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Originally published online as doi:10.1189/jlb.1107795 on March 25, 2008

Published online before print March 25, 2008
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(Journal of Leukocyte Biology. 2008;83:1530-1540.)
© 2008 by Society for Leukocyte Biology

Maturation of dendritic cells for enhanced activation of anti-HIV-1 CD8+ T cell immunity

Xiao-Li Huang, Zheng Fan, LuAnn Borowski and Charles R. Rinaldo1

Graduate School of Public Health and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

1Correspondence: University of Pittsburgh Graduate School of Public Health, A419 Crabtree Hall, 130 DeSoto Street, Pittsburgh, PA 15261, USA. E-mail: rinaldo{at}pitt.edu

ABSTRACT

Maturation of dendritic cells (DC) to enhance their capacity to activate T cell immunity to HIV-1 is a key step in immunotherapy of HIV-1 infection with DC. We compared maturation of DC derived from HIV-1-uninfected subjects and infected subjects on antiretroviral therapy (ART) or ART naïve by CD40 ligand (CD40L) and combinations of TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] and inflammatory cytokines IFN-{gamma}, IFN-{alpha}, IL-1β, and TNF-{alpha}. The greatest levels of virus-specific IFN-{gamma} production by CD8+ T cells were stimulated by DC treated with CD40L, followed by DC treated with the poly(I:C)-cytokine combination. The highest levels of IL-12p70 were produced by DC treated with CD40L + IFN-{gamma}, followed by CD40L and the poly(I:C)-cytokine combination. Neutralization of IL-12p70 indicated that it was only partially involved in direct enhancement of antiviral CD8+ T cell activity. DC stimulation of antiviral CD8+ T cell reactivity was enhanced by activated CD4+ T cells at low concentrations but was suppressed at higher CD4+ T cell concentrations. Maturation of DC with CD40L obviated the need for CD4+ T cell help and overcame this suppressive activity. Finally, we showed that DC from HIV-1-infected subjects on ART, which were treated with the poly(I:C)-cytokine combination, retained the capacity to produce IL-12p70 and activate anti-HIV-1 CD8+ T cell responses after restimulation with CD40L, with or without IFN-{gamma}. Thus, DC from HIV-1-infected subjects can be engineered with CD40L or a poly(I:C)-cytokine combination for enhancing CD8+ T cell responses to HIV-1, which has potential applications in HIV-1 immunotherapy.

Key Words: immunotherapy • CD40 ligand • TLR 3 ligand • interferon • CD4 T cells

INTRODUCTION

Combination antiretroviral therapy (ART) has resulted in dramatic clinical improvement in HIV-1-infected persons. There is, however, persistence of viral infection and incomplete recovery of anti-HIV-1 T cell immunity during ART. One approach to better control HIV-1 infection during ART is immunotherapy through activation of T cells with bioengineered dendritic cells (DC) [1 2 3 ]. Such DC could provide signals that directly activate antiviral CD8+ CTL responses or act by polarizing Th1 rather than Th2 cell responses, which in turn enhance and maintain antiviral CTL immunity [4 , 5 ]. It is therefore important to define the most efficient immunomodulating regimen for inducing mature and type 1-polarizing DC that can drive potent antiviral CD8+ T cell responses.

Sequential triggers of DC are involved in downstream activation of antigen-specific T cell responses. These result in stages of DC maturation that potentiate adaptive immunity [6 ]. First, immature, steady-state DC encounter and sense pathogen-associated molecular patterns via pathogen recognition receptors, particularly TLRs. Treatment of DC with combinations of TLR agonists and inflammatory cytokines [4 , 7 8 9 ] induces potent, antigen-specific T cell responses. These "mature" DC require an additional stimulation such as that supplied by CD40 ligand (CD40L) on activated CD4+ T cells, resulting in "polarizing" DC that produce IL-12 and other cytokines that preferentially elicit Th1-type responses that lead to downstream induction of CD8+ CTL. This is being proposed for translation into clinical immunotherapy protocols for certain cancers, where the patient’s DC are treated with a combination of TLR ligands and inflammatory cytokines and loaded with appropriate antigens ex vivo [4 ]. These engineered DC would then be injected into the patient, with the hypothesis that CD40L and IFN-{gamma} expressed by the resident CD4+ T cells will trigger the DC to activate Th1 cell-polarizing signals and thereby enhance anti-tumor immunity.

Differential activation and maturation of DC from an immature state by a combination of TLR ligands and inflammatory cytokines have not been systematically assessed in HIV-1 infection. Therefore, we studied combinations of several immune-modulating factors with known effects on DC, i.e., polyinosinic:polycytidylic acid [poly(I:C)], a dsRNA and TLR3 agonist [10 ], which induces IL-12 production [11 , 12 ] and is Th1-polarizing [13 ]; IFN-{alpha}, which enhances DC generation, IL-15 production and cross-priming [14 ]; and inflammatory mediators IL-1β, TNF-{alpha}, IFN-{gamma} [4 , 15 ], and CD40L, which increase expression of costimulatory molecules and production of IL-12, IL-15, TNF-{alpha}, and other T cell activators [16 ]. We have recently reported that DC from subjects with hepatitis C virus (HCV) infection, with or without HIV-1 coinfection, can produce high levels of IL-12p70 after stimulation with CD40L plus IFN-{gamma} [17 ]. In the present study, the effects of these two immune-modulating agents on DC function, as well as those of poly(I:C) and the three other proinflammatory cytokines, were assessed using DC derived from uninfected subjects and HIV-1-infected subjects who were or were not receiving ART. The results show DC derived from uninfected and HIV-1-infected subjects and treated with CD40L or a combination of poly(I:C) and the four proinflammatory cytokines were potent inducers of antiviral T cell responses. Moreover, DC retained their responsiveness to sequential treatments with these maturation factors, which may have implications for the design of DC-based therapeutic vaccines.

MATERIALS AND METHODS

Study subjects
Twenty HIV-1-seropositive and HCV-seronegative homosexual men were studied from the Multicenter AIDS Cohort Study (Pittsburgh, PA, USA), an investigation of the natural history of HIV-1 infection. Fourteen were on ART (CD4+ T cell count: median, 423/µl, range, 89–981/µl; HIV-1 RNA: median, <40, range, <40–13,825 copies/ml), and six subjects were not on ART (CD4+ T cell count: median, 458, range, 310–745/mm3; HIV RNA: median, 12,168, range, 2683–48,102 copies/ml). Fifteen HIV-1-seronegative persons were controls.

DC cultures
To obtain immature DC, CD14+ monocytes were positively selected from PBMC using anti-CD14, mAb-coated magnetic microbeads (StemCell Technologies, Vancouver, Canada) to a purity of >96%, cultured for 5–6 days in AIM V medium (Gibco, Grand Island, NY, USA) containing 1000 U/ml recombinant human (rh)IL-4 (R & D Systems, Minneapolis, MN, USA) and 1000 U/ml rGM-CSF (Amgen, Seattle, WA, USA). Fresh hIL-4 and GM-CSF were added every other day. The DC were treated with CD40L (0.5–1 µg/ml, Amgen), poly(I:C) (dsRNA complex; 20 µg/ml, Sigma Chemical Co., St. Louis, MO, USA), IFN-{alpha} (1000 U/ml, Strathmann Biotech, Hannover, Germany), IFN-{gamma} (1000 U/ml, R & D Systems), IL-1β (25 ng/ml, R & D Systems), and TNF-{alpha} (50 ng/ml, BD Biosciences, Bedford, MA, USA) for 40 h to induce DC maturation. The number of viable DC was determined by typical DC morphology in trypan blue dye-stained preparations. The maturation status of the DC was determined by flow cytometry as percent-positive cells and mean fluorescent intensity of expression of MHC class II (HLA-DR), MHC class I (HLA ABC), CD80, CD86, and CD83. The mean percent ± SE yield of these DC was 3.1 ± 1.2% (n=15) for the uninfected subjects, 2.9 ± 1.1% (n=14) for HIV-1-infected subjects on ART, and 2.0 ± 1.5% (n=6) for HIV-1-infected subjects not on ART; P> 0.05. The DC from all subjects displayed a characteristic DC morphology and cell surface marker expression.

Induction of IL-12p70 and IL-2
Supernatants were collected from untreated and treated DC cultures at 0, 4, 24, and 40 h. These samples were frozen at –70°C for later assay of IL-12p70 by ELISA (R & D Systems). In some experiments, culture supernatants were collected from an overnight ELISPOT assay and were frozen at –70°C for later assay of IL-12p70 and IL-2 by ELISA (R & D Systems).

Synthetic peptides
Synthetic peptides represented dominant MHC class I CTL epitopes for HIV-1 LAI/IIIB, human CMV strain AD169, EBV, and influenza A virus (Flu) (prepared by the Protein Research Lab, University of Illinois, Chicago, IL, USA) and a HIV-1 Gag peptide pool (241–371; consecutive 15-mers overlapping by 11 amino acids, clade B consensus sequences) and a pool of four CMV, 15 EBV, and 12 Flu (CEF) peptides (1 µg/ml), representing a cross-section of MHC class I-dominant epitopes [18 ], provided by National Institutes of Health AIDS Research and Reference Reagent Program (Germantown, MD, USA; Table 1 ).


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  		Table 1. Peptides and HLA Restriction Alleles

Elispot assay
The Elispot assay was used to determine single-cell IFN-{gamma} production. The untreated and treated DC were added to Elispot plates and loaded with peptides (10 µg/ml) for 2 h. CD8+ (96–98% pure) cells were positively selected from PBMCs using anti-CD8, mAb-coated, magnetic microbeads (StemCell Technologies). The responder cells (CD8+ cells) were stimulated overnight with peptide-loaded DC at a ratio of 10:1. After processing, the spots were counted with an Elispot reader system (Cell Technology, Columbia, MD, USA). Data were expressed as the net spot-forming cells (SFC) per 106 cells, i.e., the number of spots/106 responder cells stimulated with HIV-1 antigen or non-HIV-1 antigen expressing DC minus the number of spots/106 responder cells stimulated with antigen-negative DC. To confirm the effects of IL-12, rIL-12 (0.1–100 ng/ml) was added to responder cells in Elispot plates for overnight assay. For the inhibition of IFN-{gamma} production, anti-IL-12 mAb (1 µg/ml, PeproTech, Rocky Hill, NJ, USA) and IgG (1 µg/ml, PeproTech) were cocultured with responders and stimulators in Elispot overnight assay. To confirm the neutralization by anti-IL-12 mAb, the supernatants were collected from the overnight assay and frozen at –70°C for later assay of IL-12p70 by ELISA (R & D Systems). In other experiments, the CD4+ T cells (96–98% pure) were negatively selected by magnetic microbeads (StemCell Technologies) and activated with OKT-3 (200 ng/ml, Ortho, Raritan, NJ, USA) and rhIL-2 (100 U/ml, Chiron, Emeryville, CA, USA) for 4 days. Then, CD8+ T cells were mixed at 1:0.125, 1:0.5, and 1:2 ratios with resting or activated CD4+ T cells as responder cells in the Elispot assay. The supernatants collected from overnight Elispot were frozen at –70°C for later assay of IL-2 by ELISA (R & D Systems).

T cell blastogenesis assay
CD8+ T cells were stimulated with HIV-1 peptide-loaded DC and cocultured with resting or activated CD4+ T cells at 1:0.125, 1:0.5, and 1:2 ratios in triplicate wells in 96-well plates for 6 days. Cells were harvested after labeling of DNA with 0.5 µCi/well [3H]-thymidine (ICN Pharmaceuticals, Irvine, CA, USA) for the final 6 h, and the radioactivity was measured by a scintillation counter (Topcount, Packard, Meriden, CT, USA).

Restimulation of IL-12 production and IFN-{gamma} production by CD8+ T cells by DC
Twenty-four hours after the first immunomodulating stimulation [None, poly(I:C)+IFN-{alpha}+IFN-{gamma}+IL-1β+TNF-{alpha}], DC were exposed to a second stimulation (None, CD40L, CD40L+IFN-{gamma}) for an additional 24 h. DC were washed thoroughly before the second stimulation and compared with that without wash. Supernatants were collected from untreated and maturation factor-treated DC cultures at 24 h after the first and second stimulation. Samples were frozen at –70°C for later assay of IL-12p70 by ELISA. At 24 h after the first and second stimulation, DC were harvested, loaded with peptides, and mixed with CD8+ T cells for overnight Elispot assay.

Statistics
The data were compared for significance (P<0.05) by ANOVA with Scheffe multiple comparison test, Student’s t-test, and Wilcoxon’s signed rank test.

RESULTS

IFN-{gamma} production by CD8+ T cells stimulated by viral peptide-loaded DC
We first investigated the ability of DC derived from HIV-1-infected subjects and treated with the various maturation factors to stimulate IFN-{gamma} production by CD8+ T cells to MHC class I immunodominant, HIV-1 peptides (Table 1) . For comparison, we studied T cell reactivity induced by DC from HIV-1-negative persons that were loaded with immunodominant, non-HIV-1 viral peptides. Cumulative mean (+SE) and representative results (Fig. 1A and 1B , respectively) show that immature DC from the three groups of study subjects (HIV-1-negative, HIV-1 infected on ART, and HIV-1 infected not on ART), which were loaded with the viral peptides, stimulated greater CD8+ T cell reactivity than CD8+ T cells without DC. More importantly, DC from each group of study subjects, which were treated with CD40L, induced greater antiviral CD8+ T cell reactivity compared with the untreated, immature DC. This enhancing effect was a result of expansion of CD8+ T cell reactivity to the individual viral peptides (Fig. 1B) and was not increased by treatment of the DC with IFN-{gamma} in addition to CD40L (Fig. 1A and 1B) .


Figure 1
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  		Figure 1. Maturation of DC for stimulation of HIV-1-specific and non-HIV-1 viral-specific IFN-{gamma} production by autologous CD8+ T cells. (A) Cumulative results show that within each group, peptide-loaded, untreated, immature DC stimulated greater CD8+ T cell responses than did CD8+ T cells, to which peptide was added without DC [P<0.05 (•) for the three groups of study subjects]. Compared with immature DC, DC treated with CD40L alone or CD40L + IFN-{gamma} induced the greatest CD8+ T cell reactivity in HIV-1-negative persons [n=6–8; CD40L: P=0.05 (*); CD40L+IFN-{gamma}: P=not significant (ns)], HIV-1-infected subjects on ART [n=13–19; CD40L: P<0.01 ({triangleup}); CD40L+IFN-{gamma}: P<0.01 ({triangleup})], and HIV-1-infected subjects not on ART [n=7–13; CD40L: P<0.05 (*); CD40L+IFN-{gamma}: P<0.05 (*)]. There were no differences compared with immature DC in virus-specific IFN-{gamma} production by CD8+ T cells by DC stimulated by a combination of poly(I:C) and the four cytokines (P=ns). Analysis across the three groups showed there were no differences in the levels of IFN-{gamma} produced by CD8+ T cells from HIV-1-infected or HIV-1-negative subjects (P=ns). (B) Representative data of IFN-{gamma} production by CD8+ T cells stimulated with viral peptide-loaded, autologous DC from a HIV-1-negative person (left panel), a HIV-1-infected subject on ART (middle panel), and a HIV-1-infected subject not on ART (right panel). The DC were examined for stimulation of IFN-{gamma} production by CD8+ T cells in response to CEF peptides or HIV-1 peptides.

There were no significant differences in mean levels of antiviral T cell reactivity to DC treated with poly(I:C) or the various cytokines alone (Fig. 1A) or with less than the full combination of these five maturation factors (data not shown) compared with untreated, immature DC from uninfected persons and HIV-1-infected subjects on ART or not on ART. However, assessing the number of positive antiviral T cell responses induced by DC revealed that DC treated with the complete combination of poly(I:C) and the four cytokines stimulated a greater number of positive antiviral T cell responses than did immature DC from uninfected subjects or HIV-1-infected subjects not on ART (Table 2 ). Indeed, DC from the HIV-1-infected subjects not on ART and treated with poly(I:C) or IFN-{alpha} alone (Table 2) or any combination including poly(I:C) or IFN-{alpha} (data not shown), induced more positive T cell responses than did immature DC. In contrast, there were no differences in the number of positive T cell responses induced by DC from the HIV-1-infected subjects on ART that were treated with any combination of poly(I:C) and the four cytokines (Table 2) . Similar results were noted using a series of 15mer peptides overlapping by 11 aa, which contained these minimal 9mer- immunodominant sequences and therefore required proteolysis for antigen presentation (data not shown).


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  		Table 2. Virus-Specific IFN-{gamma} Production in CD8+ T Cells Stimulated by Peptide-Loaded DC

Thus, DC treated with CD40L induced the greatest levels of activation of antiviral CD8+ T cells in uninfected and HIV-1-infected persons on ART or not on ART. The DC stimulated with the full combination of poly(I:C) and four proinflammatory cytokines induced significant antiviral reactivity in CD8+ T cells from uninfected persons and HIV-1-infected persons not on ART but not in HIV-1-infected persons on ART.

IL-12p70 production by DC treated with CD40L and other maturation factors
It is known that CD40L stimulates robust production of IL-12p70 by DC [19 ], with even greater amounts being induced by costimulation with IFN-{gamma} [17 , 20 ]. This biologically active form of IL-12 can in turn directly activate antigen-specific CD8+ T cell responses [21 ], in addition to acting indirectly on CD8+ T cells by polarizing Th1 responses [4 ]. For this study, we investigated the role of IL-12 in direct activation of anti-HIV-1 CD8+ T cell responses. First, we showed that the highest levels of IL-12p70 production by DC were induced by CD40L + IFN-{gamma} in all three groups of study subjects (Fig. 2A ), i.e., peak amounts at 24–48 h from HIV-1-negative subjects = 6663–7791 pg/ml (P<0.001 compared with amounts produced by untreated DC), HIV-1-infected subjects on ART = 4127–5389 pg/ml (P<0.001), and HIV-1-infected subjects not on ART = 1752–1893 pg/ml (P=0.05). In response to CD40L + IFN-{gamma}, DC from HIV-1-negative subjects and HIV-1-infected subjects on ART produced higher levels of IL-12p70 than DC from HIV-1-infected subjects not on ART (P<0.05). DC treated with CD40L alone produced the next-highest amounts of IL-12p70; i.e., peak levels by 24–48 h = 1959–2532 pg/ml for HIV-1-negative subjects (P<0.05), 1243–1577 pg/ml for HIV-1-infected subjects on ART (P<0.01), and 543–1302 pg/ml for HIV-1-infected subjects not on ART (P=ns).


Figure 2
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  		Figure 2. IL-12p70 production by DC from HIV-1-negative persons, HIV-1-infected subjects on ART, and HIV-1-infected subjects not on ART. (A) IL-12p70 production by untreated, immature DC or DC treated with CD40L, CD40L + IFN-{gamma}, or IFN-{gamma}. Analysis within each group showed that DC from all three groups that were treated with CD40L + IFN-{gamma} produced more IL-12p70 than untreated DC [P=0.05 (*), and P<0.01 (**)]. DC from HIV-1-negative subjects and HIV-1-infected subjects on ART that were treated with CD40L alone produced larger amounts of IL-12p70 than untreated DC [P<0.01 (**)]. Analysis across the three groups showed that levels of IL-12p70 produced by DC treated with CD40L + IFN-{gamma} were comparable among the three groups of study subjects (P=ns). The levels of IL-12p70 produced by DC treated with CD40L from the HIV-1-infected subjects not on ART were lower than the HIV-1-negative subjects and HIV-1-infected subjects on ART [P<0.05 (#)], but there was no difference between HIV-1-negative persons and HIV-1-infected subjects on ART (P=ns). (B) IL-12p70 production by untreated, immature DC or DC treated with poly(I:C), IFN-{alpha}, IFN-{gamma}, IL-1β, or TNF-{alpha} alone or a combination of poly(I:C) and the four cytokines. Analysis within each group showed that the combination of poly(I:C) and the four cytokines induced greater amounts of IL-12p70 in HIV-1-negative persons and HIV-1-infected subjects on ART compared with untreated, immature DC [P<0.01 (**)]. Treatment of DC from HIV-1-infected subjects not on ART with the full poly(I:C)-cytokine combination did not induce more IL-12p70 than immature DC (P=ns). Analysis across the three groups showed that levels of IL-12p70 produced by DC treated with the full poly(I:C)-cytokine combination were comparable between the HIV-1-uninfected subjects and HIV-1-infected subjects on ART (P=ns). The levels of IL-12p70 produced by DC treated with full poly(I:C)-cytokine combination from the HIV-1-infected subjects not on ART were lower than the HIV-1-negative subjects and HIV-1-infected subjects on ART [P<0.05 (#)]. HAART, Highly active ART.

Treatment of DC with the TLR3 ligand poly(I:C) or any of the four inflammatory cytokines alone did not induce more IL-12p70 than the low levels produced by untreated, immature DC (10–90 pg/ml by 48 h; P=ns; Fig. 2B ). However, treatment of DC with the full combination of poly(I:C) and the four proinflammatory cytokines stimulated production of IL-12p70 above that in untreated, immature DC cultures derived from HIV-1-negative subjects and HIV-1-positive subjects on ART, i.e., 278–557 pg/ml produced within 48 h (P<0.05; Fig. 2B ). The full poly(I:C)-cytokine combination induced lower levels of IL-12 in DC from the HIV-1-infected subjects not on ART compared with the two other groups of subjects (P<0.01, Fig. 2B ).

Taken together, these results show that DC derived from uninfected subjects, HIV-1-infected subjects on ART, and HIV-1-infected persons not on ART, which were treated with CD40L, with or without IFN-{gamma}, produce the greatest amounts of IL-12. Treatment of DC from the HIV-1-uninfected and HIV-1-infected subjects on ART, but not DC from HIV-1-infected subjects not on ART, with a combination of poly(I:C) and the four proinflammatory cytokines, induced significant, albeit lower, levels of IL-12.

Effect of IL-12 on IFN-{gamma} production by CD8+ T cells stimulated with viral peptide-loaded DC
To determine if IL-12 played a direct role in the effect of CD40L on activation of this antiviral CD8+ T cell reactivity, we added rIL-12 to the DC–T cell cultures. We found that CD8+ T cell IFN-{gamma} responses to the viral peptides were enhanced by rIL-12 in the absence of DC or when CD8+ T cells were mixed with peptide-loaded, immature DC from HIV-1-negative persons and HIV-1-infected subjects on ART (Supplemental Fig. 1). This was dependent on the concentration of rIL-12, with ≥1 ng/ml being required for an enhancing effect (P<0.05). This amount of IL-12p70 was within the range of levels of the IL-12p70 produced by DC treated with the CD40L and poly(I:C)-cytokine combinations (Fig. 2A and 2B) . There was, however, no enhancement of antiviral T cell reactivity induced by peptide-loaded DC that were treated with rIL-12 and CD40L alone or CD40L + IFN-{gamma}.

We next determined whether neutralization of endogenously produced IL-12 in the DC cultures with anti-IL-12 mAb inhibited their capacity to activate MHC class I-restricted, viral peptide-specific IFN-{gamma} production by CD8+ T cells. For these studies, we compared T cell reactivity to DC from HIV-1-negative persons and HIV-1-infected persons on ART. The concentration of anti-IL-12p70 mAb that we used neutralized over 95% of IL-12p70 for all of the groups of study subjects, as indicated by an ELISA on supernatants collected from the overnight Elispot assays (data not shown). Neutralization of IL-12p70 in cultures of CD8+ T cells alone, untreated DC + CD8+ T cells, and IFN-{gamma}-treated DC + CD8+ T cells decreased CD8+ T cell reactivity to EBV and HIV-1 peptides by 65–93% in HIV-1-negative and HIV-1-infected subjects on ART, respectively (Fig. 3A and 3B ). In contrast, neutralization of IL-12p70 had little or no effect on antiviral reactivity of CD8+ T cells from HIV-1-negative persons or HIV-1-infected persons on ART in response to viral peptide-loaded, CD40L-treated DC or CD40L + IFN-{gamma}-treated DC. In HIV-1-negative subjects, the percent inhibition of the CD8+ T cell response to viral peptides without DC was 92%, P < 0.05; with untreated DC, 38%, P < 0.01; with IFN-{gamma}-treated DC, 79%, P< 0.01; with DC + CD40L, 7%, P< 0.05; and with DC + CD40L + IFN-{gamma}, 0%, P = ns (Fig. 3A) . A similar pattern was noted using DC from HIV-1-infected subjects on ART; i.e., percent inhibition of CD8+ T cell response to viral peptide with no DC was 93%, P < 0.01; DC, 86%, P < 0.01; DC + IFN-{gamma}, 65%, P < 0.01; DC + CD40L, 6%, P = ns; and DC + CD40L + IFN-{gamma}, 0%, P = ns (Fig. 3B) .


Figure 3
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  		Figure 3. Effect of anti-IL-12 mAb on IFN-{gamma} production by DC-stimulated CD8+ T cells. (A) Anti-IL-12 mAb inhibited DC stimulation of MHC class I, EBV peptide-specific, IFN-{gamma} production by CD8+ T cells from three HIV-1-negative subjects, with the exception of CD8+ T cells stimulated by DC treated with CD40L + IFN-{gamma}. (B) A similar pattern was seen in two HIV-1-infected subjects on ART [P<0.01 ({Delta}); P<0.05(*)].

These results support that the small amounts of endogenous IL-12p70 induced by viral peptides in CD8+ T cells alone, in immature DC, or in DC treated with IFN-{gamma} (10–20 pg/ml, Fig. 2 ) have a direct enhancing effect on antiviral CD8+ T cell responses. However, these data also suggest that the much higher levels of IL-12p70 produced by DC treated with CD40L, with or without IFN-{gamma} (>1000 pg/ml, Fig. 2 ), are not essential for direct enhancement of antiviral CD8+ T cell responses, as neutralization of this IL-12 does not decrease the stimulation of antiviral CD8+ T cells by the DC.

Effect of CD4+ T cells on IFN-{gamma} production in CD8+ T cell cultures stimulated by viral peptide-loaded DC
CD4+ T cells can have helper and suppressor effects on antigen-specific CD8+ T cell reactivity [22 ]. To define the effect of CD4+ T cells on CD8+ T cell reactivity to viral peptide-loaded DC in our system, CD8+ T cells from HIV-1-negative persons and HIV-1-infected subjects on ART were mixed with resting or activated, autologous CD4+ T cells at ratios of 1:0.125, 1:0.5, and 1:2. Addition of resting CD4+ T cells had no effect on anti-HIV-2 specific, IFN-{gamma} production by CD8+ T cells (Fig. 4A1 ). Addition of activated CD4+ T cells (Fig. 4A2) to CD8+ T cells without DC at a 1:2 ratio enhanced HIV-1 viral peptide-specific IFN-{gamma} production in HIV-1-infected subjects (P<0.05) and non-HIV-1 virus-specific T cell reactivity in HIV-1-negative persons (data not shown; P<0.05). However, addition of resting CD4+ T cells had no effect or a mild, suppressive effect on peptide-specific IFN-{gamma} production in the various CD8+ T cell–DC cultures as compared with cultures without CD4+ T cells (suppression: immature DC, 5–11%, P<0.05; DC treated with IFN-{gamma}, 14–28%, P<0.05; DC treated with CD40L, 0–11%, P<0.001; DC treated with CD40L+IFN-{gamma}, 0–3%, P=ns). Addition of activated CD4+ T cells had a stronger suppressive effect, especially at the 1:2 ratio of CD8:CD4 T cells (suppression: DC, 5–57%, P<0.01; DC+IFN-{gamma}, 4–23%, P<0.05; DC+CD40L, 12–84%, P<0.001; DC+CD40L+IFN-{gamma}, 2–82%, P<0.001). Similar results were found using cells from HIV-1-negative persons (data not shown). The cell viability in these cultures was 92%, which indicated that the suppressive effects were not related to cell death.


Figure 4
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  		Figure 4. Effect of CD4+ T cells on CD8+ T cell responses to viral antigen-loaded DC. (A) Effect of resting CD4+ T cells (A1) and activated CD4+ T cells (A2) at a1:2 ratio enhanced peptide-specific IFN-{gamma} production by CD8+ T cells. (B) Effect of CD4+ T cells on IL-2 production in cultures of DC and CD8+ T cells. Addition of resting CD4+ T cells (B1) or activated CD4+ T cells (B2).

We further examined this DC–T cell system in HIV-1-infected subjects on ART by ELISA assay of supernatants collected from the overnight Elispot assay for IL-2 production. No IL-2 was detected in the cultures of CD8+ T cells from HIV-1-infected persons or resting or activated, autologous CD4+ T cells without DC. However, there was an increase in IL-2 production in the resting CD4+ T cell–CD8+ T cell–DC cultures, which was CD4+ T cell concentration-dependent (Fig. 4B1) . This effect of the resting CD4+ T cells was greater than that associated with activated CD4+ T cells (Fig. 4B2) . Similar results were observed using non-HIV-1 viral peptides and cells from HIV-1-negative persons (data not shown).

For a third measure of the effects of CD4+ T cells on antiviral CD8+ T cell function, CD8+ T cells were mixed at the 1:0.125, 1:0.5, and 1:2 ratios with resting or activated CD4+ T cells in a blastogenesis assay in response to viral, peptide-loaded, autologous DC. HIV-1 peptide-specific, CD8+ T cell blastogenesis was significantly enhanced in a concentration-dependent manner by resting CD4+ T cells (Fig. 4C1) compared with activated CD4+ T cells (Fig. 4C2) in cocultures with HIV-1 9mer peptide-loaded DC. Similar effects were found for non-HIV-1 viral peptides using cells from HIV-1-uninfected persons (data not shown).

Taken together, these results suggest that in the presence of viral peptide-loaded DC, resting CD4+ T cells had no effect on activation of antiviral CD8+ T cell reactivity (Fig. 4A1) but appeared to provide helper function for IL-2 production by T cells (Fig. 4B1) and for T cell blastogenesis (Fig. 4C1) .

CD8+ T cell response to restimulation with viral peptide-loaded DC
A key issue in the clinical efficacy of DC-based immunotherapy for HIV-1 infection is whether the ex vivo-engineered DC will be exhausted and unable to activate antiviral CD8+ T cells in vivo after re-infusion [20 ]. To avoid exhaustion of DC to be used in immunotherapy clinical trials, DC are being treated with the full combination of poly(I:C) and the four proinflammatory cytokines ex vivo and then injected back into the patients [4 ]. It is hypothesized that activated CD4+ T cells in the local lymphatics will then stimulate these DC via CD40L–CD40 interactions. To test this in our DC-antiviral T cell system, 24 h after the first stimulation with poly(I:C) + IFN-{alpha} + IFN-{gamma} + IL-1β + TNF-{alpha}, DC from a HIV-1-negative person and three HIV-1-infected subjects on ART were given a second stimulation with CD40L or CD40L + IFN-{gamma} for an additional 24 h as surrogates for activated CD4+ T cells. These DC were washed thoroughly before the second stimulation to remove excess maturation factors and to mimic the procedures for their use as an immunotherapy in clinical trials. We observed that a second stimulation of the DC with CD40L or CD40L + IFN-{gamma} induced greater levels of IL-12p70 than found after the first stimulation (Fig. 5 ). A second stimulation of these DC with CD40L or CD40L + IFN-{gamma} maintained but did not enhance virus-specific CD8+ T cell responses above the untreated, control DC (Fig. 6 ). A comparable pattern of IL-12p70 production and stimulation of antiviral T cell activity by DC was noted using DC that were not washed between stimulations (data not shown).


Figure 5
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  		Figure 5. IL-12p70 production by DC from a HIV-1-negative subject (A) and three HIV-1-positive subjects on ART (B–D). For this, 24 h after the first immunomodulating stimulation, DC were washed three times and restimulated for an additional 24 h. DC treated with poly(I:C) + IFN-{alpha} + IFN-{gamma} + IL-1β + TNF-{alpha}, washed, and then treated with CD40L or CD40L + IFN-{gamma} produced greater levels of IL-12 after the second stimulation compared with untreated DC (None). DC that were not washed before the second stimulation had similar results (data not shown).


Figure 6
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  		Figure 6. IFN-{gamma} production by CD8+ cells after stimulation of DC from a HIV-1-negative subject (A) and three HIV-1-infected subjects on ART (B–D). After the first stimulation with poly(I:C) + IFN-{alpha} + IFN-{gamma} + IL-1β + TNF-{alpha}, the DC were washed and treated with CD40L or CD40L + IFN-{gamma}. As compared with untreated DC (None) for the first simulation, DC treated with the full poly(I:C)-cytokine combination induced higher production of IFN-{gamma} in CD8+ T cells for a HIV-1-negative person and two of three HIV-1-infected persons [P<0.05 (#)]. DC from four subjects that were stimulated first with the full poly(I:C)-cytokine combination and then with CD40L or CD40L + IFN-{gamma} maintained their capacity to induce IFN-{gamma} production by CD8+ T cells (P=ns compared with untreated DC). For comparison, DC from a HIV-1-negative person and two of three HIV-1-infected persons who were not treated first with the full poly(I:C)-cytokine combination were able to stimulate increased levels of IFN-{gamma} by CD8+ T cells after stimulation with CD40L or CD40L + IFN-{gamma} [P<0.01 ({triangleup})].

Taken together, these results support that DC treated with poly(I:C) and the four proinflammatory cytokines retained their capacity to produce increased amounts of IL-12p70 after a second stimulation with CD40L or CD40L + IFN-{gamma}. Although DC treated with poly(I:C)-cytokine also retain their capacity to induce antiviral CD8+ T cell responses after a second, boosting treatment with CD40L or CD40L + IFN-{gamma}, the levels of these antiviral T cell responses were not increased as compared with mock-restimulated DC.

DISCUSSION

Based on the emerging inter-relationships of innate and adaptive immunity that are critical to the control and prevention of viral infections and cancers, DC are the most potent APC that can be targeted to elicit a prolonged, elevated, and broadly reactive T and B cell immune response to pathogens. This has led to clinical trials based on the hypothesis that ex vivo-engineered, autologous DC could act as a potent immunotherapy for HIV-1 infection and cancers [23 , 24 ]. Notably, Lu et al. [1 ] reported that immunotherapy with DC loaded with inactivated HIV-1 had inhibitory effects on disease progression. Of importance is that the immunomodulating agents used to mature DC for such clinical therapies can, to a large degree, dictate the capacity of the DC to enhance anti-HIV-1 cellular immunity. In particular, production of IL-12p70 by DC is critical for driving antiviral CTL and Th1 cell-dependent immune responses [19 ]. CD40L and IFN-{gamma} provided by activated CD4+ T cells are key molecules that stimulate production of IL-12 by DC [25 ]. This can be through direct stimulation of the CD8+ T cells or by a polarizing effect on CD4+ Th1 cells that produce cytokines, particularly IFN-{gamma}, which in turn activate the CD8+ T cells [21 ]. However, CD40L is not currently available for clinical use. Alternatively, maturation with a combination of poly(I:C), IFN-{alpha}, IFN-{gamma}, IL-1β, and TNF-{alpha} generates fully mature, Th1-polarizing DC, termed {alpha} type 1-polarized DC [4 ]. These DC have produced IL-12 and are able to stimulate CTL reactivity to immunodominant melanoma peptides. We therefore compared this complete cocktail and various combinations of these stimuli for eliciting antiviral CD8+ T cell immune responses and IL-12 production by DC from HIV-1-infected subjects to DC that were treated with CD40L, with or without IFN-{gamma}.

Our study shows that stimulation of CD8+ T cells with viral peptide-loaded, immature DC from the three groups of HIV-1-negative and HIV-1-infected subjects induced greater IFN-{gamma} production than did direct stimulation of CD8+ T cells with these peptides. Although this difference could be related to the lack of APC in the purified CD8+ T cells subsets, we have previously shown a similar enhancing effect of DC compared with the common method of stimulating PBMC directly with antiviral peptides [26 ]. We further demonstrated in the present study that DC from HIV-1-infected persons on ART, which were treated with CD40L, induced the greatest levels of IFN-{gamma} production by CD8+ T cells to HIV-1-immunodominant peptides compared with immature DC. This T cell-enhancing effect was also observed with DC from HIV-1-infected subjects not on ART, supporting our previous study [27 ]. A similar enhancing effect was noted with DC and CD8+ T cells from HIV-1 negative persons to CMV, EBV, and Flu peptides. Antiviral T cell responses were not further enhanced by treatment of the DC with a combination of CD40L and IFN-{gamma}.

Importantly, DC treated with the full combination of TLR3 ligand poly(I:C) and inflammatory cytokines IFN-{alpha}, IFN-{gamma}, IL-1β, and TNF-{alpha} significantly increased antiviral T cell responses above the responses to immature DC in HIV-1-negative persons and in HIV-1-infected subjects not on ART. The lack of an enhancing effect of poly(I:C) alone on stimulation of antiviral CD8+ T cell reactivity by DC is in contrast to the report of Lore et al. [7 ]. The basis for the difference in our results is not clear. The only major difference in techniques was that our study used higher concentrations of poly(I:C), which were not toxic to the DC. Although we found that the complete combination of poly(I:C) and the four cytokines was necessary to elicit the enhancing effect in DC from the HIV-1-negative subjects, the T cell-enhancing effects were shown to be related to poly(I:C) and IFN-{alpha} in the DC maturation cocktail in the HIV-1-infected subjects not on ART. Interestingly, we did not find an enhancing effect on T cell reactivity of DC treated with poly(I:C) and the proinflammatory cytokines as compared with immature DC in HIV-1-infected subjects on ART. This could relate to the lower CD8+ T cell reactivity in persons on ART, which is associated with their lower HIV-1-antigenic burden [28 ] and requires further study.

We observed that DC treated with the complete combination of poly(I:C) and the four proinflammatory cytokines produced higher levels of the biologically active p70 form of IL-12 than did immature DC in uninfected persons and HIV-1-infected subjects on ART. These levels of IL-12 were lower than those induced by CD40L alone or CD40L and IFN-{gamma}. None of the various combinations of maturation factors increased production of IL-12 by DC from HIV-1-infected persons not on ART. Previous studies have shown that the TLR3 ligand poly(I:C) can stimulate DC to produce more IL-12 if given in combination with other immunomodulators [4 , 8 ]. Indeed, addition of several inflammatory mediators and TLR agonists along with poly(I:C) has recently been reported to enhance the capacity of DC to stimulate antiviral T cell responses [9 ]. However, the use of poly(I:C) in an immunomodulating cocktail can alter RNA expression, which could be an issue when using RNA constructs as a source of antigen in DC [9 ]. Thus, consideration of which form of DC maturation is best for immunotherapy of HIV-1 infection should include additional parameters of DC function, such as effects on expression of T cell costimulatory molecules, processing of different forms of HIV-1 antigens, and chemotaxis.

In the present study, we examined this model for a direct role of IL-12 in activation of antiviral CD8+ T cell reactivity. We found that as previously reported [17 , 20 ], DC treated with CD40L + IFN-{gamma} produced the greatest amounts of IL-12p70, followed by CD40L alone. Notably, however, neutralization of IL-12 in CD40L-treated or CD40L- and IFN-{gamma}-treated DC with anti-IL-12 mAb did not significantly reduce the capacity of these DC to stimulate viral peptide-specific IFN-{gamma} production by CD8+ T cells. Thus, other T cell-activating mechanisms of DC are presumably operative in enhancing the antiviral memory T cell responses in these DC–T cell cultures. Indeed, we have previously reported that expression of T cell costimulatory molecules CD80, CD86, and CD83 is up-regulated in a normal manner in CD40L-modulated DC from HIV-1-infected subjects on ART [17 , 26 ]. Alternatively, these DC could mediate enhanced antiviral T cell reactivity through IL-23 and IL-27 cytokines of the IL-12 family [29 ] or IL-18 of the IL-1 family, which in combination with IL-12, induces IFN-{gamma} in Th1 cells [30 ].

Given the requirement for CD4+ Th cells in maintenance of anti-HIV CD8+ CTL reactivity [31 , 32 ], we examined whether CD4+ T cells could help in stimulation of virus-specific CD8+ T cell responses by our immunomodulated DC. We found that addition of autologous resting or activated CD4+ T cells to CD8+ T cells in the absence of DC enhanced peptide-specific IFN-{gamma} production by CD8+ T cells. However, addition of resting CD4+ T cells had no effect or a mild-suppressive effect on peptide-specific IFN-{gamma} production in CD8+ T cells cultured with immature DC or DC treated with CD40L, with or without IFN-{gamma}. Interestingly, there was greater suppression of viral antigen-specific CD8+ T cell production of IFN-{gamma} when autologous activated CD4+ T cells were added to the CD8+ T cell–DC cultures at a 2:1, CD4:CD8 cell ratio, similar to that present in normal blood. We found that activated CD4+ T cells could also suppress proliferation of antiviral CD8+ T cells by peptide-loaded, CD40L-modulated DC. A similar, suppressive effect was associated with the presence of T regulatory cells in HIV-1-negative persons and HIV-1-infected subjects on ART (unpublished results) and requires further study.

The requirement of CD4+ T cell help for CD8+ T cell activation can be replaced by CD40 agonists [33 34 35 ]. DC activated by CD40L-expressing CD4+ T cells act as a link between the responses of CD4+ and CD8+ T cells [36 ]. In our study, the requirement for CD4+ T cell help in antiviral CD8+ T cell responses could be replaced by CD40L. Therefore, CD40L-treated DC could be the most important pathway to elicit T cell-immune responses maximally and efficiently, overcoming defects in CD4+ T cells in HIV-1-infected subjects. Although a potent, clinical grade of the soluble form of CD40L is not currently available for clinical use, agonistic, nondepleting CD40 antibodies that are being tested clinically in the oncology setting may have the potential to address the promise of CD40L signaling in the future.

There is concern that DC stimulated in vitro with CD40L, with or without IFN-{gamma}, will be exhausted and hyporesponsive to further activation signals in vivo after therapeutic injection [20 ]. Indeed, optimal production of IL-12 occurs when the poly(I:C)-, IFN-{alpha}-, IFN-{gamma}-, IL-1β-, and TNF-{alpha}-treated DCs are subsequently stimulated with CD40L [4 ]. These DC, termed {alpha} DC1, produce moderate amounts of IL-12 but require an additional stimulation such as that supplied by CD40L on activated CD4+ T cells to become fully activated, mature DC. Our present data indicate that DC from HIV-1-negative persons or HIV-1-infected subjects on ART that had been stimulated with poly(I:C) and these four proinflammatory cytokines had an enhanced capacity for production of IL-12p70 in response to restimulation with CD40L or CD40L plus IFN-{gamma}. However, although DC retained their capacity to activate antiviral CD8+ T cell responses after restimulation with CD40L or CD40L plus IFN-{gamma}, the antiviral T cell responses were not enhanced above those induced by restimulation with mock-treated DC. Nevertheless, a similar approach has recently been reported for treatment of early breast cancers with HER-2/neu peptide-loaded DC that were preconditioned in vitro with the TLR4 agonist LPS and IFN-{gamma} [37 ]. This effect could occur in vivo in HIV-1-infected patients receiving preconditioned DC as immunotherapy and a second burst of IL-12 production by the injected DC or resident DC occurring at the site of T cell sensitization. Such an approach could be highly relevant to the design of DC-based immunotherapeutic vaccines for HIV-1 infection.

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Allergy and Infectious Diseases grants P01 AI-055794, U01 AI-35041, and R37 AI-41870. We thank P. Kalinski for helpful comments, K. Phalmer and K. Picha (Amgen, Seattle, WA, USA) for providing the CD40L, W. Jiang and P. Zhang for technical assistance, W. Buchanan for clinical assistance, and the volunteers of the Pitt Men’s Study (the Pittsburgh site of the Multicenter AIDS Cohort Study).

Received November 27, 2007; revised February 20, 2008; accepted February 26, 2008.

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