DNA vaccination is an invaluable approach for immune therapy in that it lacks vector interference and thus permits repeated vaccination boosts. However, by themselves, DNA-based vaccines are typically poor inducers of Ag-specific immunity in humans and non-human primates. Cytokines, such as IL-12 and IL-15, have been shown to be potent adjuvants for the induction and maintenance of cellular immune responses, in particular during HIV infection. In this study, we examined the ability of therapeutic vaccination with SIV-DNA+IL-12 or IL-15 as molecular adjuvants to improve DNA vaccine potency and to enhance memory immune responses in SIV-infected macaques. Our results demonstrate that incorporating IL-12 into the vaccine induces SIV-specific CD8 effector memory T cell (TEM) functional responses and enhances the capacity of IFN-γ-producing CD8 TEM cells to produce TNF. Lower levels of PD-1 were expressed on T cells acquiring dual function upon vaccination as compared with mono-functional CD8 TEM cells. Finally, a boost with SIV-DNA+IL-15 triggered most T cell memory subsets in macaques primed with either DNA-SIV or placebo but only CD8 TEM in macaques primed with SIV-DNA+IL-12. These results indicate that plasmid IL-12 and IL-15 cytokines represent a significant addition to enhance the ability of therapeutic DNA vaccines to induce better immunity.

Human immunodeficiency virus-1 pathogenesis can be delayed with the use of effective preventive strategies for opportunistic infections and aggressive antiretroviral therapy (ART)4 to suppress viral production and its attendant rapid CD4 lymphocyte population turnover (1). However, prolonged highly active antiretroviral therapy treatment has not been successful in eradicating virus in as much as infectious HIV-1 persists in cellular reservoirs (2, 3). Therapeutic vaccination represents an important tool with the potential for decreasing patient’s dependence on antiretroviral drugs by inducing potent T cell responses, thereby allowing for continued suppression of viral replication (4, 5, 6).

Several studies have confirmed that strong host immune responses correlate with the containment of HIV-1 replication. Studies of individuals who were long-term non-progressors indicated their ability to mount and maintain a robust polyclonal HIV-1-specific CD4 T cell proliferative response against HIV, characterized by intact CD4 memory for HIV epitopes and persistent CD8 cytotoxic activity (7, 8, 9, 10). Those patients appeared to mount cellular immune responses that were able to suppress HIV replication and hence delayed the progressive damage to their immune system. Furthermore, subjects who were identified within the first few weeks to months of HIV infection and treated aggressively with highly active ART often maintained CD4 recognition of HIV epitopes and preserved CD8-lymphocyte cytotoxic activity against cells expressing HIV Ags. By the same token, those cells showed a strong capacity to control viral rebound following cessation of therapy (11, 12, 13). Similar results have been observed in an SIV structured-treatment interruption study in Rhesus macaques (14). It would thus appear that early treatment helps protect the cellular immune system activated by HIV infection from being consumed by the rapid viral production and the ensuing destruction of memory T cells.

Although Ag-specific immune responses were triggered by DNA immunization (15, 16, 17, 18), the levels of these responses need to be significantly improved. Cellular immune responses to DNA vaccines were shown to be enhanced when codelivering DNA plasmids expressing immune modulators (19, 20, 21). In particular, it has been demonstrated that coimmunization with Th1-type cytokines triggers a Th1 cellular immune response while Th2-type cytokines can prompt Ab responses. Of particular interest, IL-12, a dendritic cell (DC)-produced cytokine, is a strong stimulator of NK cells as well as of T lymphocyte activity. IL-12 supports the differentiation of Ag-specific CD4 T cells to produce Th1 cytokines and triggers the expansion of Ag-specific CD8 T cells to express cytotoxic mediators, such as granzyme B/perforin and IFN-γ, respectively (22, 23, 24, 25, 26, 27, 28). Coadministration of an IL-12 expression vector with the DNA immunogen led to the expansion of precursor CTL and the increase of their lytic activity as compared with immunization with the DNA immunogen alone (29). The use of IL-12 as an immune adjuvant has proven effective at improving immune priming ex vivo as well as in animal models (29, 30, 31, 32, 33, 34). Coimmunization of HIV-infected chimpanzees with IL-12 expressing plasmids led to transient boosting of the proliferative response to gag; however, the impact on viral load was minimal (35). Chattergoon et al. (36) have shown that priming mice with a DNA vaccine for the flu Ag together with IL-12 led to a greater frequency of Ag-specific CD8 T cells immediately following immunization and to a better control of flu virus when challenged up to 6 mo after the last immunization. As such, they proposed that IL-12 increased the frequency of memory cells generated to the vaccine Ag, thereby improving long-term vaccine efficacy. Moreover, IL-12 plasmids enhanced the SIVgag-specific T cell response to a SIVgag DNA vaccine (19, 20).

Among cytokines that are of particular interest for generating an efficacious HIV-1 vaccine are those that share the common γ receptor chain subunit and are implicated in the generation and persistence of memory T cells (37, 38, 39). Among them, IL-2, IL-4, IL-7, IL-15, and IL-21 are all involved in enhancing or modulating T cell responses (38, 40, 41, 42, 43, 44). IL-15, an important survival factor, exerts its impact primarily on memory CD8 T cells (45, 46) by stimulating their proliferation and preventing their apoptosis, thereby promoting their persistence (41, 44, 47, 48). Moreover, memory CD8 T cells increased significantly in the peripheral lymphoid tissue of IL-15 transgenic mice, while their numbers declined in IL-15- and IL-15R-deficient mice (41). Therefore, cumulative evidences from many studies suggested that IL-15 would be used as an effective adjuvant in enhancing and sustaining memory CD8 T cell responses (49, 50).

In this study, we tested the ability of DNA vaccines encoding SIV structural proteins gag, pol, and env either alone or in combination with a plasmid expressing IL-12 or IL-15 to enhance the immune responses of chronically infected Rhesus macaques treated with anti-retrovirals. We observed that the codelivery of plasmid IL-12/IL-15 along with an SIV-DNA vaccine was able to significantly enhance its ability to induce SIV-specific cellular immune responses.

The 32 Indian Rhesus macaques used in this study were housed at the Southern Research Institute in Frederick, MD. These facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care International and meet National Institutes of Health standards. The macaques were tissue typed for their MHC class I simian leukocyte Ag allele genotype (51, 52) and inoculated i.v. with 100 MID50 (50% macaque infectious dose) of highly pathogenic SIVmac251. All macaques were infected and reached peak viral loads by wk 2 and set point by wk 12. Treatment was initiated in infected macaques at wk 14 with 2,3-dideoxy-5-fluoro-3′-thiacytidine (FTC) (50 mg/kg/day) and 9-[9(R)-2(phosphonomethoxy)propyl]adenine (PMPA) (20 mg/kg/day) once daily by s.c. injection. Control of viral replication was already observed with 4 wk of therapy start up. Eleven animals, however, were not able to suppress viral replication and were thus eliminated from the study. The SIV vaccine plasmid constructs (SIVgag, SIVenv, and SIVpol) were produced, mixed together, and formulated with 0.25% bupivicaine-HCL for a final concentration of 3 mg/ml of each plasmid. Macaque IL-12 and IL-15 plasmid constructs were used in this study. IL-12 constructs are two promoter constructs that drive each chain of IL-12 (p35 and p40) individually which then associate to form the bioactive molecule. Supercoiled plasmids do not include any CpG sequences that could trigger macaque TLR-9 (19). Macaques were immunized with 3 mg of each DNA at weeks 26, 32, 36, and 41. IL-12 or IL-15 adjuvant constructs were mixed with the SIV plasmid constructs such that 3 mg IL-12 (or IL-15) adjuvant plasmid was delivered per injection.

Ninety-six well polyvinylidene diflouride backed plates (Millipore) were coated with 15 μg/ml of anti-IFN-γ mAb (Mabtech) and incubated overnight at 4°C. The plates were then washed with PBS, and PBMCs were added at a concentration of 2 × 105 cells per well. Pools of SIVgag, pol, and env peptides from the AIDS Reagent Repository were used to assess the cellular immune response. Following 24-h incubation at 37°C-5% C02, cells were removed and the plates were washed with PBS. Biotinylated IFN-γ mAb was then added at 1 μg/ml, followed by a 2-h incubation at 4°C. After wash, spots were visualized using streptavidin alkaline phosphatase conjugate. The data are presented as the number of spot forming cells (SFC) per 1 million PBMCs. A positive response is defined as >50 SFC/million PBMCs. ELISPOT were performed in triplicates.

CD8 T cells were depleted using Dynal magnetic beads 13 wk following the final immunization. The PBMCs were then setup in a standard ELISPOT assay. The cells were added to 96-well plates at 2 × 105 cells per well and were stimulated with SIVgag, pol, and env peptides. Each spot formed represented one Ag-specific cell-secreting IFN-γ as a result of SIV peptide stimulation.

PBMCs (2 × 106 cells) were stimulated with 2 μg/ml of the cognate peptides for 6 h in RPMI containing 10% human serum in the presence of 5 μg/ml of brefeldin A (Sigma-Aldrich). Non-stimulated cells as well as cells stimulated with superantigen SEM (SEA (staphylococcal enterotoxin A) + SEB (staphylococcal enterotoxin B)) (Sigma-Aldrich) were used as controls. These cells were then stained with the following surface-markers’ specific Abs for 15 min at 4°C: CD4 (clone L200-PerCP-Cy5.5; BD Biosciences), CD8 (CD8PE-Texas Red (ECD); Cedarlane Laboratories), CD95 (clone DX2-PE-Cy5; BD Biosciences), CD28 (clone CD28.2-Pacific Blue, custom made; BD Biosciences), CCR7 (clone 3D12-PE-Cy7; BD Biosciences), and PD-1 (clone MIH4-FITC; BD Biosciences). Cells were then fixed for 10 min in 100 μl 2% paraformaldehyde at room temperature (25°C). To stain cells with Abs specific for intracellular cytokines (IFN-γ-Alexa-700, IL-2-allophycocyanin, and TNF-α-PE; BD Biosciences), we incubated the cells with Abs in 0.25% saponin (Sigma-Aldrich) for 30 min at 25°C and analyzed them using the BD LSRII flow cytometer. Between 250,000 and 1 × 106 events were acquired for each condition. Data were then analyzed using DIVA software (BD Biosciences).

We performed statistical analyses using Analyze-it for Microsoft Excel software. Differences between groups were assessed by a nonparametric Mann-Whitney U test. Differences before and after treatment within the same group were assessed using the Wilcoxon test. We used an unpaired t test assuming two-tailed distribution and two sample unequal variances for the underlying populations (see Fig. 2). An unpaired t test was also used assuming two-tailed distribution and two sample equal variances for the underlying populations (see Fig. 6).

IL-12 has recently been reported to enhance SIV-specific CD8 T cell immune responses in vaccinated macaques (19). To determine whether the plasmids encoding macaque IL-12 or IL-15 functional genes could increase the immunogenicity of SIV-DNA therapeutic vaccination, we tissue-typed 32 Indian Rhesus macaques for their MHC class I SLA (simian leukocyte Ag) allele genotype. Each macaque was inoculated i.v. with 100 MID50 of highly pathogenic SIVmac251. All the macaques were infected and reached peak viral loads by wk 2 and set point by wk 12. Viral replication was allowed to continue until wk 12 to mimic a chronic state of infection in humans. Average set point viral loads at this time ranged between 1 × 103 and 1 × 106 (Table I).

Table I.

SLA restriction and viral set points of macaques within the different groups

ControlSIV DNASIV DNA+IL-12
3468-A08 5.5 × 102 3466-A08 2.2 × 103 3467-A01 7.6 × 102 
3471-A02 2 × 105 3473-A02 5.2 × 103 3469-none 1.7 × 103 
3481-A08 2 × 103 3480-none 1.5 × 103 3474-B01 1.0 × 105 
3478-A02, 08 1.3 × 106 3482-A02, 08, B01 6.9 × 102 3477-A01, 08 5.7 × 102 
3483-A01, B01 2.9 × 103 3485-A01, B01 1.9 × 103 3484-none 1.7 × 103 
3487-A02 3.2 × 104 3490-none 1.1 × 103 3486-none 2.8 × 103 
3492-A08 8.7 × 102 3494-A01, 08 1.2 × 103 3495-A01 1.5 × 103 
ControlSIV DNASIV DNA+IL-12
3468-A08 5.5 × 102 3466-A08 2.2 × 103 3467-A01 7.6 × 102 
3471-A02 2 × 105 3473-A02 5.2 × 103 3469-none 1.7 × 103 
3481-A08 2 × 103 3480-none 1.5 × 103 3474-B01 1.0 × 105 
3478-A02, 08 1.3 × 106 3482-A02, 08, B01 6.9 × 102 3477-A01, 08 5.7 × 102 
3483-A01, B01 2.9 × 103 3485-A01, B01 1.9 × 103 3484-none 1.7 × 103 
3487-A02 3.2 × 104 3490-none 1.1 × 103 3486-none 2.8 × 103 
3492-A08 8.7 × 102 3494-A01, 08 1.2 × 103 3495-A01 1.5 × 103 

Fourteen weeks later, once the viral set point was attained, all the macaques were treated with ART (FTC (50 mg/kg/day) and PMPA (20 mg/kg/day)) once a day by s.c. injection. Viral load in most animals dropped to below detectable limits by wk 18. Eleven animals, however, were not able to suppress viral replication and were thus eliminated from the study. On wk 26, (14 wk of anti-retroviral treatment), the 21 macaques were randomly divided three groups of seven macaques each. In randomizing our animals, we distributed the animals according to peak/set point viral load, SLA, and CD4 counts at the time of ART therapy. To the best of our ability, we attempted to randomize the animals between the two vaccine regimens and the control group. The macaques were immunized i.m. with multiple DNAs at wk 26, 32, 36, and 41 (Fig. 1). At each one of these time points, all the macaques in groups 2 and 3 received 3 mg of each of SIVgag, SIVenv, and SIVpol DNA plasmids. Moreover, all the macaques in group 3 received 3 mg of macaque IL-12 encoding plasmid along with the SIV-DNA plasmids. At the same time points, group 1 macaques received 3 mg of mock DNA. The macaques were then taken off drugs at wk 54 (Fig. 1). Six months later (wk 78), randomly selected macaques from each group were again put on ART and then boosted with three rounds of DNA immunization in the presence of IL-15 instead of IL-12. Subgroups B, E, and H were immunized with the same set of SIV-DNA plasmids plus the IL-15 plasmid, while subgroups D and G received only SIV-DNA. PBMCs were collected from all macaques at wk 18 (while on ART but before immunization), 56 (2 wk following the removal of the macaques from the drug treatment; and 15 wk following the fourth immunization), 62 (2 mo following removal of the macaques from the drug treatment), 88 (2 wk following the second round of immunization (boost)), and 92 (2 wk following the removal of the macaques from the drug treatment, i.e., 6 wk after the boost). Functional characteristics of SIV-specific memory T cells were then investigated.

FIGURE 1.

Immunization scheme. Twenty-one macaques were infected with SIVmac251 at wk 0 and reached peak viral loads by wk 2 and set point by wk 12. Infected macaques were treated beginning at wk 14 with ART (FTC (50 mg/kg/day) and PMPA (20 mg/kg/day)) once daily by s.c. injection. On wk 26, 32, 36, and 41, macaques were immunized with mock DNA (group 1), SIV-DNA (encoding gag, pol, and env) (group 2), or SIV-DNA+IL-12 (group 3). On wk 54, macaques were taken off drugs (OD). Macaque subgroups B, D, E, G, and H were put again on ART and immunized with SIV-DNA (D and G) or SIV-DNA+IL-15 (B, E, and H) on wk 78, 82, and 86. On wk 90, the macaques were taken off drugs. The time points at which PBMCs from the different macaques were analyzed are highlighted at the bottom of the figure. OD: Off drugs. A-H represents macaques’ subgroups. (n): number of macaque per sub-group.

FIGURE 1.

Immunization scheme. Twenty-one macaques were infected with SIVmac251 at wk 0 and reached peak viral loads by wk 2 and set point by wk 12. Infected macaques were treated beginning at wk 14 with ART (FTC (50 mg/kg/day) and PMPA (20 mg/kg/day)) once daily by s.c. injection. On wk 26, 32, 36, and 41, macaques were immunized with mock DNA (group 1), SIV-DNA (encoding gag, pol, and env) (group 2), or SIV-DNA+IL-12 (group 3). On wk 54, macaques were taken off drugs (OD). Macaque subgroups B, D, E, G, and H were put again on ART and immunized with SIV-DNA (D and G) or SIV-DNA+IL-15 (B, E, and H) on wk 78, 82, and 86. On wk 90, the macaques were taken off drugs. The time points at which PBMCs from the different macaques were analyzed are highlighted at the bottom of the figure. OD: Off drugs. A-H represents macaques’ subgroups. (n): number of macaque per sub-group.

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When coadministered with IL-12 DNA, SIV-DNA vaccine enhanced SIVgag-specific IFN-γ production when measured by the ELISpot assay (53). In the present study, we evaluated the impact of multiple immunization regimen in the presence or absence of IL-12 on the functional activity of SIV-specific T cells. Using the ELISPOT assay, IFN-γ production in response to SIVgag, env, pol, vif, and nef peptide stimulation was monitored for total PBMCs obtained from the three macaque groups. This analysis was performed on macaque PBMCs preimmunization (Fig. 2,A) 2 wk following the first injection round (Fig. 2,B) and 2 wk following the final injection (Fig. 2,C). We monitored the magnitude, persistence, and breadth of immune responses following vaccination. Fig. 2,A shows that the magnitude of immune responses in preimmunized animals was comparable among the three groups. However, following immunization, groups 2 and 3 showed a significant increase in the number of IFN-γ producing T cells (Fig. 2, B and C). This increase was observed 2 wk following the first (Fig. 2,B) (p = 0.04 (DNA/mock), p = 0.02 (IL-12/mock)) as well as the fourth (Fig. 2,C) (p = 0.007 (DNA/mock), p = 0.01 (IL-12/mock)) round of immunization. Further analysis after the fourth immunization clearly demonstrated a significant increase in the frequency of cytokine producing cells for groups 2 (p = 0.05) and 3 (p = 0.04) following the fourth immunization compared with a single immunization, indicating that multiple exposures to the vaccine were required to generate optimal and enhanced response. Average IFN-γ SFC activity 2 wk following the fourth immunization for group 2 (3671 ± 2346, p = 0.05) or 3 (7460 ± 8214, p = 0.04) was increased significantly relative to the response observed after the first immunization (gp2 = 1409 ± 1393; gp3 = 742 ± 2072). For group 2, three out of seven macaques had 6-fold higher total responses after four immunizations as compared with a single immunization (Fig. 2,C). Other macaques had either slightly higher (0.2-fold) or lower (up to 2-fold) SIV-specific responses. However, group 3 responses were more persistent in that none of the macaques showed any decrease in their vaccine-specific responses after four immunizations. In fact, three macaques showed 7–10-fold enhancement in their SIV-specific response between the first and fourth immunizations. Moreover, analysis of SIV-specific peptide responses at the fourth immunization indicated that group 3 showed a 2-fold increase in SIVgag-specific responses compared with group 2 (Fig. 2 C). Five macaques within group 3 had gag responses higher than 2000 SFC compared with only 2 macaques among group 2. In fact, one of the macaques within group 3 (No. 3486) showed a gag response of 10,405 SFC and a pol response of 13,865 SFC. The increased frequencies of gag-specific responses in group 3 when compared with group 2 after the fourth immunization were not reproduced for other SIV Ags. Repeated vaccination, in contrast, enhanced the breadth of the SIV-specific response as well. Although responses to gag were dominant either at the first or the fourth immunization in macaque groups 2 and 3, envelope responses were also significantly boosted in both groups following the fourth immunization (group 2: 1423 ± 1689; group 3: 1381 ± 1633; p = 0.02) compared with the first (group 2: 196 ± 197; group3: 258 ± 267). In contrast to gag-specific responses, these responses were comparable between groups 2 and 3 at that time point. In both groups, responses to structural proteins were dominant compared with other non-structural proteins. Of note, although all three Mamu A01 macaques (No. 3467, 3477, and 3495) had high responses as determined by the ELISpot assay, the two macaques showing the highest gag responses at wk 43 (No. 3484 and 3486) were not Mamu A01 indicating that allele specificity did not play a major role in determining the level of responses induced by IL-12.

FIGURE 2.

Immunization with SIV-DNA+IL-12 enhanced SIV-specific CD8 T cell responses. PBMCs were isolated from macaques preimmunization and 2 wk post the first and the final DNA immunizations and setup in a standard ELISpot assay measuring IFNγ production. Cells were stimulated with SIV gag, env, pol, vif, and nef consensus peptides and the number of SFC/1 million PBMCs is plotted. Responses detected for macaques preimmunization (A) and 2 wk following the first round of DNA immunization (B). Note the significant increase in IFNγ production with SIV-DNA ± IL-12 vaccination (p = 0.04 (DNA/mock), p = 0.02 (IL-12/mock)). C, Responses detected following the fourth round of DNA immunization. Note the significant increase in IFNγ production with SIV-DNA ± IL-12 vaccination compared with responses to mock DNA (p = 0.007 (DNA/mock), p = 0.01 (IL-12/mock)) or to the first immunization (p = 0.05 (DNA), p = 0.04 (DNA+IL-12)). Env responses increased significantly at the 4th immunization compared with levels detected at the 1st round of immunization ((p = 0.02). D, SIV-specific responses on the last day of ART treatment (13 wk post the final immunization). Note the persistence of SIV responses at this time point following SIV-DNA+IL-12 but not SIV-DNA immunization (p = 0.05). E, Responses detected to SIVgag stimulation following the depletion of CD8 T cell using Dynal magnetic beads. Note the significant drop in SIVgag-specific responses for group 3 macaques following CD8 depletion (p = 0.02). The difference in total SIVgag-specific responses was significant between groups 2 and 3 before (p = 0.05), but not after (p = 0.12) CD8 depletion. ∗, p value comparing groups SIV-specific responses following the first and the last round of immunizations.

FIGURE 2.

Immunization with SIV-DNA+IL-12 enhanced SIV-specific CD8 T cell responses. PBMCs were isolated from macaques preimmunization and 2 wk post the first and the final DNA immunizations and setup in a standard ELISpot assay measuring IFNγ production. Cells were stimulated with SIV gag, env, pol, vif, and nef consensus peptides and the number of SFC/1 million PBMCs is plotted. Responses detected for macaques preimmunization (A) and 2 wk following the first round of DNA immunization (B). Note the significant increase in IFNγ production with SIV-DNA ± IL-12 vaccination (p = 0.04 (DNA/mock), p = 0.02 (IL-12/mock)). C, Responses detected following the fourth round of DNA immunization. Note the significant increase in IFNγ production with SIV-DNA ± IL-12 vaccination compared with responses to mock DNA (p = 0.007 (DNA/mock), p = 0.01 (IL-12/mock)) or to the first immunization (p = 0.05 (DNA), p = 0.04 (DNA+IL-12)). Env responses increased significantly at the 4th immunization compared with levels detected at the 1st round of immunization ((p = 0.02). D, SIV-specific responses on the last day of ART treatment (13 wk post the final immunization). Note the persistence of SIV responses at this time point following SIV-DNA+IL-12 but not SIV-DNA immunization (p = 0.05). E, Responses detected to SIVgag stimulation following the depletion of CD8 T cell using Dynal magnetic beads. Note the significant drop in SIVgag-specific responses for group 3 macaques following CD8 depletion (p = 0.02). The difference in total SIVgag-specific responses was significant between groups 2 and 3 before (p = 0.05), but not after (p = 0.12) CD8 depletion. ∗, p value comparing groups SIV-specific responses following the first and the last round of immunizations.

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We then examined the persistence of the vaccination-induced immune responses 13 wk following the last immunization (immediately before removal of the macaques off drugs). PBMCs were isolated from the 3 macaque groups on the last day of ART therapy and examined for SIV-specific responses. SIVgag, env, and pol peptides were used to stimulate T cells. As shown in Fig. 2 D, the total level of IFN-γ produced in response to the three peptide pools was higher for group 3 than for group 2. Of note, gag-specific responses in the IL-12-immunized group were significantly higher (1189 ± 905 SFC p = 0.05) than that of group 2 (440 ± 202 SFC). Altogether, these results indicated that, in contrast to vaccination with SIV-DNA alone, coadministration of IL-12 to SIV-DNA vaccine resulted in the significant enhancement of immune responses to SIVgag structural protein and, more importantly, induced SIVgag-specific T cells to persist for at least 13 wk postimmunization.

We next determined the level of immune responses induced by CD4 and CD8 T cells to the total PBMC ELISpot responses detected in the three groups upon immunization. This was investigated by depleting CD8 T cells from total PBMCs using magnetic beads. The remaining cells were then stimulated with SIVgag peptides. As illustrated in Fig. 2 E, total gag responses before depletion were statistically higher in group 3 than in group 2 (p = 0.05). However, upon CD8 T cell depletion, responses detected for SIVgag stimulation in group 3 were comparable to those of group 2 (p = 0.12), indicating that the major responses reported in group 3 were mostly contributed by CD8 T cells.

To determine the impact of SIV-DNA immunization in the presence of IL-12 on the functional activity of SIV-specific long-term memory T cells, total PBMCs from the three macaque groups were stimulated with either SIV gag or env peptide pools for 6 h and the production of Th1 cytokines, such as IFN-γ, IL-2, and TNF-α, was monitored at all time points using intracellular staining (ICS). Different CD4 and CD8 memory subsets were defined by polychromatic flow cytometry as previously described (54, 55). Naive T cells are defined as CD95CD28+/Int.CCR7+, central memory (TCM) as CD95+CD28+CCR7+, and TEM cells (56) as CD95+/Int. CD28CCR7. Using this phenotypic characterization, we investigated the functional profile of CD4 and CD8 TEM and TCM memory cells at wk 18, 56, 62, 88, and 92. Of note, the relative frequencies of total TCM and TEM were not affected in the different immunization groups throughout the immunization protocol. Importantly, the inclusion of IL-12 in the vaccine resulted in the exclusive triggering of CD8 TEM responses at wk 56 and 62 compared with responses before immunization (wk 18). In fact, we were not able to detect SIV-specific CD4 or CD8 TCM responses along with the enhanced CD8 TEM responses detected following IL-12 administration. Fig. 3,A illustrates the cytokine production from two representative macaques. IFN-γ production of SIV-specific CD8 TEM cells was significantly enhanced in group 3 macaque compared with those of group 2 macaque (Fig. 3,A). This increase in cytokine production was observed 2 wk following removal of the macaques from ART (wk 56) (Fig. 3,B). At wk 56, frequencies of cytokine producing CD8 TEM cells (accumulation of cells producing IFN-γ only, IFN-γ+TNF-α, and IFN-γ+IL-2), following gag and env peptide pools stimulation, was significantly higher for group 3 macaques compared with those detected for group 1 (p = 0.04) or group 2 (p = 0.006) macaques (Fig. 3 B). The highest response at this time point was noticed for gag peptide pool 2, where we could evidence 2–5-fold increase in total cytokine production for group 3 compared with group 2. In fact, 4 of the animals in the IL-12-immunized group had relatively high responses for gag pool 2 stimulations. Two animals were Mamu A01 and the other two expressed other alleles. Moreover, one Mamu A01 did not show any response to this peptide pool. When we excluded the Mamu A01 aminals, the statistical difference was still significant when comparing these responses to those of group 2 (p = 0.02). Although total CD8 TEM responses seem to decrease when examined 2 mo following cessation of ART (wk 62), they remained statistically higher than the response observed for group 2 macaques at the same time point (p = 0.007). When macaques in subgroups D (primed with SIV-DNA) and G (primed with SIV-DNA+IL-12) were further boosted with SIV-DNA on wk 78, total cytokine production of SIV-specific CD8 TEM cells in response to SIV peptide stimulation was again up-regulated on wk 88 and 92 in subgroup G macaques but not in macaques of subgroup D. This increase, however, did not reach statistical significance at any of these time points (p = 0.95, p = 0.74, respectively) because at these time points, only three macaques per group were compared (the rest of the macaques were boosted with SIV-DNA+IL-15 or used as control for other substudy). These results indicated that immunization in the presence of IL-12 biased the immune response toward CD8 TEM-specific responses and that these responses were further enhanced by SIV-DNA immunization.

FIGURE 3.

Immunization with SIV-DNA+IL-12 enhanced SIV-specific CD8 responses by exclusively increasing CD8 TEM responses. A, Data of representative macaques from macaque groups 2 and 3 showing high CD8 TEM response to SIVgag peptide stimulation following SIV-DNA+IL-12 but not SIV-DNA immunization. (a) CD8 TEM response to SIVgag peptide stimulation before immunization (wk 18), (b) CD8 TEM response to SIVgag peptide stimulation after immunization (wk 56). B, Average of total cytokine production of SIV gag and env specific CD8 TEM cells from the three macaque groups before (wk 18) and at different time points after immunization (wk 56, 62, 88, 92). Each dot represents total cytokine (IFNγ, TNFα, and IL-2) production of PBMCs from one macaque (the results for the 3 cytokines were compounded). Note that SIV-specific CD8 TEM responses were significantly higher for group 3 than group 1 (p = 0.04) or 2 (p = 0.006) macaques at wk 56; this difference sustained until wk 62 (p = 0.007). PBMCs analyzed after boosting macaques with SIV-DNA (on wk 88 and 92) indicated an increase in response in group 3 only, although the increase is not statistically significant due to low numbers of macaques. Data were analyzed using DIVA software. G: Gag peptide pools. E: envelope peptide pools. The numbers 1, 2, 3, above the Gag and Env represent consecutive peptide pools. (–) represent average of total cytokine production in the specified pool. ∗, p values comparing total cytokine production between SIV-DNA and SIV-DNA+IL-12 macaque groups.

FIGURE 3.

Immunization with SIV-DNA+IL-12 enhanced SIV-specific CD8 responses by exclusively increasing CD8 TEM responses. A, Data of representative macaques from macaque groups 2 and 3 showing high CD8 TEM response to SIVgag peptide stimulation following SIV-DNA+IL-12 but not SIV-DNA immunization. (a) CD8 TEM response to SIVgag peptide stimulation before immunization (wk 18), (b) CD8 TEM response to SIVgag peptide stimulation after immunization (wk 56). B, Average of total cytokine production of SIV gag and env specific CD8 TEM cells from the three macaque groups before (wk 18) and at different time points after immunization (wk 56, 62, 88, 92). Each dot represents total cytokine (IFNγ, TNFα, and IL-2) production of PBMCs from one macaque (the results for the 3 cytokines were compounded). Note that SIV-specific CD8 TEM responses were significantly higher for group 3 than group 1 (p = 0.04) or 2 (p = 0.006) macaques at wk 56; this difference sustained until wk 62 (p = 0.007). PBMCs analyzed after boosting macaques with SIV-DNA (on wk 88 and 92) indicated an increase in response in group 3 only, although the increase is not statistically significant due to low numbers of macaques. Data were analyzed using DIVA software. G: Gag peptide pools. E: envelope peptide pools. The numbers 1, 2, 3, above the Gag and Env represent consecutive peptide pools. (–) represent average of total cytokine production in the specified pool. ∗, p values comparing total cytokine production between SIV-DNA and SIV-DNA+IL-12 macaque groups.

Close modal

As reported above, addition of IL-12 to the vaccine led to the induction of immune responses of greater magnitude in CD8 TEM cells. The breadth of the CD8 TEM responses as measured by the number of SIV peptide pools eliciting responses was also increased by the addition of IL-12. Results illustrated in Fig. 4,A clearly show that the presence of IL-12 led to a 3-fold increase in the number of peptide pools inducing CD8 TEM responses compared with immunization with DNA alone (p = 0.04). In addition, IL-12 enhanced the ability of IFN-γ producing CD8 TEM cells to further produce TNF (Fig. 4, B and C). Fig. 4,B illustrates the increase in frequencies of IFN-γ+ TNF+ SIV-specific CD8 TEM cells following immunization. Cytokine production of CD8 TEM cells stimulated with SIVgag peptide pool 2 on wk 56 is shown for representative macaques from groups 2 (Fig. 4,Ba) and 3 (Fig. 4,Bb). The right panels are gated on the IFN-γ+ CD8 TEM populations shown on the left panels and hence depict frequencies of IFN-γ+TNF+ in the presence (Fig. 4,Bb) or absence (Fig. 4,Ba) of IL-12. These results clearly demonstrate an increase in the frequency of IFN-γ+TNF+ T cells in group 3 macaque (75%) as compared with group 2 macaque (42%). This increase in dual-functional SIV-specific CD8 TEM cells following immunization with SIV-DNA+IL-12 was primarily observed at wk 56. The percentages of IFN-γ+TNF+ cells significantly increased from 4.5 to 41.1% in group 3 macaques (p = 0.001) compared with a 2.5% decrease in percentage of dual-functional CD8 TEM cells in group 2 macaques (Fig. 4 C). The average level of IFN-γ+TNF+ of group 3 macaques was also significantly higher than that of group 1 macaques (p = 0.01) at the same time point (wk 56).

FIGURE 4.

Characteristics of the IL-12-induced SIV-specific CD8 TEM responses. A, Immunization with SIV-DNA+IL-12 significantly increased the breadth of CD8 TEM response compared with immunization with SIV-DNA alone. Bars represent the average of responding peptide pools for each macaque in a group. The breadth of the CD8 TEM response, as measured by the number of SIV peptide pools eliciting CD8 TEM responses, was increased by the addition of IL-12 (p = 0.04). B, IL-12 immunization enhanced the ability of IFNγ producing CD8 TEM cells to further produce TNF-α. Ba, Data of representative macaque immunized with SIV-DNA alone. Bb, Data of representative macaque immunized with SIV-DNA+IL-12. Right panels are gated on IFNγ producing cells shown on left panels. Note the increase in the frequency of IFNγ+TNF+ CD8 TEM cells in group 3 macaques compared with group 2 macaques. C, Average of IFNγ+TNF+ producing CD8 TEM cells following immunization. Note the significant increase in percentages of IFNγ+TNF+ cells in group 3 macaques compared with group 2 (p = 0.001) or group 1 (p = 0.01) macaques. ∗, p value comparing percentage of dual-functional CD8 TEM cells in group 3 between wk 18 and 56. ∗∗, p value comparing dual-functional CD8 TEM cells for groups 1 and 3 at wk 56.

FIGURE 4.

Characteristics of the IL-12-induced SIV-specific CD8 TEM responses. A, Immunization with SIV-DNA+IL-12 significantly increased the breadth of CD8 TEM response compared with immunization with SIV-DNA alone. Bars represent the average of responding peptide pools for each macaque in a group. The breadth of the CD8 TEM response, as measured by the number of SIV peptide pools eliciting CD8 TEM responses, was increased by the addition of IL-12 (p = 0.04). B, IL-12 immunization enhanced the ability of IFNγ producing CD8 TEM cells to further produce TNF-α. Ba, Data of representative macaque immunized with SIV-DNA alone. Bb, Data of representative macaque immunized with SIV-DNA+IL-12. Right panels are gated on IFNγ producing cells shown on left panels. Note the increase in the frequency of IFNγ+TNF+ CD8 TEM cells in group 3 macaques compared with group 2 macaques. C, Average of IFNγ+TNF+ producing CD8 TEM cells following immunization. Note the significant increase in percentages of IFNγ+TNF+ cells in group 3 macaques compared with group 2 (p = 0.001) or group 1 (p = 0.01) macaques. ∗, p value comparing percentage of dual-functional CD8 TEM cells in group 3 between wk 18 and 56. ∗∗, p value comparing dual-functional CD8 TEM cells for groups 1 and 3 at wk 56.

Close modal

We next hypothesized that boosting with IL-15 would help maintain responses induced during priming as had been previously suggested (50, 57, 58, 59, 60). To test this hypothesis, macaques in subgroups B (primed with mock), E (primed with SIV-DNA), and H (primed with SIV-DNA+IL-12) were boosted with SIV-DNA+IL-15 while subgroups D (primed with SIV-DNA) and G (primed with SIV-DNA+IL-12) were only boosted with SIV-DNA in the absence of IL-15. We examined the levels of IFN-γ and TNF-α cytokine production in response to gag and env peptide pools stimulation for CD4 and CD8 T cell memory subsets. These parameters were assessed 2 wk following boosting with IL-15 (wk 88) and 2 wk following removal of macaques from therapy after the IL-15 boost (wk 92). Four out of six macaques boosted with SIV-DNA+IL-15 experienced a significant enhancement in CD4 TEM and CD8 TEM total cytokine production, mostly in response to gag peptide pools, compared with the responses detected before boosting (data not shown). These results confirmed the recently reported increase in CD4 TEM responses upon immunization in the presence of IL-15 (61). Of note, boosting with SIV-DNA+IL-15 mainly enhanced previously existing responses to specific sets of peptides but failed to induce de novo responses. Moreover, IL-15 significantly triggered the dual-functionality of those already existing mono-functional memory T cell. Fig. 5 illustrates the level of IFN-γ+TNF+ SIV-specific CD4 and CD8 TEM memory subsets of subgroups B, E, and H macaques at wk 88 and 92. The level of IFN-γ+TNF+ SIV-specific CD4 TEM cells was significantly increased in the different macaque subgroups (B, E, and H) following the SIV-DNA+IL-15 boost when detected at wk 88 (p = 0.04) and 92 (p = 0.008) (Fig. 5,A). This increase of dual-functionality was also noticed for CD8 TEM cells following SIV-DNA+IL-15 boost at wk 88 (p = 0.001) and 92 (p = 0.001) (Fig. 5 B). Boosting macaques with SIV-DNA alone, however, did not significantly enhance the level of dual-functional cells (Data not shown). These results indicated that boosting in the presence of IL-15 triggered the dual-functionality of SIV-specific memory T cells.

FIGURE 5.

Enhancement of CD4 and CD8 TEM dual-functional cells upon boosting with SIV-DNA+IL-15. The frequency of dual-functional CD4 and CD8 TEM cells in response to SIV gag and env peptide pools stimulation is shown for the different macaque groups 2 wk following boosting with SIV-DNA+IL-15 (wk 88) and 2 wk following removal of macaques off ART after the IL-15 boost (wk 92). A, The relative proportion of IFNγ+TNF+ CD4 TEM cells within the total population of IFNγ producing CD4 TEM cells is shown before (wk 62) and after (wk 88 and 92) boosting with SIV-DNA+IL-15. Note the significant increase in IFNγ+TNF+ CD4 TEM cells at wk 88 (p = 0.004) and wk 92 (p = 0.008) following SIV-DNA+IL-15 immunization as compared with before boost. B, The relative proportion of IFNγ+TNF+ CD8 TEM cells within the total population of IFNγ producing CD8 TEM cells is shown before and after boosting with SIV-DNA+IL-15. Note the significant increase in IFNγ+TNF+ CD8 TEM cells at wk 88 (p = 0.001) and wk 92 (p = 0.001) following SIV-DNA+IL-15 immunization as compared with before boost. Each symbol represents the relative proportion of IFNγ+TNF+ cells within the total population of IFNγ producing TEM cells for one macaque PBMCs following gag or env peptide pool stimulation. Most responses were to gag peptide pools. p values: comparing responses before and after the IL-15 boost.

FIGURE 5.

Enhancement of CD4 and CD8 TEM dual-functional cells upon boosting with SIV-DNA+IL-15. The frequency of dual-functional CD4 and CD8 TEM cells in response to SIV gag and env peptide pools stimulation is shown for the different macaque groups 2 wk following boosting with SIV-DNA+IL-15 (wk 88) and 2 wk following removal of macaques off ART after the IL-15 boost (wk 92). A, The relative proportion of IFNγ+TNF+ CD4 TEM cells within the total population of IFNγ producing CD4 TEM cells is shown before (wk 62) and after (wk 88 and 92) boosting with SIV-DNA+IL-15. Note the significant increase in IFNγ+TNF+ CD4 TEM cells at wk 88 (p = 0.004) and wk 92 (p = 0.008) following SIV-DNA+IL-15 immunization as compared with before boost. B, The relative proportion of IFNγ+TNF+ CD8 TEM cells within the total population of IFNγ producing CD8 TEM cells is shown before and after boosting with SIV-DNA+IL-15. Note the significant increase in IFNγ+TNF+ CD8 TEM cells at wk 88 (p = 0.001) and wk 92 (p = 0.001) following SIV-DNA+IL-15 immunization as compared with before boost. Each symbol represents the relative proportion of IFNγ+TNF+ cells within the total population of IFNγ producing TEM cells for one macaque PBMCs following gag or env peptide pool stimulation. Most responses were to gag peptide pools. p values: comparing responses before and after the IL-15 boost.

Close modal

Up-regulation of PD-1 expression on CD8 T cells during chronic infections was recently associated with their functional exhaustion (62, 63, 64). We hypothesized that therapeutic immunization, through the reduction in viral load, and hence chronic TCR triggering, would decrease PD-1 expression levels on SIV-specific memory T cells, thereby leading to their activation. We examined PD-1 expression levels on SIV-specific memory T cell subsets by combining ICS technique and cell surface staining of PD-1. As illustrated in Fig. 6,A, PD-1 expression levels on total CD4 and CD8 TEM cells were significantly lower in group 3 macaques compared with group 1 macaques 2 wk following the cessation of ART (p = 0.001). In contrast, there was no significant difference in PD-1 expression levels on total CD4 and CD8 TEM cells between macaques of groups 2 and 1 (data not shown). The histograms of Fig. 6,B show PD-1 expression levels on mono-functional (as characterized by the production of IFN-γ alone after SIV peptide stimulation), and dual-functional (cells that produce IFN-γ and TNF-α after SIV peptide stimulation) memory T cells. Moreover, Fig. 6 C, D, and E demonstrate that PD-1 expression level was significantly lower on dual-functional cells compared with mono-functional cells (p < 0.0001). This was observed for CD4 and CD8 TEM cells at all time points following SIV-DNA (p = 0.0001, p < 0.0001, respectively), SIV-DNA+IL-12 (p < 0.0001 (CD8 TEM)), or SIV-DNA+IL15 (p < 0.0001 (CD8 TEM)), p = 0.03 (CD4 TEM)) immunizations. Altogether, these results demonstrated that the down-regulation of PD-1 on functional SIV-specific memory T cells was indeed correlated with the increase in their dual-functionality observed upon immunization.

FIGURE 6.

PD-1 expression on dual-functional memory T cells is significantly lower than its expression on mono-functional cells. PD-1 expression levels were determined on SIV-specific memory T-cell subsets using direct staining simultaneously with the ICS technique followed by flow-cytometry. A, PD-1 expression levels on total CD4 and CD8 TEM cells of group 1 and 3 macaques at wk 56. Note that PD-1 expression levels on total CD4 and CD8 TEM cells were significantly lower in group 3 macaques compared with group 1 macaques 2 wk following the cessation of ART (p = 0.001). B, Representative histograms indicating the PD-1 expression levels on mono- (IFNγ+) and dual-functional (IFNγ++TNF+) memory T cells. C, PD-1 expression level on functional CD8 TEM cells from SIV-DNA+IL-12 immunized macaques. D and E, PD-1 expression on functional CD4 and CD8 TEM subsets from SIV-DNA and SIV-DNA+IL-15-immunized macaques, respectively. The PD-1 expression levels were significantly lower on dual-functional cells than on mono-functional cells. This was observed for C, CD8 TEM cells at all time points following SIV-DNA+IL-12 immunization (p < 0.0001); D, CD4 and CD8 TEM cells at all time points following SIV-DNA immunization (p = 0.0001, p < 0.0001, respectively); and for E, CD4 and CD8 TEM cells at all time points following SIV-DNA+IL-15 immunization (p = 0.03, p < 0.0001, respectively).

FIGURE 6.

PD-1 expression on dual-functional memory T cells is significantly lower than its expression on mono-functional cells. PD-1 expression levels were determined on SIV-specific memory T-cell subsets using direct staining simultaneously with the ICS technique followed by flow-cytometry. A, PD-1 expression levels on total CD4 and CD8 TEM cells of group 1 and 3 macaques at wk 56. Note that PD-1 expression levels on total CD4 and CD8 TEM cells were significantly lower in group 3 macaques compared with group 1 macaques 2 wk following the cessation of ART (p = 0.001). B, Representative histograms indicating the PD-1 expression levels on mono- (IFNγ+) and dual-functional (IFNγ++TNF+) memory T cells. C, PD-1 expression level on functional CD8 TEM cells from SIV-DNA+IL-12 immunized macaques. D and E, PD-1 expression on functional CD4 and CD8 TEM subsets from SIV-DNA and SIV-DNA+IL-15-immunized macaques, respectively. The PD-1 expression levels were significantly lower on dual-functional cells than on mono-functional cells. This was observed for C, CD8 TEM cells at all time points following SIV-DNA+IL-12 immunization (p < 0.0001); D, CD4 and CD8 TEM cells at all time points following SIV-DNA immunization (p = 0.0001, p < 0.0001, respectively); and for E, CD4 and CD8 TEM cells at all time points following SIV-DNA+IL-15 immunization (p = 0.03, p < 0.0001, respectively).

Close modal

The design of a new generation of vaccines has focused on promoting the induction and persistence of memory CD8 T cells and on increasing the frequency of dual-functional T cells (65, 66, 67, 68). In this report, we have studied the impact of IL-12 and IL-15, when combined to therapeutic DNA vaccines, on enhancing memory T cell survival and function. We have showed that therapeutic vaccination with SIV-DNA+IL-12 or SIV-DNA+IL-15 enhanced immune responses in chronically SIV-infected macaques whose viral replication was suppressed with antiretroviral drugs. Although recent reports have examined the use of IL-12 as an adjuvant during prophylactic DNA vaccination, this is the first report, to our knowledge, that examines therapeutic vaccination of SIV chronically infected macaques using DNA vaccination in the presence of the cytokine adjuvants IL-12 and IL-15.

IL-12 induces Th1-biased memory responses that usually lead to a heightened cytolytic effector phenotype (69). In this report, we have shown that there was a significant increase in the magnitude and breadth (induction of responses to env in addition to gag peptides) of total immune responses at the fourth immunization compared with the first immunization, indicating that not only the magnitude, but also the quality of the response was improved with repeated immunizations. In addition, immunization with SIV-DNA+IL-12 induced a significantly higher SIVgag immune responses compared with immunization with SIV-DNA alone. The high cellular immune responses observed in our study are likely due to multiple reasons. It is possible that the efficacy of the immune response translates anamnestic responses. In addition, our primates were treated with antiretroviral drugs within 14 wk of infection. This allowed us to immunize animals that did not have severe immune deficiency. In fact, we have previously shown that early treatment leads to restoration and preservation of immune responses in HIV specific CD4 T cells (70). In addition, the use of a DNA vaccine allowed for continued immunization without immune responses to vector suppressing the SIV-specific response. Tracking these responses 13 wk following the fourth, and last, immunization (just before the removal of animals from ART treatment) indicated that priming with SIV-DNA in the presence of IL-12 enhanced the persistence of SIV-specific CD8 memory responses compared with priming with SIV-DNA alone. In fact, the persistence of SIV-specific memory T cells has been recently shown to be the best predictor of survival outcome in SIV-vaccinated and -challenged macaques (71).

A significant enhancement in the frequency of cytokine producing SIV-specific CD8 TEM cells in group 3 compared with group 2 macaques was detected 2 wk following the cessation of ART. Moreover, when macaques in subgroups D (primed with SIV-DNA) and G (primed with SIV-DNA+IL-12) were further boosted with SIV-DNA on wk 78, total cytokine production of specific CD8 TEM cells was again up-regulated on wk 88 and 92 in subgroup G macaques compared with responses of subgroup D at the same time points (Fig. 3,B). These results suggested that memory cells that have received DNA+IL-12 show better persistence of the SIV-specific response than memory cells which have not received IL-12. Furthermore, the breadth, as determined by the number of responding peptide pools, and the quality of the response, were significantly higher for CD8 TEM cells of macaques primed in the presence of IL-12 compared with those of macaques primed with SIV-DNA alone. IL-12 drives CD8 TEM cells to a dual-functional state by enhancing TNF production by IFN-γ producing cells (Fig. 4). This concurs with a recent report by Chong et al. (21) who showed that prophylactic immunization of macaques with SIVgag DNA in the presence of IL-12 enhanced the level of dual-functional T cells (in this case IFN-γ+ IL-2+). In contrast, we were not able to detect CD4 or CD8 TCM responses along with the enhanced CD8 TEM responses detected following IL-12 administration. This may indicate that IL-12 could either: 1) exclusively stimulate CD8 TEM cells either directly or indirectly without affecting CD8 TCMs, or 2) trigger CD8 TCM cells to quickly differentiate to TEM.

Blachère et al. (72) recently showed that exogenous IL-12 acts directly on memory CD8 T cells and overcomes the lack of CD4 T cell help. Snyder et al. (73) have also reported that exogenous rIL-12 increases CD40L expression on primary human PBMC cultures resulting in their enhanced activation. It has also been suggested that when used as an adjuvant, IL-12 enhances the OX-40 (CD134) receptor (OX-40R)/OX-40 ligand (OX-40L) interaction, which has been shown to be critical for the generation of a functional antitumor immune response (74). OX-40R (expressed on T cells), when binding to OX-40L (expressed on DCs), was shown to exert significant costimulatory effects on T cell proliferation, survival, and cytokine production (75). These findings may suggest that the enhancement of immune responses observed when priming in the presence of IL-12 could result from the increase in these receptor-ligand interactions and thus improved activation of T cells by DC. In addition, Li et al. (76) have recently reported that brief IL-12 exposure of naive TCR transgenic CD8 cells during Ag stimulation leads to transient phosphorylation of STAT4, which then regulate the expression of transcriptional factors that promote the survival of these CD8 cells. This may explain the persistence of the induced specific-memory CD8 TEM cells observed following immunization in the presence of IL-12. In contrast, although macaques immunized with DNA+IL-12 acquired an increased capacity to control viral rebound at the first 21 days following cessation of ART (data not shown), these results did not reach statistical significance. Studies with larger animal groups are needed to determine impact of IL-12 on control of viral rebound.

Vaccination in the presence of IL-15 has been recently reported to boost immune responses especially for CD4 TEM cells (61). Based on these findings, we hypothesized that boosting with SIV-DNA in the presence of IL-15 would enhance the magnitude of Ag-specific memory T cells induced during primary immunization. Accordingly, we have shown that boosting with IL-15 not only amplified the SIV-specific responses, but also rescued dual-functional responses (Fig. 5). Moreover, IL-15 was extensively shown to enhance the survival of memory T cells (77, 78). It is unfortunate that we were not able to follow the maintenance of memory T cells as well as the survival rate of the macaques following IL-15 immunization due to technical problems beyond our control.

The up-regulation of PD-1 expression on specific CD8 T cells was recently correlated with their exhaustion (62, 63, 64). We examined the effect of therapeutic vaccination on PD-1 expression levels on specific memory CD4 and CD8 T cells. Initially, when investigating PD-1 expression levels on total CD4 and CD8 TEM cells on wk 57 (2 wk following the cessation of ART), we observed that PD-1 expression was significantly lower in macaques immunized with SIV-DNA+IL-12 compared with macaques receiving mock DNA (Fig. 6,A). In contrast, there was no significant difference in PD-1 expression levels on CD4 and CD8 TEM cells in the SIV-DNA immunized group compared with the mock-immunized group (data not shown). In fact, when examining PD-1 level on total CD8 TEM cells among the different macaque groups on wk 61, we noted that PD-1 levels were lower on cells from group 3 macaques compared with the other groups, although this decrease was not statistically significant (Data not shown). The median viral load of the mock group at this time point was 0.4 log higher than that of the SIV-DNA+IL-12 immunized group. Moreover, there was a significant correlation between PD-1 expression levels and the ability of SIV-specific memory T cells to produce cytokines. PD-1 expression on dual-functional memory T cells was significantly lower than that on mono-functional cells (Fig. 6, B–E). This observation is supported by results of Wherry’s et al. (79) indicating that cells producing IFN-γ+TNF are less exhausted than cells producing IFN-γ alone. Our results suggest that immunization with SIV-DNA in the presence of IL-12 may rescue DC signals resulting in stimulating a broad dual-functional immune response that will lead to lower PD-1 levels on SIV-specific T cell. Alternatively, IL-12 may act directly on down-regulating PD-1 level of expression on SIV-specific T cells resulting in their activation and dual-functionality. However, confirmation of this hypothesis requires further investigation at the molecular level.

In summary, these data demonstrate that therapeutic vaccination of chronically SIV-infected macaques with SIV-DNA+IL-12 specifically triggered CD8 TEM immune responses. Boosting with SIV-DNA+IL-15 further enhanced existing responses. Our results indicate that the mechanisms behind this enhancement of the immune system can in part be explained by the ability of these two cytokines to down-regulate PD-1 expression levels on SIV-specific memory T cells and, thereby, induce their activation. The inclusion of these cytokines with therapeutic DNA vaccines appears to represent a significant addition to promote their ability to induce better immune responses.

We thank Naglaa Shoukry for critically reviewing the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants awarded to R.H. from the Canadian Institutes of Health Research and to R.-P.S. from the U.S. National Institutes of Health, the Canadian Institutes of Health Research, the Canadian Network for Vaccine and Immunotherapeutics, Genome Québec, Genome Canada, and the Fonds de la recherche en Santé du Québec (FRSQ) AIDS network. R.-P.S. is the Canada Research Chair in Human Immunology.

4

Abbreviations used in this paper: ART, antiretroviral therapy; SEM, SEA+SEB; SFC, spot forming cells; TEM, effector memory T cell; TCM, central memory T cell; SLA, simian leukocyte Ag; DC, dendritic cell; PMPA, 9-[9(R)-2(phosphonomethoxy) propyl]adenine; ICS, intracellular staining; int, intermediate.

1
Powderly, W. G., A. Landay, M. M. Lederman.
1998
. Recovery of the immune system with antiretroviral therapy: the end of opportunism?.
J. Am. Med. Assoc.
280
:
72
-77.
2
Finzi, D., M. Hermankova, T. Pierson, L. M. Carruth, C. Buck, R. E. Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, et al
1997
. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.
Science
278
:
1295
-1300.
3
Wong, J. K., M. Hezareh, H. F. Gunthard, D. V. Havlir, C. C. Ignacio, C. A. Spina, D. D. Richman.
1997
. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.
Science
278
:
1291
-1295.
4
Allen, T. M., A. D. Kelleher, J. Zaunders, B. D. Walker.
2002
. STI and beyond: the prospects of boosting anti-HIV immune responses.
Trends Immunol.
23
:
456
-460.
5
Fagard, C., A. Oxenius, H. Gunthard, F. Garcia, M. Le Braz, G. Mestre, M. Battegay, H. Furrer, P. Vernazza, E. Bernasconi, et al
2003
. A prospective trial of structured treatment interruptions in human immunodeficiency virus infection.
Arch. Intern. Med.
163
:
1220
-1226.
6
Kovacs, A., M. Connors.
2004
. HIV-1 and immune control: can we change the course of HIV-1?.
Lancet
363
:
833
-834.
7
Cao, Y., L. Qin, L. Zhang, J. Safrit, D. D. Ho.
1995
. Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection.
N. Engl. J. Med.
332
:
201
-208.
8
Rosenberg, E. S., J. M. Billingsley, A. M. Caliendo, S. L. Boswell, P. E. Sax, S. A. Kalams, B. D. Walker.
1997
. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia.
Science
278
:
1447
-1450.
9
Pantaleo, G., S. Menzo, M. Vaccarezza, C. Graziosi, O. J. Cohen, J. F. Demarest, D. Montefiori, J. M. Orenstein, C. Fox, L. K. Schrager, et al
1995
. Studies in subjects with long-term nonprogressive human immunodeficiency virus infection.
N. Engl. J. Med.
332
:
209
-216.
10
Pinto, L. A., J. Sullivan, J. A. Berzofsky, M. Clerici, H. A. Kessler, A. L. Landay, G. M. Shearer.
1995
. ENV-specific cytotoxic T lymphocyte responses in HIV seronegative health care workers occupationally exposed to HIV-contaminated body fluids.
J. Clin. Invest.
96
:
867
-876.
11
Hoen, B., B. Dumon, M. Harzic, A. Venet, B. Dubeaux, C. Lascoux, Y. Bourezane, J. M. Ragnaud, A. Bicart-See, F. Raffi, et al
1999
. Highly active antiretroviral treatment initiated early in the course of symptomatic primary HIV-1 infection: results of the ANRS 053 trial.
J. Infect. Dis.
180
:
1342
-1346.
12
Corey, L., M. M. Berrey.
1999
. Antiretroviral therapy in primary HIV.
Adv. Exp. Med. Biol.
458
:
223
-227.
13
Ortiz, G. M., D. F. Nixon, A. Trkola, J. Binley, X. Jin, S. Bonhoeffer, P. J. Kuebler, S. M. Donahoe, M. A. Demoitie, W. M. Kakimoto, et al
1999
. HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy.
J. Clin. Invest.
104
:
R13
-R18.
14
Lori, F., M. G. Lewis, J. Xu, G. Varga, D. E. Zinn, Jr, C. Crabbs, W. Wagner, J. Greenhouse, P. Silvera, J. Yalley-Ogunro, et al
2000
. Control of SIV rebound through structured treatment interruptions during early infection.
Science
290
:
1591
-1593.
15
von Gegerfelt, A. S., M. Rosati, C. Alicea, A. Valentin, P. Roth, J. Bear, G. Franchini, P. S. Albert, N. Bischofberger, J. D. Boyer, et al
2007
. Long-lasting decrease in viremia in macaques chronically infected with simian immunodeficiency virus SIVmac251 after therapeutic DNA immunization.
J. Virol.
81
:
1972
-1979.
16
Luckay, A., M. K. Sidhu, R. Kjeken, S. Megati, S. Y. Chong, V. Roopchand, D. Garcia-Hand, R. Abdullah, R. Braun, D. C. Montefiori, et al
2007
. Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques.
J. Virol.
81
:
5257
-5269.
17
Laddy, D. J., D. B. Weiner.
2006
. From plasmids to protection: a review of DNA vaccines against infectious diseases.
Int. Rev. Immunol.
25
:
99
-123.
18
Calarota, S. A., D. B. Weiner.
2004
. Approaches for the design and evaluation of HIV-1 DNA vaccines.
Expert Rev. Vaccines
3
:
S135
-S149.
19
Boyer, J. D., T. M. Robinson, M. A. Kutzler, R. Parkinson, S. A. Calarota, M. K. Sidhu, K. Muthumani, M. Lewis, G. Pavlakis, B. Felber, D. Weiner.
2005
. SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques.
J. Med. Primatol.
34
:
262
-270.
20
Egan, M. A., S. Y. Chong, S. Megati, D. C. Montefiori, N. F. Rose, J. D. Boyer, M. K. Sidhu, J. Quiroz, M. Rosati, E. B. Schadeck, et al
2005
. Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus.
AIDS Res. Hum. Retroviruses
21
:
629
-643.
21
Chong, S. Y., M. A. Egan, M. Kutzler, S. Megati, A. Masood, V. Roopchard, D. Garci-Hand, D. C. Montefiori, J. Quiroz, M. Rosati, et al
2007
. Comparative ability of plasmid IL-12 and IL-15 to enhance cellular and humoral immune responses elicited by a SIVgag plasmid DNA vaccine and alter disease progression following SHIV(89.6P) challenge in rhesus macaques.
Vaccine
25
:
4967
-4982.
22
Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri.
1989
. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.
J. Exp. Med.
170
:
827
-845.
23
Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O'Garra, K. M. Murphy.
1993
. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.
Science
260
:
547
-549.
24
Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O'Garra.
1995
. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells.
J. Immunol.
154
:
5071
-5079.
25
Hunter, C. A., R. Chizzonite, J. S. Remington.
1995
. IL-1 β is required for IL-12 to induce production of IFN-γ by NK cells: a role for IL-1 β in the T cell-independent mechanism of resistance against intracellular pathogens.
J. Immunol.
155
:
4347
-4354.
26
Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani.
1993
. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells.
J. Exp. Med.
177
:
1199
-1204.
27
Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, G. Trinchieri.
1995
. Interleukin-12 is required for interferon-γ production and lethality in lipopolysaccharide-induced shock in mice.
Eur. J. Immunol.
25
:
672
-676.
28
Trinchieri, G..
1998
. Interleukin-12: a cytokine at the interface of inflammation and immunity.
Adv. Immunol.
70
:
83
-243.
29
Kim, J. J., V. Ayyavoo, M. L. Bagarazzi, M. A. Chattergoon, K. Dang, B. Wang, J. D. Boyer, D. B. Weiner.
1997
. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen.
J. Immunol.
158
:
816
-826.
30
Du, D. W., Z. S. Jia, G. Y. Li, Y. Y. Zhou.
2003
. HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection.
World J. Gastroenterol.
9
:
108
-111.
31
Triccas, J. A., L. Sun, U. Palendira, W. J. Britton.
2002
. Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis.
Immunol. Cell Biol.
80
:
346
-350.
32
Katae, M., Y. Miyahira, K. Takeda, H. Matsuda, H. Yagita, K. Okumura, T. Takeuchi, T. Kamiyama, A. Ohwada, Y. Fukuchi, T. Aoki.
2002
. Coadministration of an interleukin-12 gene and a Trypanosoma cruzi gene improves vaccine efficacy.
Infect. Immun.
70
:
4833
-4840.
33
Weiner, D..
2000
. Plasmid interleukin 12.
AIDS
14
:
1759
-1760.
34
Gherardi, M. M., J. C. Ramirez, M. Esteban.
2000
. Interleukin-12 (IL-12) enhancement of the cellular immune response against human immunodeficiency virus type 1 env antigen in a DNA prime/vaccinia virus boost vaccine regimen is time and dose dependent: suppressive effects of IL-12 boost are mediated by nitric oxide.
J. Virol.
74
:
6278
-6286.
35
Boyer, J. D., A. D. Cohen, K. E. Ugen, R. L. Edgeworth, M. Bennett, A. Shah, K. Schumann, B. Nath, A. Javadian, M. L. Bagarazzi, et al
2000
. Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.
AIDS
14
:
1515
-1522.
36
Chattergoon, M. A., V. Saulino, J. P. Shames, J. Stein, L. J. Montaner, D. B. Weiner.
2004
. Co-immunization with plasmid IL-12 generates a strong T-cell memory response in mice.
Vaccine
22
:
1744
-1750.
37
Tagaya, Y., R. N. Bamford, A. P. DeFilippis, T. A. Waldmann.
1996
. IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels.
Immunity
4
:
329
-336.
38
Schluns, K. S., L. Lefrancois.
2003
. Cytokine control of memory T-cell development and survival.
Nat. Rev. Immunol.
3
:
269
-279.
39
Waldmann, T., Y. Tagaya, R. Bamford.
1998
. Interleukin-2, interleukin-15, and their receptors.
Int. Rev. Immunol.
16
:
205
-226.
40
D'Souza, W. N., K. S. Schluns, D. Masopust, L. Lefrancois.
2002
. Essential role for IL-2 in the regulation of antiviral extralymphoid CD8 T cell responses.
J. Immunol.
168
:
5566
-5572.
41
Schluns, K. S., K. Williams, A. Ma, X. X. Zheng, L. Lefrancois.
2002
. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells.
J. Immunol.
168
:
4827
-4831.
42
Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois.
2000
. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo.
Nat. Immunol.
1
:
426
-432.
43
Lefrancois, L., D. Masopust.
2002
. T cell immunity in lymphoid and non-lymphoid tissues.
Curr. Opin. Immunol.
14
:
503
-508.
44
Sprent, J., A. D. Judge, X. Zhang.
2002
. Cytokines and memory-phenotype CD8+ cells.
Adv. Exp. Med. Biol.
512
:
147
-153.
45
Kanai, T., E. K. Thomas, Y. Yasutomi, N. L. Letvin.
1996
. IL-15 stimulates the expansion of AIDS virus-specific CTL.
J. Immunol.
157
:
3681
-3687.
46
Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack.
2000
. Control of homeostasis of CD8+ memory T cells by opposing cytokines.
Science
288
:
675
-678.
47
Judge, A. D., X. Zhang, H. Fujii, C. D. Surh, J. Sprent.
2002
. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8+ T cells.
J. Exp. Med.
196
:
935
-946.
48
Becker, T. C., E. J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, R. Ahmed.
2002
. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells.
J. Exp. Med.
195
:
1541
-1548.
49
Moroz, A., C. Eppolito, Q. Li, J. Tao, C. H. Clegg, P. A. Shrikant.
2004
. IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15, and IL-21.
J. Immunol.
173
:
900
-909.
50
Zeng, R., R. Spolski, S. E. Finkelstein, S. Oh, P. E. Kovanen, C. S. Hinrichs, C. A. Pise-Masison, M. F. Radonovich, J. N. Brady, N. P. Restifo, et al
2005
. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function.
J. Exp. Med.
201
:
139
-148.
51
Friedrich, T. C., E. J. Dodds, L. J. Yant, L. Vojnov, R. Rudersdorf, C. Cullen, D. T. Evans, R. C. Desrosiers, B. R. Mothe, J. Sidney, et al
2004
. Reversion of CTL escape-variant immunodeficiency viruses in vivo.
Nat. Med.
10
:
275
-281.
52
Knapp, L. A., E. Lehmann, L. Hennes, M. E. Eberle, D. I. Watkins.
1997
. High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.
Tissue Antigens
50
:
170
-177.
53
Schadeck, E. B., M. Sidhu, M. A. Egan, S. Y. Chong, P. Piacente, A. Masood, D. Garcia-Hand, S. Cappello, V. Roopchand, S. Megati, et al
2006
. A dose sparing effect by plasmid encoded IL-12 adjuvant on a SIVgag-plasmid DNA vaccine in rhesus macaques.
Vaccine
24
:
4677
-4687.
54
Pitcher, C. J., S. I. Hagen, J. M. Walker, R. Lum, B. L. Mitchell, V. C. Maino, M. K. Axthelm, L. J. Picker.
2002
. Development and homeostasis of T cell memory in rhesus macaque.
J. Immunol.
168
:
29
-43.
55
Vaccari, M., C. J. Trindade, D. Venzon, M. Zanetti, G. Franchini.
2005
. Vaccine-induced CD8+ central memory T cells in protection from simian AIDS.
J. Immunol.
175
:
3502
-3507.
56
Hazenberg, M. D., S. A. Otto, D. Hamann, M. T. Roos, H. Schuitemaker, R. J. de Boer, F. Miedema.
2003
. Depletion of naive CD4 T cells by CXCR4-using HIV-1 variants occurs mainly through increased T-cell death and activation.
AIDS
17
:
1419
-1424.
57
Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh.
2002
. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells.
J. Exp. Med.
195
:
1523
-1532.
58
Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma.
1998
. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation.
Immunity
9
:
669
-676.
59
Wherry, E. J., T. C. Becker, D. Boone, M. K. Kaja, A. Ma, R. Ahmed.
2002
. Homeostatic proliferation but not the generation of virus specific memory CD8 T cells is impaired in the absence of IL-15 or IL-15Rα.
Adv. Exp. Med. Biol.
512
:
165
-175.
60
Marrack, P., J. Kappler.
2004
. Control of T cell viability.
Annu. Rev. Immunol.
22
:
765
-787.
61
Picker, L. J., E. F. Reed-Inderbitzin, S. I. Hagen, J. B. Edgar, S. G. Hansen, A. Legasse, S. Planer, M. Piatak, Jr, J. D. Lifson, V. C. Maino, et al
2006
. IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates.
J. Clin. Invest.
116
:
1514
-1524.
62
Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, G. J. Freeman, R. Ahmed.
2006
. Restoring function in exhausted CD8 T cells during chronic viral infection.
Nature
439
:
682
-687.
63
Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette, M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al
2006
. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction.
Nat. Med.
12
:
1198
-1202.
64
Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy, E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al
2006
. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression.
Nature
443
:
350
-354.
65
Goonetilleke, N., S. Moore, L. Dally, N. Winstone, I. Cebere, A. Mahmoud, S. Pinheiro, G. Gillespie, D. Brown, V. Loach, et al
2006
. Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes.
J. Virol.
80
:
4717
-4728.
66
Laddy, D. J., J. Yan, N. Corbitt, D. Kobasa, G. P. Kobinger, D. B. Weiner.
2007
. Immunogenicity of novel consensus-based DNA vaccines against avian influenza.
Vaccine
25
:
2984
-2989.
67
Calarota, S. A., D. B. Weiner, F. Lori, J. Lisziewicz.
2007
. Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine.
Vaccine
25
:
3070
-3074.
68
Stemberger, C., K. M. Huster, D. H. Busch.
2006
. Defining correlates of T cell protection against infection.
Discov. Med.
6
:
148
-152.
69
Portielje, J. E., J. W. Gratama, H. H. van Ojik, G. Stoter, W. H. Kruit.
2003
. IL-12: a promising adjuvant for cancer vaccination.
Cancer Immunol. Immunother.
52
:
133
-144.
70
Younes, S. A., L. Trautmann, B. Yassine-Diab, L. H. Kalfayan, A. E. Kernaleguen, T. O. Cameron, R. Boulassel, L. J. Stern, J. P. Routy, Z. Grossman, et al
2007
. The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells.
J. Immunol.
178
:
788
-797.
71
Letvin, N. L., J. R. Mascola, Y. Sun, D. A. Gorgone, A. P. Buzby, L. Xu, Z. Y. Yang, B. Chakrabarti, S. S. Rao, J. E. Schmitz, et al
2006
. Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys.
Science
312
:
1530
-1533.
72
Blachere, N. E., H. K. Morris, D. Braun, H. Saklani, J. P. Di Santo, R. B. Darnell, M. L. Albert.
2006
. IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation.
J. Immunol.
176
:
7288
-7300.
73
Snyder, J. T., J. Shen, H. Azmi, J. Hou, D. H. Fowler, J. A. Ragheb.
2007
. Direct inhibition of CD40L expression can contribute to the clinical efficacy of daclizumab independently of its effects on cell division and Th1/Th2 cytokine production.
Blood
109
:
5399
-5406.
74
Kuriyama, H., S. Watanabe, J. Kjaergaard, H. Tamai, R. Zheng, A. D. Weinberg, H. M. Hu, P. A. Cohen, G. E. Plautz, S. Shu.
2006
. Mechanism of third signals provided by IL-12 and OX-40R ligation in eliciting therapeutic immunity following dendritic-tumor fusion vaccination.
Cell. Immunol.
243
:
30
-40.
75
Croft, M..
2003
. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?.
Nat. Rev. Immunol.
3
:
609
-620.
76
Li, Q., C. Eppolito, K. Odunsi, P. A. Shrikant.
2006
. IL-12-programmed long-term CD8+ T cell responses require STAT4.
J. Immunol.
177
:
7618
-7625.
77
Pulle, G., M. Vidric, T. H. Watts.
2006
. IL-15-dependent induction of 4–1BB promotes antigen-independent CD8 memory T cell survival.
J. Immunol.
176
:
2739
-2748.
78
Sato, N., H. J. Patel, T. A. Waldmann, Y. Tagaya.
2007
. The IL-15/IL-15Rα on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells.
Proc. Natl. Acad. Sci. USA
104
:
588
-593.
79
Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, R. Ahmed.
2003
. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment.
J. Virol.
77
:
4911
-4927.