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The Journal of Immunology, 2006, 177: 8531-8541.
Copyright © 2006 by The American Association of Immunologists, Inc.

Local and Systemic Effects of Intranodally Injected CpG-C Immunostimulatory-Oligodeoxyribonucleotides in Macaques1

Natalia Teleshova2,*, Jessica Kenney*, Gary Van Nest{dagger}, Jason Marshall{dagger}, Jeffrey D. Lifson{ddagger}, Irving Sivin*, Jason Dufour§, Rudolf Bohm§, Agegnehu Gettie and Melissa Robbiani3,*

* Center for Biomedical Research, Population Council, New York, NY 10021; {dagger} Dynavax Technologies, Berkeley, CA 94710; {ddagger} AIDS Vaccine Program, Science Applications International Corporation, National Cancer Institute, Frederick, MD 21702; § Tulane National Primate Research Center, Tulane University, Covington, LA 70433; and Aaron Diamond AIDS Research Center, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunostimulatory CpG-C oligodeoxyribonucleotides (ISS-ODNs) represent a promising strategy to enhance vaccine efficacy. We have shown that the CpG-C ISS-ODN C274 stimulates macaque blood dendritic cells (DCs) and B cells and augments SIV-specific IFN-{gamma} responses in vitro. To further explore the potential of C274 for future vaccine studies, we assessed the in vivo effects of locally administered C274 (in naive and healthy infected macaques). Costimulatory molecules were marginally increased on DCs and B cells within cells isolated from C274-injected lymph nodes (LNs). However, cells from C274-injected LNs exhibited heightened responsiveness to in vitro culture. This was particularly apparent at the level of CD80 (less so CD86) expression by CD123+ plasmacytoid DCs and was further boosted in the presence of additional C274 in vitro. Notably, cells from C274-injected LNs secreted significantly elevated levels of several cytokines and chemokines upon in vitro culture. This was more pronounced when cells were exposed to additional stimuli in vitro, producing IFN-{alpha}, IL-3, IL-6, IL-12, TNF-{alpha}, CCL2, CCL3, CCL5, and CXCL8. Following C274 administration in the absence of additional SIV Ag, endogenous IFN-{gamma} secretion was elevated in LN cells of infected animals, but SIV-specific responses were unchanged. Endogenous and SIV-specific responses decreased in blood, before the SIV-specific responses rebounded by 2 wk after C274 treatment. Elevated IFN-{alpha}, CCL2, and CCL5 were also detected in the plasma after C274 injection. Thus, locally administered C274 has local and systemic activities, supporting the potential for CpG-C ISS-ODNs to boost immune function to enhance anti-HIV vaccine immunogenicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)4 are immune system sentinels that respond to pathogens and orchestrate innate and adaptive immunity (1, 2, 3, 4). Pathogen-derived components and/or proinflammatory stimuli induce DCs to mature and migrate into lymphoid tissue or lymph nodes (LNs) where, as interdigitating DCs, they form a network in the parafollicular T cell-rich areas and orchestrate immune responses. Increasing evidence indicates how DCs and B cells also communicate with each other to encourage potent Th1 immunity (5, 6, 7, 8, 9, 10, 11, 12). Strategies that boost DC and B cell functions should improve prophylactic and therapeutic vaccines against pathogens like HIV.

SIV challenge of rhesus macaques is the preferred animal model for the evaluation of vaccine approaches for the prevention of AIDS (13). As in humans, there are two major subsets of DCs present in macaque blood and LNs, myeloid CD11c+CD123– (myeloid DCs; MDCs) and plasmacytoid CD11c–CD123+ (plasmacytoid DCs; PDCs) DCs (14, 15, 16, 17). DCs readily capture SIV and HIV (18, 19) and immunodeficiency viruses can trigger innate PDC responses (17, 20). However, interaction of these viruses with DCs is a double-edged sword. DCs play a critical role in initiating and coordinating antiviral immune responses, with mature DCs typically inducing stronger virus-specific responses (17, 21, 22, 23, 24). In contrast, these viruses seem to have developed replication strategies that subvert DC biology (19, 25, 26). DCs efficiently take up particulate material and interact with T cells to present antigenic peptides and promote cellular activation. These processes are effectively hijacked by the viruses, which are taken up by immature DCs (in which they can replicate to a limited extent), and then effectively transmitted to T cells, with the T cell activation provided by the DCs serving to amplify the infection (18, 19). Approaches to shift the balance of this two-sided process in favor of effective immune responses are thus clearly needed.

A number of observations point to derangement of DC function as a contributing factor to HIV immunopathogenesis. Reduced numbers of circulating DCs have been reported in HIV-infected people, correlating with increased virus load (27, 28, 29, 30, 31), suggesting possible redistribution of DCs from the periphery to the lymphoid tissues. However, the number of interdigitating DCs is reduced in patients with AIDS (32). The expression of costimulatory molecules CD80 and CD86 on LN DCs during HIV infection might be insufficient for induction of adequate antiviral responses (32). Macaque studies also demonstrated that DC maturation and frequency in LNs are affected during SIV infection (33, 34). Adding to this, several reports have indicated that B cell function is impaired in HIV-infected individuals (35, 36, 37, 38, 39, 40, 41, 42, 43, 44).

One attractive approach to enhance B cell and DC activities is through stimulation with immunostimulatory CpG-C oligodeoxyribonucleotides (ISS-ODNs) (45, 46, 47). ISS-ODNs trigger expression of a variety of cytokines and induce potent Th1, B cell, and cytotoxic T cell responses and are well-tolerated in vivo (48). CpG-C ISS-ODN-activated human PDCs also drive B cell activation and plasma cell differentiation (47). Previous studies showed that CpG-B ISS-ODNs boosted vaccine efficacy in nonhuman primates (49, 50, 51, 52, 53, 54, 55), but until recently there were no reports of CpG-C ISS-ODN usage in macaques (56). We found that the CpG-C ISS-ODN C274 activates macaque blood-derived DCs (17) and B cells (57) in vitro. This is supported by a recent report from Abel et al. (58), documenting the responses of macaque blood and lymphoid PDCs to the different classes of CpG ISS-ODNs. Of note, systemic administration of CpGs can induce systemic increases in cytokine level and septic shock-like side effects (59, 60). In contrast, intralymphatic administration of CpGs not only markedly decreases the effective dose needed to achieve the adjuvant effect as compared with systemic administration, it avoids potential side effects of systemic administration (61). Thus, local administration of CpG-C ISS-ODNs represents an appealing strategy to explore. In this study, we show that a single intranodal administration of C274 exhibited local and systemic effects on LN cell (LNC) functions (enhanced DC, cytokine, and chemokine responses). The results suggest that local administration of CpG-C ISS-ODNs may be helpful in boosting innate functions to improve the immunogenicity and protective efficacy of vaccines and in augmenting other forms of immunotherapy to limit HIV.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and treatment

Adult male and female Indian rhesus macaques (Macaca mulatta) were bred and housed at the Tulane National Primate Research Center (TNPRC; Covington, LA). At the start of these studies, all naive animals tested negative by PCR for simian type D retroviruses, simian T cell leukemia virus-1, and SIV. Macaques infected with simian HIV (SHIV) 162P variants (62, 63, 64, 65, 66) were used as a model for infected individuals with low level plasma viremia with the goal to follow virus-specific responses upon intranodal injection with C274. This would provide the first evidence as to whether such treatment would boost existing SIV-specific responses that (in the long-term and likely with repeated treatments coadministered with a boosting vaccine) might prolong the control of virus in such individuals. Macaques BD19 and BE72 were infected intravaginally with SHIV162P3 on 1/14/02, AM40 and P311 were exposed intravaginally to SHIV162P4 on 10/9/00 and 1/13/00, respectively, and T122 was i.v. infected with SHIV162P4 on 2/8/99. T cells from these animals secrete IFN-{gamma} in response to chemically inactivated SIV (aldrithiol-2 (AT-2) SIV) in vitro (our unpublished observation) (17).

Animals were anesthetized with ketamine/HCl (10 mg/kg) before all procedures. Heparinized blood was taken (10 ml/kg/month/animal) and superficial LN biopsies taken from the inguinal and axillary regions using standard survival surgery procedures. Twenty-four hours before removal, the superficial LNs of naive macaques were directly injected with 500 µg (50 µl) of the CpG-C ISS-ODN C274 or CpG-B ISS-ODN 1018, respectively; 500 µg of the inactive control ODNs C661 and 1040 was injected in the contralateral LNs. SHIV162P-infected macaques were intranodally injected with 500 µg of C274 and 500 µg of C661 in the contralateral LN. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the TNPRC. Animal care procedures were in compliance with the regulations detailed under the Animal Welfare Act (67) and in the Guide for the Care and Use of Laboratory Animals (68).

ODNs

The ODNs used in this study were as follows: 1018, 5'-TGACTGTGAACGTTCGAGATGA (CpG-B ISS-ODN); C274, 5'-TCGTCGAACGTTCGAGATGAT (CpG-C ISS-ODN); 1040, 5'-TGACTGTGAACCTTAGAGATGA (CpG-B ODN control); and C661, 5'-TGCTTGCAAGCTTGCAAGCA (CpG-C ODN control) (45). ODNs were provided by Dynavax Technologies.

Tissue and cell preparation

After ODN injection, LNs were surgically removed and total LNC suspensions were obtained by mechanical disruption of the tissue (69, 70). LNCs were cultured for 24 h, 7 days, and 13 days. Peripheral blood was obtained from the SHIV-infected macaques on the day LNs were injected with indicated ODNs (before injections) as well as 1, 7, and 14 days after the injections. PBMCs were isolated using Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech).

FACS analysis

To characterize DCs, three- or four-color flow cytometry was used (17). CD123– (MDC-containing) cells and CD123+ PDCs were identified within Lin–HLA-DR+ APC populations using FITC-conjugated anti-Lin Abs (anti-CD3, clone SP34 (BD Biosciences); anti-CD8, SK1 (BD Biosciences); anti-CD11b, clone F6.2 (Ex{alpha} Biologicals); anti-CD14, clone M5E2 (BD Pharmingen); anti-CD20, clone L27 (BD Biosciences)) with allophycocyanin-conjugated anti-HLA-DR (clone G46-6; BD Pharmingen) and PE-conjugated anti-CD123 (clone 7G3; BD Pharmingen). Cy-conjugated anti-CD80 (clone L307.4; BD Pharmingen) or anti-CD86 (clone 2331 FUN1; BD Pharmingen) were used to monitor CD80 and CD86 expression by Lin–HLA-DR+CD123– cells and Lin–HLA-DR+CD123+ PDCs. B cells were identified as CD20+ cells within the total mononuclear cell gate. PE-conjugated anti-CD80 (clone L307.4), anti-CD86 (clone IT2.2; BD Biosciences), anti-CD40 (clone 5C3; BD Biosciences), or anti-CD38 (HB7; BD Biosciences) were used to monitor the CD20+ B cell phenotype. Isotype Ig controls for each fluorochrome were included in all experiments and typically gave mean fluorescence intensities (MFIs) of <1 log. All samples were acquired on a FACSCalibur (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star).

Medium and reagents

Cells were cultured in 1 ml at 2 x 106 cells/well in 48-well plates (BD Biosciences) or in 250 µl at 5 x 105 cells/well in 96-well plates (Microtest U-Bottom; BD Biosciences) in complete medium (RPMI 1640; Cellgro; Fisher Scientific) containing 2 mM L-glutamine (Invitrogen Life Technologies), 10 mM HEPES (Invitrogen Life Technologies), 50 µM mercaptoethanol (Sigma-Aldrich), 100 U/ml penicillin/100 µg/ml streptomycin (Invitrogen Life Technologies), and 1% heparinized human plasma. Based on previous titration data (17), ODNs were used at 5 µg/ml for all in vitro cultures and human CD40L (R&D Systems) at 1 µg/ml. AT-2-treated SIVmneE11S, SIVmac239, SIVmac239 {Delta}V1V2 were provided by the AIDS Vaccine Program (Science Applications International Corporation-Frederick, National Cancer Institute, Frederick, MD). Virus content was determined with an Ag capture immunoassay for SIV gag p27 (AIDS Vaccine Program) and/or by HPLC analysis (71). Virus stocks were diluted to 3 µg of p27/ml in PBS containing 1% BSA (Intergen) and stored as aliquots at –80°C. Thawed aliquots were kept at 4°C ≤1 wk and AT-2 SIV used at final concentrations of 30–300 ng of p27/ml. Because highly purified virions and virus preparations contain proteins derived from the cells in which the virus was produced (72) a no-virus microvesicle (MV) control, prepared from the uninfected cells of the same cell lines in which the viruses were grown, was included to confirm the viral Ag specificity against the different isolates. SIVmne E11S was grown in Hut78 cells, SIVmac239 in SUPT1 cells, and SIVmac239 {Delta}V1V2 in CEM x 174 cells. MV from each cell line were normalized on the total protein content (for each batch of AT-2 SIV).

Cell cultures

LNCs and PBMCs were cultured with the indicated stimuli. Stimuli were added in the beginning of the cultures as well as on day 7 for 13 day LNC cultures. After 24 h (PBMCs and LNCs), 7 or 13 days (LNCs), cell-free supernatants were collected and stored at –20°C before ELISA and Luminex fluorescent bead assay analysis (Beadlyte Human 22-plex cytokine detection system recognizing macaque IL-1beta, -4, -5, -6, -8, -12 p40, GM-CSF, MCP-1/CCL2, MIP-1{alpha}/CCL3, TNF-{alpha}, RANTES/CCL5; Upstate Biotechnology). Although not listed as cross-reactive with macaque IL-3 by the manufacturers (after PBMC stimulation with PHA/PMA), IL-3 responses were detected upon C274 stimulation on the LNCs and PBMCs (57). Cell-free supernatants were analyzed for the presence of IL-12 p70 and the free p40 subunit and IFN-{alpha} by ELISA (BioSource International). Cultured cells were collected and assayed by flow cytometry.

IFN-{gamma} ELISPOT

The numbers of IFN-{gamma} spot-forming cells (SFC) were measured by ELISPOT (17, 73). LNCs and PBMCs (2 x 105 cells in 100 µl) were cultured in triplicate for 24 h in the presence of AT-2 SIV (300 ng/ml p27) or MV. In each experiment, 5 x 104 cells were also cultured with medium vs 5 µg/ml PHA (BD Biosciences) to control for cell functionality and assay integrity. Spots were counted using an AID ELISPOT reader (Cell Technology) using once optimized settings.

Statistical analyses

The limited number of animals from which cells were derived, coupled with the slender likelihood that the resulting distributions would be Gaussian, required use of nonparametric statistical analyses. Two-sided sign tests examined whether stimuli produced increased responses over controls. In comparing results from groups with cells from different animals or animals with different infection status, Kruskal-Wallis ANOVA of ranks was used. Wilcoxon’s matched pairs analysis or sign tests calculated probabilities for comparisons based on cells from the same animals. Differences, ratios, or rank tests were considered significant when p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immediate in vivo effects of C274 on DC and B cell frequency and phenotype

To explore the in vivo activity of ISS-ODNs on macaque leukocytes, we directly injected the LNs with ISS-ODNs and monitored DCs, B cells, and cytokine/chemokine responses upon isolation of cells from tissue biopsied 24 h later. ODNs were injected at different sites in the same animal to allow comparisons between C274 and 1018 ISS-ODNs in naive animals and then to focus on the more promising C274 in the SHIV-infected monkeys. Direct intranodal injection was used to enable controlled examination of local vs systemic effects and so that we could use smaller amounts of the ODNs without the potential side effects of systemic administration. No evidence of inflammation was detected at any of the injected sites in all animals. There was no obvious effect of the ISS-ODN injection on total viable cell numbers recovered (data not shown).

Fig. 1 shows representative flow cytometry plots demonstrating how we identified DCs and B cells. Four-color staining was used to define the Lin–HLA-DR+ DC-containing subsets (irregular gates, left panels), allowing the examination of CD80 (middle panels, rectangular gates) or CD86 (data not shown) expression by the Lin–HLA-DR+CD123– (MDC containing) and Lin–HLA-DR+CD123+ PDC fractions within the LNCs (17). The Lin–HLA-DR+ DC populations included HLA-DR dim to bright cells to ensure that we included all DC subsets. The example shown highlights the small, ill-defined (compared with blood) subset of Lin–HLA-DR+ cells often detected in LNs (16, 17) comprising the Lin–HLA-DR+CD123– and Lin–HLA-DR+CD123+ subsets. Separate CD20 staining was used to delineate B cells and similarly monitor costimulatory molecule expression (Fig. 1, rectangular gates, right panels).


Figure 1
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  		FIGURE 1. Identification and characterization of lymphoid DCs and B cells. The LNs of a SHIV-infected macaque (BE72) were injected with 500 µg of C661 or C274 and 24 h later, the LNs were removed and LNC suspensions were prepared. Cells from the C661- (upper row) vs C274-injected (lower row) LNs were stained with Abs to identify DCs and B cells. Four-color staining with FITC-conjugated anti-lineage Abs, allophycocyanin-conjugated anti-HLA-DR, Cy-conjugated anti-CD80 (or -CD86; data not shown), and PE-conjugated anti-CD123 was used to identify the Lin–HLA-DR+ DC-containing fractions (irregular gates, left panels). CD80 and CD123 expression by DCs within the Lin–HLA-DR+ gate are shown in the middle panels and the rectangular gates were used to define the CD123– and CD123+ subsets. In separate samples, CD20+ B cells were identified by two-color staining with FITC-conjugated anti-CD20, as indicated by the rectangular gates in the right panels, and CD80/CD86 expression was monitored. The expression of CD80/CD86 was determined on the entire populations to ensure that even small increases in expression were documented. Isotype Ig controls were included in each experiment and exhibited <1 log of staining.

 
Immediate analysis of the isolated cells indicated that there was minimal impact of ISS-ODN injection on the recovered percentages of DCs and B cells within the LNC suspensions (Table I). Fig. 1 is an example of one of the greatest increases in the percentage of Lin–HLA-DR+ cells induced by C274 treatment. But, overall there was negligible change in the percentage of Lin–HLA-DR+ cells and, despite a tendency to be higher, there was no significant change in the percentage of Lin–HLA-DR+CD123+ PDCs in the LNCs from tissue injected with C274. When data from the naive and SHIV-infected animals were pooled, the percentage of Lin–HLA-DR+CD123+ PDCs was significantly greater in cells from C274-injected (vs C661-injected) LNs (p < 0.05). There was no change in the B cell frequency following in vivo exposure to ISS-ODNs. The absolute numbers of leukocytes isolated from each LN varied significantly between animals (independent of the treatment) and the numbers of DCs and B cells actually tended to be lower in the ISS-ODN-injected LNs (compared with control ODN-injected LNs, Table I).


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  		Table I. DC and B cell frequency in the LNs following in vivo C274 exposure

 
There were marginal surface immunophenotypic and more dramatic functional modifications in response to in vivo ISS-ODN treatment. CD80 and CD86 levels were slightly elevated on Lin–HLA-DR+CD123+ PDCs as well as on Lin–HLA-DR+CD123– cells within the freshly isolated cells from the ISS-ODN-injected LNs (Fig. 1, Table II). The differences in CD80 and CD86 expression by DCs within C274 vs C661-injected LNCs were not statistically significant, likely due to analyses being limited by the animal numbers (and because we gated more conservatively on the entire CD123+ population). Only the CD80 expression by Lin–HLA-DR+CD123+ PDCs and CD86 by Lin–HLA-DR+CD123– cells was significantly increased in the 1018-injected LNCs compared with the 1040-injected control (p < 0.05, Table II, numbers in bold). The MFIs of CD80 and CD86 on Lin–HLA-DR+ cells within the control ODN-treated LNCs from the SHIV-infected animals were higher than those of the naive animals, suggestive of infection-induced activation in vivo. CD80 levels showed a significant increase on CD20+ B cells from C274-treated LNs when data from naive and SHIV-infected animals were pooled (p < 0.05).


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  		Table II. CD80 and CD86 expression by DC and B cells isolated from ODN-injected LNs

 
In vivo C274 exposure heightens cellular activation upon in vitro culture

Simply culturing these LNCs overnight in medium revealed more definitive evidence of C274 activation in vivo. Overnight culture of cells up-regulated the expression of CD80 and CD86, especially on CD123+ PDCs (compare Table II and Fig. 2A). The levels of CD80 and CD86 on Lin–HLA-DR+CD123+ PDCs after in vitro culture were higher in cells isolated from LNs injected with C274 (compared with C661-injected controls). Although not significant, the C274 effect was consistent in all animals from both groups, despite the cells from infected animals having higher levels of expression to start with. Less dramatic increases were seen in the Lin–HLA-DR+CD123– fraction, as expected for bystander activation of MDCs by the ISS-ODNs. There was little difference in the CD40, CD80, or CD86 levels on B cells from the different tissues upon overnight culture in medium (data not shown; Fig. 3). Analysis of the subset frequencies after overnight culture (Table III) revealed that in vivo exposure to C274 had no significant impact on the percentages of Lin–HLA-DR+CD123– cells, Lin–HLA-DR+CD123+ PDCs, or CD20+ B cells.


Figure 2
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  		FIGURE 2. Immediate effects of in vivo ISS-ODN treatment on lymphoid DC phenotype and functional activation. LNCs isolated from the differently treated LNs of three to five naive or four to five SHIV-infected animals were cultured overnight in medium. Each symbol indicates the in vivo treatment. A, CD80 and CD86 levels (mean MFIs) expressed by the Lin–HLA-DR+CD123– (CD123–) and Lin–HLA-DR+CD123+ (CD123+) cells (gated as described in Fig. 1) are shown for the naive (left panel) and SHIV-infected (right panel) animals. B, Cell-free supernatants collected from the cultured cells were analyzed for the presence of various cytokines and chemokines using the fluorescent bead Luminex assay. The mean (±SEM) fold increased production of each factor by cells from the C274- and 1018-injected LNs above that produced by cells from the C661 and 1040 control ODN-treated LNs, respectively, are summarized. Data for the C274/C661-treated LNs comes from naive and SHIV-infected macaques and the 1018/1040-treated LN data are from naive animals.

 

Figure 3
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  		FIGURE 3. Costimulatory molecule up-regulation on DCs and B cells after in vitro stimulation. Cells were isolated from 1040-, 1018-, C661-, and C274-treated LNs of naive and SHIV-infected monkeys. LNCs from three to five naive and four to five SHIV-infected animals were cultured for 24 h in the presence of 5 µg/ml C274 (+) or C661 (–). The different in vivo treatments are marked by the distinct symbols. Costimulatory molecule expression (MFI) was measured on CD123+ and CD123– cells within the Lin–HLA-DR+ cell gate and on the CD20+ B cells within the total leukocyte gate (Fig. 1). MFIs of CD80 and CD86 expression by CD123– APCs (CD123–), CD123+ PDCs (CD123+), and CD20+ B cells (CD20+) are shown for naive and SHIV-infected animals together. PDC responses to AT-2 SIV as well as B cell and MDC responses to CD40L were also observed in each of the differently treated LNC populations (typically highest in the ISS-ODN-treated samples; data not shown).

 

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  		Table III. PDC and B cell percentages following in vitro culture

 
We also measured whether there were functional differences between the cells from ISS-ODN- vs control ODN-injected LNs. Although the amounts of each cytokine/chemokine produced differed between donors, the overall pattern of higher production by cells from ISS-ODN-treated LNs was apparent across the board. Fig. 2B shows the fold greater chemokine/cytokine production for all animals injected with C274 vs C661 (left panel, naive and infected) and 1018 vs 1040 (right panel, naive). The responses driven by C274 and 1018 were similar, although there were some differences in the factors released. Both ISS-ODNs induced CCL2 (higher after C274 treatment), CCL3, CCL5 (much higher for 1018-treated LNs), CXCL8, IL-1beta, IL-6, IL-12 p40, GM-CSF, and TNF-{alpha}, while neither induced detectable levels of IL-4. CCL2 (p = 0.039) and IL-6 (p = 0.0039) levels released by the cultured LNCs were significantly greater following C274 injection. In vivo C274 (not 1018) treatment also primed the LNCs to secrete IL-3 upon culture (see medium-cultured cells in Fig. 5). Little, if any, IFN-{alpha} was detected upon culturing these cells in medium (see medium-cultured cells in Fig. 4).


Figure 5
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  		FIGURE 5. Enhanced cytokine and chemokine release in response to ISS-ODN stimulation. Cell-free supernatants collected from the cultures described in Fig. 4 were also analyzed for the presence of other cytokines and chemokines using the Luminex assay. Responses stimulated in vitro by CD40L, AT-2 SIV (300 ng/ml), and C274 (vs the respective controls; x axes) are summarized (mean picograms per milliliter for LNCs from differently treated LNs of all animals, denoted with different symbols).

 

Figure 4
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  		FIGURE 4. IFN-{alpha} and IL-12 production by in vitro-stimulated LNCs. LNCs from five naive (1040-, 1018-, C661-, or C274-treated) and five SHIV-infected (C661- or C274-treated) animals were cultured for 24 h in the presence of medium (Med), CD40L, 300 or 30 ng (p27 equivalents, SIV 30 or SIV 300) of AT-2 SIV (vs the "300 ng" equivalent for the MV control), or 5 µg/ml 1018 (vs 1040) or C274 (vs C661). In vitro stimuli are noted on the x axes and the in vivo treatments denoted with the different symbols. Cell-free supernatants were collected and the amounts of IFN-{alpha} (upper panel) and IL-12 (lower panel) released by the cells were measured by ELISA. Mean picogram per milliliter values are shown for the results from both naive and infected animals together.

 
Up-regulation of costimulatory molecules on DCs and B cells upon in vitro stimulation of in vivo ISS-ODN-treated LNCs

Having demonstrated that in vivo exposure to C274 (and 1018) activated cells within the LNs, we investigated whether DCs and B cells from the differently treated LNs were responsive to in vitro stimulation. LNCs were cultured with ISS-ODNs, CD40L, or AT-2 SIV, stimuli that trigger macaque blood-derived MDC and PDC (17) as well as B cell (57) activation. This allows a better appreciation of the leukocyte function after in vivo ISS-ODN treatment and provides the first indication as to whether the cells remain responsive to repeated stimulation as would be needed to adjuvant a vaccine.

Comparable results were obtained for cells from naive and SHIV-infected macaques (using Kruskal-Wallis two-sided tests), and the data from all animals are pooled for presentation in Fig. 3. Just as for blood cells (17, 57), in vitro exposure to C274 induced the most prominent up-regulation of CD80 and (less so) CD86 on Lin–HLA-DR+CD123+ PDCs and B cells in the LNC cultures (Fig. 3). Significant increases in the levels of CD80 expression by Lin–HLA-DR+CD123+ PDCs and Lin–HLA-DR+CD123– cells upon C274 stimulation in vitro were only detected in cells from the control (C661) treated LNs (p = 0.00769). In vitro exposure to C274 resulted in a significant increase in the expression of CD40 by B cells from C661- and C274-treated LNs (data not shown) and of CD86 in B cells from C661-treated LNs (p = 0.0039 for each). In vitro CD40L stimulation also increased CD40, CD80, and CD86 expression by B cells in all LNC preparations (data not shown), with CD40 (p = 0.0039 and 0.0215) and CD86 (p = 0.039 and 0.002) being significantly increased in cells from LNs injected with C661 and C274, respectively. AT-2 SIV induced minimal changes in B cell phenotype (data not shown; as in blood; Ref. 57). CD80 and CD86 were highest in cells from ISS-ODN-injected LNs. Beyond the culture-induced activation of the HLA-DR+CD123– cells there was limited influence of C274 on their phenotype.

Boosted cytokine and chemokine responses by in vitro-stimulated, in vivo ISS-ODN-treated lymphoid cells

With the knowledge that C274 and, even more strikingly, AT-2 SIV induce macaque blood leukocytes to secrete IFN-{alpha} (17), we measured IFN-{alpha} responses of in vitro-stimulated LNCs to further gauge the functional implications of in vivo C274 treatment. We first examined the similarity of responses by cells from the naive and SHIV-infected groups by Kruskal-Wallis rank tests. All LNC preparations from naive and infected macaques released IFN-{alpha} in response to AT-2 SIV and C274 in vitro (Fig. 4, upper panel). The large amounts of IFN-{alpha} induced by 300 ng/ml AT-2 SIV (vs MV) were significant in both the C274-treated LNCs (p = 0.002) and in the C661-treated LNCs (p = 0.039), and were similar in magnitude (p < 0.05). Although produced in significantly lower amounts, the IFN-{alpha} was also greatly increased in response to the 30 ng/ml AT-2 SIV dose for both C274- and C661-treated LNCs (p = 0.0156 for each). Although the limited number of animals exposed to 1018 in vivo did not permit statistical differentiation, both doses of AT-2 SIV-induced IFN-{alpha} release from in vivo 1018-treated cells in vitro. In vitro C274-induced IFN-{alpha} production was significantly increased in both C274- and C661-treated LNC cultures (p = 0.039 for both). As in macaque blood (17), no IFN-{alpha} was produced by any of the LNCs in response to in vitro exposure to 1018. Earlier work in macaque blood confirmed that the IFN-{alpha}-producing cells fall within the Lin–HLA-DR+ cells (like those shown in Fig. 1) and that enrichment of these cells increases the amounts of IFN-{alpha} detected upon C274 stimulation (17).

In vitro exposure to either ISS-ODN induced IL-12 p70 responses (Fig. 4, lower panel). In vitro C274 stimulation of cells from C274-treated LNs resulted in increased IL-12 production (p = 0.0039). The highest amounts of IL-12 were detected in cells from C274-treated LNs after in vitro 1018 stimulation. Low-level IL-12 was induced by CD40L (most prominently by LNCs exposed to C274 in vivo, compared with control ODN-treated LNs; p = 0.0215). The IL-12 responses to AT-2 SIV were similar for each LNC preparation, with a significant increase in IL-12 being produced upon AT-2 SIV (300 ng/ml) stimulation of C661-treated LNCs (p = 0.0391).

Because IFN-{alpha} and IL-12 responses were consistently induced by in vitro C274 exposure (compared with in vitro 1018 stimulation), more extensive analyses of the cytokine and chemokine profiles elicited by in vitro stimulation with C274 were performed (Fig. 5). In vitro stimulation with C274, CD40L, or AT-2 SIV induced cytokine (IL-3, IL-6, TNF-{alpha}, and even low levels of GM-CSF (data not shown)) and chemokine (CCL2, CCL3, CCL5, and CXCL8) responses above those secreted simply upon culture in medium (and the respective controls; Figs. 2 and 5). Statistically significant increases in IL-3 (p = 0.0156), TNF-{alpha} (p = 0.0156), and IL-6 (p = 0.0039) release were detected upon in vitro C274 stimulation of in vivo C274-treated LNCs. In addition, in vitro C274 stimulation induced statistically significant production of CCL2 (p = 0.0078), CCL5 (p = 0.0078), and IL-6 (p = 0.0039) by in vivo C661-treated LNCs. Cells from C274-injected LNs also produced significant amounts of TNF-{alpha} (p = 0.0156), CCL5 (p = 0.0391), CCL3 (p = 0.0391), and IL-6 (p = 0.0215) in response to AT-2 SIV stimulation. AT-2 SIV stimulation induced TNF-{alpha} (p = 0.0391), CCL2 (p = 0.0391), and IL-6 (p = 0.0391) responses by in C661-treated LNCs. CD40L induced IL-6 in both C661- (p = 0.031) and C274-treated (p = 0.0039) LNC cultures.

In some instances, the responses of LNCs from tissues already treated with C274 in vivo did not increase above those induced by the respective controls. Combined exposure to the stimuli in vitro had no synergistic effects on these 24 h responses (data not shown). We also monitored cultures maintained for 7–13 days and observed no shift in the patterns observed after 24 h (data not shown). All of these responses were comparable for cells from naive and SHIV-infected animals.

Effect of a single C274 dose in vivo on SIV-specific IFN-{gamma} responses

We have reported previously that the LNs and blood of SIV- or SHIV-infected macaques contain SIV-specific T cells that can be activated by AT-2 SIV in vitro to secrete IFN-{gamma} (17, 22). Moreover, C274-activated blood DCs augment the responses to AT-2 SIV in vitro (17). To ascertain whether the intranodal injection of C274 influenced the levels of SIV-specific IFN-{gamma} release in vitro in response to AT-2 SIV, we monitored the responses in blood before and after C274 injection and compared the responses in the C274- vs C661-injected LNs from the same SHIV-infected animal.

Blood samples were taken before as well as 1, 7, and 14 days after intranodal injection of C274 (and C661). SIV-specific IFN-{gamma} responses to three different AT-2 SIV isolates (two wild-type isolates and an isolate with a deletion in the V1V2 regions of envelope) were measured by ELISPOT. The results from five different animals are shown in Fig. 6A. Background or endogenous IFN-{gamma} release upon culture with MV were comparable to those seen when the cells were cultured in medium (data not shown) and decreased after ODN injection (Fig. 6B, blue squares). Varying levels of SIV-specific IFN-{gamma} release were seen in each animal before C274 injection, reacting comparably against each of the AT-2 SIV isolates. Unlike four of the five animals, the SIV-specific responses of cells from animal T122 immediately increased following C274 administration. However, the medians of the data from all five animals show that the SIV-specific responses initially decreased post ODN injection, but then rebounded to preinjection levels by day 14 (Fig. 6B, black triangles). Coincidently, increased levels of IFN-{alpha}, CCL2, and CCL5 were detected in the plasma immediately after C274 administration (Fig. 6C), providing further support for the systemic effect of ISS-ODN treatment. Plasma viral loads remained undetectable at these time points after ODN injection (data not shown).


Figure 6
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  		FIGURE 6. Impact of in vivo C274 treatment on in vitro-activated SIV-specific IFN-{gamma} responses. A, PBMCs from five SHIV-infected animals (BD19, AM40, BE72, P311, and T122) were isolated before (Pre) and 1, 7, or 14 days (D1, D7, D14) after the LNs were injected with C661 or C274. PBMCs were cultured with the appropriate MV control for each AT-2 SIV stimulus; AT-2 SIV E11S (E11S), SIVmac239 (239), or SIVmac239 {Delta}V1V2 (V1V2). The numbers of SFC per 2 x 105 cells releasing IFN-{gamma} are shown (mean ± SEM for triplicate wells) for each animal. B, Relative to the pretreatment responses, the median fold differences in SIV-specific (left axis, black triangles) and background MV-induced (right axis, blue squares) IFN-{gamma} responses are shown for all animals. All SIV-specific or background MV-induced SFC numbers at 1, 7, and 14 days posttreatment were divided by the numbers of SIV-specific or background SFCs detected before ODN injection. Values for the pretreatment time point are set at 1. C, Plasma collected before and 1 day after ODN injection from three of the five animals were monitored for cytokine and chemokine levels. Mean picogram per milliliter (±SEM) are shown. IFN-{alpha}, CCL2, and CCL5 were back to baseline levels at the 7 and 14 day time points. No other factors were detected at any of the time points. D, C661- vs C274-injected LNs (from the same animal) were harvested 24 h after injection and the spontaneous IFN-{gamma} released upon in vitro culture of the LNCs with MV (or medium; comparable to MV, data not shown) was measured by ELISPOT. Mean IFN-{gamma} SFC (±SEM) from replicate cultures of cells from C661- vs C274-injected LNs of three animals are shown. E, LNCs collected 24 h after intranodal injection of C661 vs C274 were stimulated with the indicated AT-2 SIV isolates (vs MV controls) and the IFN-{gamma} release was measured. Mean (±SEM) SIV-specific responses against the three SIV isolates (above the MV controls) for the cells from the C661- vs C247-treated LNs of three infected animals are summarized (left panel). The mean fold differences (±SEM) between the SIV-specific responses and the endogenous IFN-{gamma} production are shown for these three animals (right panel).

 
Paralleling these experiments, LNCs from sites injected with C661 vs C274 24 h earlier were assayed for their ability to release IFN-{gamma}. LNCs from the C274-injected site exhibited enhanced IFN-{gamma} release upon culture in the absence of additional Ag (Fig. 6D). Just as with the blood leukocytes (Fig. 6A), LNCs responded to all isolates of AT-2 SIV, but there was no difference between the SIV-specific responses of cells from C661- and C274-injected LNs (Fig. 6E, left panel). The fold difference between the SIV-specific and the endogenous IFN-{gamma} release by the C274-injected LNCs was in fact lower than that of the C661-injected LNs (Fig. 6E, right panel), due to the elevated endogenous IFN-{gamma} production in the former (Fig. 6D). Thus, a single dose of C274 injected into the LNs enhanced DC and B cell activity, cytokine and chemokine production, and endogenous IFN-{gamma} release, but this was insufficient to dramatically augment responses against Ag added in vitro.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A successful therapy against HIV will probably have to elicit robust mucosal and systemic immunity, involving T and B cells as well as innate responses (74, 75, 76, 77). Triggering both innate and adaptive activities, ISS-ODNs promote the activation of potent B and T cell responses, largely through the stimulation of DCs and B cells (45, 46, 47, 48, 78, 79) and represent a promising adjuvant to boost vaccine efficacy and augment mucosal responses (48, 51). Furthermore, a more recently defined class of ISS-ODN, CpG-C, was shown to possess the ability to stimulate both DCs and B cells, affording a more effective way to enhance immunity (45, 46, 47).

Prior macaque studies described that CpG-B ISS-ODNs augmented Ab responses in animals treated with anthrax (51, 52), Leishmania (53), hepatitis B (49, 50, 51, 55), or HIV (80) vaccines, and were also effective in SIV-infected animals (50, 53). Although vaginally applied CpG-A ISS-ODN induced local IFN-{alpha} responses, this was not sufficient to protect against vaginal challenge (81). Having previously demonstrated that CpG-C ISS-ODNs stimulate macaque blood-derived DCs (17) and B cells (57) in vitro (more effectively than the CpG-B ISS-ODN 1018), we now provide evidence that the CpG-C ISS-ODN C274 functions in vivo and effectively stimulates macaque lymphoid DCs and B cells as well as potent cytokine and chemokine responses. This supports other recent reports illustrating the ability of CpG-C ISS-ODNs to activate primate APCs (56, 58) and to boost the T cell responses to a recombinant adenovirus/gag vaccine regimen in macaques (82). This sets the stage for additional studies to use CpG-C ISS-ODNs to enhance T and B cell responses in macaques to improve anti-HIV vaccine efficacy.

In addition to demonstrating the in vivo local and systemic activity of C274 following local administration in macaques, we wanted to compare its effectiveness in naive as well as healthy infected animals. The latter allows us to monitor the impact of C274 on SIV-specific immunity while also gaining evidence for the potential use of such a strategy to boost existing responses to help maintain control of infection. Contrasting what we observed in the blood where there were more PDCs in PBMCs from SHIV-infected monkeys (17), the percentages of PDCs were comparable in LNCs from naive and SHIV-infected animals. HIV reportedly infects blood-derived PDCs, increasing cell viability (83, 84), and this may be what we have observed in the blood but not LNs of the SHIV-infected animals. Whether this reflects inherent differences between PDCs in the blood and lymphoid tissues and/or differences in the distribution of DC subsets between blood and tissues needs to be elucidated. Earlier studies also reported alterations in LN DC numbers and phenotype during SIV infection (33, 34). We did not detect any significant differences between the numbers of DCs in the LNs of naive vs SHIV-infected animals (at this time point of infection), but the DCs from the infected animals did express higher levels of CD80 and CD86, indicative of at least partial activation. This complements earlier studies examining the immunophenotypes of lymphoid DCs in situ (33, 34).

Some reports suggest that DC (27, 28, 30, 31, 85, 86) and B cell (35, 36, 37, 38, 39, 40, 41, 42, 44) functions are altered during HIV infection. This emphasizes the need to identify ways to boost DC and B cell activities that will work in both naive and infected settings to maximally enhance the efficacy of prophylactic and therapeutic vaccines and more effectively limit HIV spread. We were able to demonstrate local and systemic effects of in vivo C274 treatment in both groups. Intranodal injection of C274 had limited impact on the frequency of DCs and B cells within the LNs and the absolute numbers of cells isolated were not enhanced in ISS-ODN-injected LNs. However, there tended to be a higher percentage of PDCs within the LNC suspensions after C274 injection compared with the control C661-injected LNs (p < 0.05 when data from naive and SHIV-infected animals were pooled together). Any increase in PDC frequency could reflect migration of PDCs into the tissues in response to C274 because of the chemokine production (87, 88) or enhanced survival due to direct antiapoptotic effects of ISS-ODNs (89). Cells from C274-treated LNs (from either infected or uninfected animals) secreted IL-3 upon in vitro culture, which could also contribute to the improved PDC survival (90).

Although the immediate effects of C274 were not striking within the freshly isolated cells, the impact of in vivo C274 treatment became more apparent upon subsequent in vitro culture of the isolated LNCs. PDCs within the in vitro-cultured cells from C274-injected LNs up-regulated CD80 (and to a lesser extent CD86) more dramatically than PDCs within the C661-injected LNCs. Some bystander effects on the CD123– MDC-containing fraction were also observed. More strikingly, cells from ISS-ODN-injected LNs spontaneously secreted elevated levels of cytokines and chemokines (upon culture) that would promote DC and B cell activation in addition to the recruitment of leukocytes into the lymphoid tissues to favor cell-cell communication critical for effective immune activation (48, 91, 92, 93, 94, 95, 96). This is similar to the report in humans describing elevated serum chemokine levels following s.c. injection of a CpG-B ISS-ODN (97).

Vaccine strategies that incorporate boosting chemokine responses have been proposed as advantageous in the fight against HIV (54, 98, 99, 100). Cytokine and chemokine responses triggered by ISS-ODNs also induce secondary effects, including NK cell activation (101, 102). Such innate antiviral responses would add to the virus-specific adaptive responses elicited by a coadministered vaccine, increasing the chances of effective virus control. Even though we did not detect significant changes in the LNC numbers isolated from the ISS-ODN vs control ODN-injected macaque LNs, there may be selective recruitment of leukocytes into the tissues over longer time periods that will require extensive examination of larger amounts of tissue than were available in this study. Increased IFN-{alpha} production following C274 injection also represents important innate response that might help limit HIV infection via direct and indirect means. Solid IFN-{alpha} and IL-12 responses (deficient in HIV infection (27, 28, 103, 104, 105, 106)) that are induced by C274 would be expected to further encourage DC-B cell interactions as well as improved Th1 stimulatory capacity (5, 6, 7, 8, 9, 10, 11, 47). Although we did not observe plasma cell differentiation or Ab secretion in vitro under these conditions, future studies will need to explore whether C274 can augment macaque plasma cell differentiation (in vitro or in vivo) in combination with Ag to trigger the specific BCR (47).

Using an established assay to measure SIV-specific IFN-{gamma} responses (17, 22) allowed us to evaluate what impact a single intranodal injection of C274 had on these responses within the tissue, as well as systemically. We showed previously with in vitro studies using PBMCs from SHIV162P-infected monkeys that C274-activated DCs induced stronger SIV-specific IFN-{gamma} release in response to AT-2 SIV, but C274 did not increase the endogenous IFN-{gamma} secretion in the absence of AT-2 SIV when directly added to the cultures (17). In the present studies, increased endogenous IFN-{gamma} release was detected upon culture of cells from the C274-injected LNs, coincident with decreased endogenous IFN-{gamma} responses in the PBMCs after injection. This might reflect migration of the IFN-{gamma}-secreting cells from the periphery to the C274-injected LN in response to chemokines triggered by C274. This paralleled an increase in the levels of IFN-{alpha}, CCL2, and CCL5 in the plasma. Except for the blood cells of one animal, the SIV-specific responses in the PBMCs and LNCs decreased after ODN injection and normalized back to preinjection levels after 14 days in the blood. This suggests that the increased endogenous activity in the LNCs masked any increase in the SIV-specific responses. Greater responses may be present at later time points in the LNs and would likely require the coadministration of specific Ag like AT-2 SIV to effectively expand SIV-specific T (and B) cells (as for a therapeutic vaccine), but this was impossible to assess in this initial study. The preliminary observation that C274 administration did not alter plasma viremia is encouraging for its application to boost immunity that will help control infection.

In summary, we have documented that CpG-C ISS-ODN C274 works in vivo and readily activates leukocytes (including DCs and B cells) in the LNs of both naive and chronically infected macaques. By augmenting DC and B cell functionality in the lymphoid tissues, stronger (innate and adaptive) immunity should result upon administration of vaccine Ag to prime or boost the immune system. Increased endogenous cytokine and chemokine activity in the lymphoid tissues as a result of C274 exposure might be especially important to help control virus replication within these tissues. Moreover, cellular activation and numerous cytokine and chemokine responses were detected upon specific restimulation of the C274-treated LNCs, indicating that these cells were still (sometimes more) responsive to additional stimulation. Although we did not perform repeated in vivo treatments with C274, verifying maintained responsiveness after treatment has important implications for the repeated treatments that would likely be needed for the most effective results in vivo. Additional studies comparing dosages and dosing intervals to achieve optimal immune responses including possible coadministration of CpG-C ISS-ODN C274 with Ag are warranted.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 National Institutes of Health Grants R21 AI060405 and R01s AI040877 and DE016256 (to M.R.), as well as by funds from the Elizabeth Glaser Pediatric AIDS Foundation, and the Tulane National Primate Research Center National Institutes of Health Base Grant RR00164. M.R. is an Elizabeth Glaser Scientist. This work was also funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-12400. Back

2 Current address: Division of Infectious Diseases, Mt. Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Back

3 Address correspondence and reprint requests to Dr. Melissa Robbiani, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021. E-mail address: mrobbiani{at}popcouncil.org Back

4 Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; MDC, myeloid DC; PDC, plasmacytoid DC; ISS-ODN, immunostimulatory CpG-C oligodeoxyribonucleotide; SHIV, simian HIV; LNC, LN cell; MFI, mean fluorescence intensity; AT-2, aldrithiol-2; MV, microvesicle; SFC, spot-forming cell. Back

Received for publication December 12, 2005. Accepted for publication October 2, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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