphatidylserine on the outer leaflet of apoptotic cell membranes using annexin V-fluorescein staining (Boehringer Mannheim). MDDCs were also counterstained for HLA-DR. The numbers in upper right corner represent percentage of annexin V-positive MDDCs per total viable MDDCs. Data are taken from HIV-1-uninfected subject 2 and are representative of experiments with MDDCs derived from HIV-1-infected and HIV-1-uninfected individuals. Representative data of three independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We also demonstrated particularly in PBMCs taken from HIV-1-uninfected individuals, that TNF- or RANKL can cooperate with CD40L to induce maximal maturation and expansion of EBV-specific CTL. Given that the effects of TNF- or RANKLT alone were minimal to moderate on DC activation suggests that they may have more of a cooperative and synergistic role in DC maturation and CTL induction. These observations also suggest that multiple signals are required to properly induce DC to be optimally activated. For example, a DC could encounter TNF- from activated monocytes or RANKL from activated T cells in combination with CD40L from activated CD4⁺ T cells to optimally prime CTL responses.

The mechanisms whereby TNF- or RANKL can synergize with CD40L were investigated in this study. Although CD40L induced the greatest degree of costimulatory molecule and HLA expression on MDDCs, both TNF- and RANKL could further increase expression of these molecules, which would allow a more intense activation of virus-specific CD8⁺ T cells. TNF- enhanced the ability of CD40L to induce IL-12 in DCs, which was not observed with RANKL. Thus, TNF- may cooperate with CD40L by also synergizing IL-12 production. RANKL was a potent inhibitor of MDDC apoptosis, even in the presence of other TNF family molecules. This suggests that RANKL may enhance the effects of CD40L on CTL responses by increasing the survival of CD40L-stimulated DCs. Cytokines including IL-15 and IL-10 produced by MDDCs may also play important roles in expanding or down-regulating CTL responses, respectively. Interestingly, we observed that CD40L induced high levels of both IL-10 and IL-15 from MDDCs. We were, however, unable to demonstrate further IL-15 production or a decrease in IL-10 production in conditions in which TNF- or RANKLT or both were added to CD40LT. In summary, the mechanisms whereby TNF- and RANKL cooperate with CD40L may differ in part, but have the same overall effect by enhancing CTL responses. Elucidating the signaling pathways in which CD40 and TNFR ligation induce IL-12 production and RANK ligation inhibits apoptosis will be important for future studies.

Surprisingly, when studying PBMCs taken from HIV-1-infected individuals, TNF- or RANKL did not always cooperatively expand HIV-1-specific CTL responses in the presence of CD40L, despite observing enhanced expression of costimulatory molecules on DCs. These findings suggest either dysregulation of activated DC or an inability of HIV-1-specific CD8⁺ T cells to be primed by maximally activated DCs. We have previously noted a defect in HIV-1-specific CD8⁺ T cells in some HIV-1-infected individuals to maximally respond to matured DCs (19). In the current study, we were unable to correlate a lack of synergy between CD40LT and TNF- or RANKLT with IL-10 or IL-15 production. Thus, aberrant or deficient cytokine production by MDDCs cannot explain these findings. An alternative possibility is that circulating HIV-1-specific CD8⁺ T cell memory cells in HIV-1 infection are already activated due to constant virus production, and may not require fully activated MDDCs to be expanded. This hypothesis will require further study.

We occasionally observed paradoxical effects if DCs were conditioned with all three TNF family molecules, TNF-, RANKL, and CD40L, on CTL responses in PBMCs taken from certain individuals (subject 2, patient 1, and patient 3), either HIV-1 infected or uninfected. That is, we did not always see greater expansion of CTL when DCs were maximally stimulated by all three TNF family molecules together if compared with CD40L stimulation alone. This suggests that TNF family molecules may also induce inhibitory effects on immune responses at certain stages of DC maturation. Excess costimulation has been shown to induce activation-induced nonresponsiveness in CD8⁺ T cells (34). In the latter situation, activation-induced nonresponsiveness was characterized by an inability of CD8⁺ T cells to produce IL-2 and subsequently proliferate. Other potential mechanisms may include increased IL-10 production by MDDCs or to the induction of counterregulatory CD4⁺ and/or CD8⁺ T cells (35). We were unable to correlate paradoxical decreases in CTL with IL-10 levels in supernatants. Further studies are warranted to investigate this phenomenon in our coculture system.

CD4⁺ T cells have been shown to help CTL responses by at least two mechanisms: 1) expression of CD40L (8, 10, 11), and 2) production of soluble lymphokines, namely IL-2 (36). In this study, we have compared the effects of TNF family-conditioned DCs on CD8⁺ T cells in both CD4⁺ T cell-containing and CD4⁺ T cell-depleted conditions. Interestingly, we generally observed greater expansion of virus-specific CTL in CD4⁺ T cell-containing conditions, even in the presence of exogenous TNF family molecules. Because the addition of exogenous trimeric CD40L or RANKL did not equalize the CTL responses between CD4⁺ T cell-containing and -depleted conditions, it is likely that CD4⁺ T cell help was also supplied independently of these TNF family molecules in our coculture system. In this regard, Lu et al. (36) recently demonstrated using a similar in vitro murine model that CD4⁺ T cell help comprised at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4⁺-CD8⁺ T cell communication. Interestingly, in HIV-1-infected patient 1, who was a long-term nonprogressor, we observed that CTL responses were not always higher in CD4⁺ T cell-containing conditions (Fig. 3). We have previously observed this phenomenon in PBMC from long-term nonprogressors (19), which suggests that CTL responses in some long-term nonprogressors may expand well without CD4⁺ T cell help. Further study in this regard is clearly warranted.

Our findings have important implications for vaccine design and immunotherapeutics. For example, DCs conditioned in the presence of CD40L plus TNF- or RANKL may be more effective than DC conditioned with CD40L alone when designing autologous DC-based vaccines. In addition, the inclusion of CD40L plus RANKL or TNF- in the design of DNA-based vaccines may allow for greater immunogenicity than a vaccine expressing CD40L alone. In the case of therapeutic DNA vaccines targeted toward HIV-1-infected individuals, CD40L expression alone may be sufficient to augment vaccine-induced immune responses.

	Acknowledgments

 
We thank our patients for their time and commitment.

	Footnotes

 
¹ This study was funded through grant support supplied by American Foundation for AIDS Research and Aventis-Pasteur. Q.Y. and M.A.O. are career scientists of The Ontario HIV Treatment Network.

² Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Clinical Sciences Division, Room 6271, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S 1A8. E-mail address: m.ostrowski{at}utoronto.ca

³ Abbreviations used in this paper: CD40L, CD40 ligand; B-LCL, B-lymphoblastoid cell line; CD40LT, trimeric CD40L; DC, dendritic cell; iMDDC, immature MDDC; MDDC, monocyte-derived DC; RANK, receptor activator of NF-B; RANKL, RANK ligand; RANKLT, trimeric RANKL.

Received for publication May 17, 2002. Accepted for publication December 6, 2002.

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