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(Journal of Leukocyte Biology. 2006;79:257-267.)
© 2006 by Society for Leukocyte Biology

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques

N. Teleshova*, J. Kenney*, V. Williams*, G. Van Nest{dagger}, J. Marshall{dagger}, J. D. Lifson{ddagger}, I. Sivin*, J. Dufour§, R. Bohm§, A. Gettie and M. Pope*,1

* Center for Biomedical Research, Population Council, New York, New York;
{dagger} Dynavax Technologies, Berkley, California;
{ddagger} AIDS Vaccine Program, Science Applications International Corporation-Frederick, National Cancer Institute at Frederick, Maryland;
§ Tulane National Primate Research Center, Tulane University, Covington, Louisiana; and
Aaron Diamond AIDS Research Center, New York, New York

1 Correspondence: Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021. E-mail: mpope{at}popcouncil.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20+ B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3–7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-{alpha}] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1{alpha}/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

Key Words: B lymphocyte • immunostimulation • primate • SIV


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of effective prophylactic or therapeutic human immunodeficiency virus (HIV) vaccines will likely entail the activation of broad humoral and cellular responses, with antibody responses especially important to limit mucosal transmission [1 2 3 4 5 6 7 8 9 ]. Identifying how to achieve the proper immune stimulation in infected and uninfected settings is a critical step in this task.

B cell activation requires binding of the antigen to the B cell surface immunoglobulin (Ig; B cell receptor) and costimulation by antigen-specific T cells through CD40-CD40 ligand (CD40L) interactions and T cell-derived cytokines (reviewed in refs. [10 , 11 ]). Appropriately activated B cells proliferate and differentiate into plasma cells or long-lived memory cells. HIV infection leads to progressive immune dysfunction in multiple cell types, including B cells that exhibit aberrant hyper-reactivity [12 13 14 15 16 17 18 19 ]. Despite evidence of polyclonal B cell activation against HIV and nonviral antigens [20 ], humoral responses to immunizations in HIV-infected individuals are impaired [21 ]. The basis of inadequate B cell responses during HIV infection might be a result of direct effects of virus on B cells [22 ], impaired T cell–B cell interactions [23 ], and/or a changed cytokine milieu [24 , 25 ]. Strategies to overcome this impairment and boost B cell responses (directly or indirectly) may play an important role in advancing vaccines/therapies aimed at curbing HIV infection.

Immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) are synthetic oligonucleotides containing certain cytosine-phosphate-guanine (CpG) motifs, which act as potent adjuvants for inducing strong antigen-specific T helper cell type 1 (Th1) responses [26 27 28 29 ]. They represent a promising new avenue for enhancing vaccine efficiency [30 , 31 ]. ISS-ODNs act via Toll-like receptor 9, and thus, cells bearing this receptor, including plasmacytoid dendritic cells (PDCs) [32 ] and B cells [33 ], can be powerfully stimulated by ISS-ODNs [30 , 31 , 34 35 36 37 38 39 ]. In response to ISS-ODNs, PDCs secrete enormous amounts of interferon-{alpha} (IFN-{alpha}) [40 41 42 ], driving the differentiation and activation of myeloid DCs (MDCs) [43 44 45 46 47 48 ] and PDCs [49 , 50 ] biasing toward robust Th1 immunity. DCs are also critical in B cell activation [51 52 53 54 55 56 ], interacting with B cells to provide survival and differentiation signals for effective B cell responses [57 ]. IFN-{alpha} stimulation of DCs is a requisite for this [53 ], triggering DCs to drive B cell activation and differentiation [58 ]. One study also revealed that virus-activated PDCs and CD40L (or T cells) can stimulate B cell differentiation into plasma cells via an IFN-{alpha}/ß/interleukin-6 (IL-6)-dependent pathway [59 ]. Recent human studies compared different classes of ISS-ODNs and showed that unlike the more extensively studied CpG-A and CpG-B ISS-ODNs, CpG-C ISS-ODNs stimulate B cells and PDCs (with sustained IFN-{alpha} release) [37 , 60 ]. In addition, CpG-C ISS-ODN-stimulated PDCs fostered B cell differentiation and plasma cell development [61 ].

Using the simian immunodeficiency virus (SIV)-macaque model, which provides a unique system to test anti-HIV prophylactic and therapeutic vaccines [62 ], we recently demonstrated that the CpG-C ISS-ODN C274 effectively activates macaque PDCs [63 ]. Herein, we provide evidence that C274 also readily stimulates macaque B cells isolated from the blood of naïve as well as healthy immunodeficiency virus-infected animals. These data underscore the potential use of CpG-C ISS-ODNs to bolster DC and B cell activities. Additional in vivo studies in nonhuman, primate models will help to assess the use of CpG-C ISS-ODNs to enhance the efficacy of prophylactic and therapeutic vaccination approaches to prevent immunodeficiency virus infection and AIDS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Adult male and female Indian rhesus macaques (Macaca mulatta) were bred and housed at the Tulane National Primate Research Center (TNPRC; Covington, LA). All naïve animals tested negative by polymerase chain reaction for simian type D retroviruses, simian T cell leukemia virus-1, and SIV. Macaques chronically infected with simian-HIV (SHIV)162P variants [3 , 64 65 66 67 ] were also used for these studies. Macaques T820 and T122 were infected on February 8, 1999, with SHIV162P4 via intravaginal and intravenous (i.v.) routes, respectively. Macaques T635 and T660 were i.v.-infected with SHIV162P1 on April 9, 1998; and T373 and T528 were i.v.-infected with SHIV162P on March 23, 1998. Macaques M010 and R228 were intravaginally infected with SHIV162P4 on January 13, 2000; and J526 and AP85 were infected with SHIV162P4 on January 20, 2000, and October 9, 2000, respectively. Macaque BE72 was intravaginally infected with SHIV162P3 on January 14, 2002. SHIV162P infection is not pathogenic, and none of the animals has shown evidence of SIV-associated disease during the 2–6 years of post-challenge follow-up. Previous studies confirmed that the DCs and T cells from these SHIV-infected animals are functional and can be used to monitor the presentation of SIV antigens by DCs to stimulate SIV-specific IFN-{gamma} release by the T cells [63 ]. Animals were anesthetized with ketamine-HCl (10 mg/kg) prior to heparainized blood samples being taken (no more than 10 ml/kg/month/animal). 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 in the Animal Welfare Act and in the "Guide for the Care and Use of Laboratory Animals."

Cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech AB, Uppsala, Sweden). PBMCs were used as total cell suspensions or to isolate B cells. To obtain purified B cell preparations, PBMCs were incubated with anti-CD20 beads (nonhuman primate, Miltenyi Biotec, Auburn, CA) followed by collection of the CD20+ B cell fraction [68 ] using the DepleteS program of the Miltenyi AutoMACS system (Miltenyi Biotec). The basic guidelines provided by the manufacturer were followed, except that the buffer used was cold phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA; Intergen, New York, NY).

Fluorescein-activated cell sorter (FACS) analysis
To determine the percentage of B cells in PBMCs and to determine the purity of B cells in the B cell-enriched populations, cells were monitored by flow cytometry. B cells were identified using fluorescein isothiocyanate (FITC)-conjugated anti-CD20 (clone L27, BD PharMingen, San Diego, CA). The expression of CD80 (clone L307.4, BD PharMingen), CD86 (clone IT2.2, BD PharMingen), CD40 (clone 5C3, BD PharMingen), and CD38 (clone HB7, BD Immunocytometry Systems, Franklin Lakes, NJ) was monitored using phycoerythrin (PE)-conjugated monoclonal antibodies against these markers. Staining was performed on freshly isolated cells as well as on cells cultured with the indicated stimuli in vitro (below). PE- and FITC-conjugated isotype Ig controls were included in all experiments and typically gave signals <1 log of fluorescence. Gates were set to include all mononuclear leukocytes based on the forward- and side-scatter characteristics (excluding any contaminating neutrophils). The gates used to define the CD20+ cells were determined based on the isotype controls. All samples were acquired on a FACSCalibur (BD Immunocytomerty Systems) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

Medium and reagents
Cells were cultured in RPMI 1640 (Cellgro, Fisher Scientific, Springfield, NJ) containing 2 mM L-glutamine (Gibco-BRL Life Technologies, Grand Island, NY), 10 mM HEPES (Gibco-BRL Life Technologies), 50 µM mercaptoethanol (Sigma Chemical Co., St. Louis, MO), penicillin (100 U/ml), streptomycin (100 µg/ml, Gibco-BRL Life Technologies), and 1% heparinized human plasma. The ODNs studied have the phosphorothioate backbone and included 1018: 5'-TGA CTG TGA ACG TTC GAG ATGA (CpG-B ISS-ODN); C274: 5'-TCG TCG AAC GTT CGA GAT GAT (CpG-C ISS-ODN); 1040: 5'-TGA CTG TGA ACC TTA GAG ATGA (CpG-B control ODN); and C661: 5'-TGCTTGCAAGCTTGCAAGCA (CpG-C control ODN) [37 ]. ISS-ODNs were provided by Dynavax Technologies (Berkeley, CA) and used at a final concentration of 5 µg/ml. As in our earlier DC work [63 ], some of the initial studies were done comparing C274 and 1018 with the CpG-B control ODN 1040. The CpG-C control ODN C661 was used routinely as the control for C274 in subsequent studies; results were generally comparable for control ODNs, CpG-B 1040, and CpG-C C661 (e.g., see Fig. 1A ). Human CD40L (R&D Systems, Minneapolis, MN) was used at a final concentration of 1 µg/ml. In initial comparative dose titration assays, 5 µg/ml ISS-ODNs (1–20 µg/ml compared) and 1 µg/ml CD40L (0.3–10 µg/ml compared) were found to be sufficient to reproducibly induce the strongest B cell proliferation by each stimulus (not shown). These doses were used routinely for all experiments. Chemically inactivated, noninfectious SIVmneE11S [aldrithiol-2 (AT-2) inactivated SIV] was prepared as described previously [69 ] and provided by the AIDS Vaccine Program [Science Applications International Corporation-Frederick, National Cancer Institute (NCI) at Frederick, MD]. Virus stocks were diluted in 1% BSA in PBS and stored as aliquots (3 µg p27 equivalents per ml) at –80°C. Thawed aliquots were kept at 4°C up to 1 week, and AT-2 SIV was used at a final concentration of 300 ng p27/ml (a dose known to induce the strongest IFN-{alpha} responses by PBMCs [63 ]). As even highly purified virions and virus preparations contain proteins derived from the cells in which the virus was produced [70 ] a no-virus microvesicle (MV) control, prepared from the uninfected cells of the same cell line in which the virus was grown, was included to exclude host cell-driven responses. The MV control was normalized on the total protein content (for each batch of AT-2 SIV).


Figure 1
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  		Figure 1. Macaque B cell proliferation in response to CpG-C ISS-ODN stimulation. (A) B cells were isolated from the PBMCs of three to six SHIV-infected ({blacksquare}) and three to four naïve ({triangleup}) macaques. Cells were cultured at 2 x 105 in a 96-well round-bottomed plate in the presence of medium (Med) versus CD40L (1 µg/ml), control ODN 1040 versus 1018, or control ODN C661 versus C274 (all ODNs at 5 µg/ml). After 3 days culture, cells were pulsed with 3H-TdR, and the amount incorporated [Inc.; counts per minute (cpm)] during the last 8 h of culture was measured. Proliferative responses (mean cpm±SEM) of B cells from SHIV versus naïve animals are shown. (B) The stimulus-specific responses induced by CD40L (six animals), 1018 (eight animals), or C274 (10 animals) are summarized for SHIV-infected and naïve animals together (mean cpm±SEM, after subtracting the cpm of the background responses induced by the respective controls). (C) To determine the kinetics of C274 activation (vs. 1040 control ODN), B cells from two naïve animals were cultured (as in A) for 1–3 days, and the 3H-TdR uptake was determined at each time-point (D1, D2, D3). (D) PBMCs from five SHIV-infected animals (solid symbols) and five naïve animals (open symbols) were cultured at 5 x 105 cells per well in 96-well flat-bottomed plates with C274 versus control ODN or CD40L versus medium. After 1 (five naïve, five SHIV), 3 (two SHIV), or 7 (five naïve, three SHIV) days, cells were stained with FITC-conjugated anti-CD20 to determine the percentage of B cells present. The percentage of CD20+ B cells (within the total leukocyte gate) in the stimulated conditions (C274 and CD40L) was divided by the percentage of CD20+ B cells in the respective control cultures (control ODN and medium) to obtain the fold increase (mean±SEM) in B cells induced by C274 (triangles) versus CD40L (squares).

 
Analysis of cultured PBMCs and B cells
B cells and PBMCs were cultured at 2 x 105 cells per well in 96-well round-bottomed plates (MicrotestTM U-Bottom, Becton Dickinson, Franklin Lakes, NJ) in 100 µl medium. PBMCs were also cultured at 5 x 105 cells per well (in 250 µl) in 96-well flat-bottomed plates (BD Falcon Multiwell flat-bottomed plates, Becton Dickinson) or in 48-well plates (BD Falcon Multiwell flat-bottomed plates, Becton Dickinson) at 1 x 106 cells in 1 ml. PBMCs or B cells were cultured with the specified stimuli for 24 h, 7 days, and 13 days. Stimuli were added again on Day 7 of the 13-day cultures. After culture, cell-free supernatants were collected and stored at –20°C. The supernatants were analyzed using the Luminex, Beadlyte®Human 22-plex cytokine detection kit (Upstate Biotechnology, Lake Placid, NY). This assay tested the supernatants for various cytokines and chemokines, which included: IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXC chemokine ligand 8 (CXCL8), IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IFN-inducible protein 10/CXCL10, eotaxin/CC chemokine ligand 11 (CCL11), IFN-{gamma}, granulocyte macrophage-colony stimulating factor (GM-CSF), monocyte chemoattractant protein-1/CCL2, macrophage-inflammatory protein-1{alpha}/CCL3, tumor necrosis factor {alpha} (TNF-{alpha}), and regulated on activation, normal T expressed and secreted/CCL5. The following factors are reported by Upstate Biotechnology to be cross-reactive with rhesus macaque factors [based on phytohemagglutinin (PHA)- or phorbol 12-myristate 13-acetate (PMA)-stimulated PBMCs]: IL-1ß, IL-4, IL-5, IL-6, CXCL8, IL-12 (p40), IFN-{gamma}, GM-CSF, CCL2, CCL3, TNF-{alpha}, and CCL5. Despite not being listed as recognizing macaque IL-3 when PBMCs are stimulated with PHA/PMA, IL-3 was reproducibly detected in supernatants from C274-stimulated PBMCs. The cultured cells were collected and assayed by flow cytometry (above) to observe phenotypic changes in response to the various stimuli.

B cell proliferation
To determine the proliferative responses of PBMCs or B cells to the CpG-C ISS-ODN C274, 1 x 105 cells were cultured with 5 µg/ml C274, 1018, or the control ODNs (C661 or 1040) versus 1 µg/ml CD40L in 96-well round-bottomed plates. The cultures were left at 37°C for 1–3 days before being pulsed with 1 µCi tritiated thymidine (3H-TdR) per well. After 8 h at 37°C, the cells were then harvested using a Brander harvester (Brander, Gaithersburg, MD). Incorporation of 3H-TdR into newly synthesized DNA was measured as an index of cell proliferation using a 1450 microbeta Wallac jet liquid scintillation and luminescence counter (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA).

Statistical analyses
Because of the limited number of animals from which cells were derived, nonparametric tests were used to examine their differential responses to stimuli and controls. Ratios of responses to stimuli and control from cells of each animal were computed from the data shown for each pairing and analyzed by the Kruskall-Wallis ANOVA of ranks. Mann-Whitney tests comparing the responses of cells from naïve versus SHIV-infected animals indicated that there was no significant difference in the fold increases, enabling the joint analysis of naïve and infected animal data. P values less than 0.05 were taken as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Macaque B cells proliferate strongly in response to C274
In prior studies, we described how the CpG-C ISS-ODN C274 readily activated PDCs found in the blood of healthy as well as chronically SIV- or SHIV-infected macaques [63 ]. To test whether macaque B cells are also responsive to C274 stimulation, we first monitored proliferative responses. Purified B cells isolated from the blood of naïve ({triangleup}) or SHIV-infected ({blacksquare}) macaques proliferated most strongly in response to C274 (compared with the proliferation induced by CD40L or the CpG-B ISS-ODN 1018; Fig. 1A ). Responses to the control 1040 and C661 ODNs were minimal, and the levels of 3H-TdR incorporated were only slightly greater than that seen for cells cultured in medium. The stimulus-induced responses from all animals are summarized in Figure 1B . Kruskall-Wallis ANOVA of ranks confirmed that the differences in the responses induced by C274 versus CD40L and 1018 were statistically significant (P=0.002 and P=0.03, respectively). Proliferative responses to C274 were readily detected after 3 days of culture, and less proliferative activity was at earlier time-points (Fig. 1C) . Similar patterns of proliferation were observed upon culture of PBMCs with these stimuli, but the magnitudes of 3H-TdR incorporated were lower than those seen upon B cell culture (not shown). This is not surprising, as CD20+ B cells made up ~10% of the PBMCs (mean percentages of CD20+ B cells±SEM of 9.64±1.94 for five naïve and 10.94±4.16 for five infected animals) compared with the ~90% CD20+ B cells in the purified preparations (87.51±2.1 from 10 animals; see representative example FACS plots in Fig. 2 ).


Figure 2
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  		Figure 2. Flow cytometric analysis of macaque B cell activation. PBMCs and B cells were isolated from the blood of a naïve macaque (T613). The cells were cultured for 24 h in the presence of medium (Med), CD40L (1 µg/ml), the MV control (normalized to the total protein concentration in the AT-2 SIV-stimulated cultures), AT-2 SIV (SIV, 300 ng p27/ml), C661 control ODN (5 µg/ml), or C274 (5 µg/ml). After 24 h, the cells were collected and stained with FITC-conjugated anti-CD20 (x-axes), in combination with PE-conjugated anti-CD40, -CD80, or -CD86 (y-axes). Cells were gated on total leukocytes, and the CD20+ subset was defined using the rectangular gate indicated. The MFIs of CD40, CD80, and CD86 expression were determined for the total CD20+ cells in each preparation and are indicated by the numbers in each dot plot. PE- and FITC-conjugated Ig controls exhibited <1 log of staining for each population.

 
FACS analyses of CD20+ B cells within the cultured PBMCs revealed that exposure to C274 maintained B cell numbers and even expanded the B cell subset within the mixed populations, compared with cells cultured with the control ODN C661 (Fig. 1D) . The effect of C274 on B cell numbers (1.5- to 2-fold more CD20+ B cells than those in the C661-stimulated cultures) was most apparent after 3–7 days of culture in cells from naïve and SHIV-infected macaques. In contrast, the percentages of CD20+ B cells in cultures stimulated by CD40L (Fig. 1D) or the CpG-B ISS-ODN 1018 (not shown) were comparable with those detected in the medium- or control ODN 1040-cultured controls (respectively). This mirrors the lower B cell proliferative responses seen with these stimuli (Fig. 1A and 1B) .

CpG-C ISS-ODN C274 up-regulates costimulatory molecule expression by B cells
Having confirmed that macaque B cells proliferated more robustly in response to C274 (compared with 1018), we then focused on monitoring the ability of C274 to up-regulate expression of important costimulatory molecules on macaque B cells. C274-induced responses were compared with those stimulated by CD40L (used as a standard B cell stimulus). We also compared the B cell phenotypes after culture with AT-2 SIV, which is known to mimic live virus when interacting with DCs [71 ] and also triggers macaque PDCs to secrete IFN-{alpha} [63 ]. Thus, any direct effects of whole virus on B cells as well as indirect effects through SIV-driven responses by PDCs (and other leukocytes) that might favor DC–B cell interactions could be assessed.

Prior to culture, the levels of CD40, CD80, and CD86 expressed by CD20+ B cells from naïve and SHIV-infected animals were equivalent [CD40 mean fluorescence intensities (MFIs) of 48.85±23.5 and 22.98±9.49, CD80 MFIs of 14.93±2.55 and 22.6±7.07, and CD86 MFIs of 23.09±6.72 and 21.03±5.58; mean MFIs±SEM by B cells from six naïve and seven infected animals, respectively]. Increased expression of CD40, CD80, and CD86 by B cells was detected within 1 day of culture of purified B cells or PBMCs with C274 or CD40L (Fig. 2) . Increased expression of CD80 and CD86 by cells within the CD20– subset of the PBMC cultures was apparent in response to CD40L, AT-2 SIV, or C274 (vs. their respective controls). This likely represents CD20– DCs [63 ] and/or monocytes within this fraction responding to these stimuli. Comparable B cell responses were seen between purified B cell and PBMC cultures. Thus, for more extensive analyses of the B cell phenotypic responses to these stimuli, we initially concentrated mainly on total PBMC mixtures, allowing more detailed comparisons of the cultured cells using smaller volumes of blood from each animal. Moreover, examining the unfractionated PBMCs also ensured that critical cell–cell communication involved in B cell activation could occur. The cell-surface immunophenotypic changes in B cells from SHIV-infected versus naïve animals induced by C274, CD40L, or AT-2 SIV (and their respective controls) are summarized in Figure 3 . These data show the MFI of each marker on the total CD20+ B cell population (left y-axes, black, solid symbols), the MFI of each marker on double-positive B cells (left y-axes, black, open symbols (i.e., CD40, CD80, or CD86 MFIs on those CD20+ cells also expressing CD40, CD80, or CD86 above the gate set on the Ig-PE-stained controls of CD20+ cells), as well as the percentages of double-positive cells (right y-axes, blue, open symbols).


Figure 3
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  		Figure 3. CpG-C ISS-ODN stimulation up-regulates costimulatory molecule expression on macaque B cells. PBMCs obtained from seven to 11 SHIV-infected and four to five naïve macaques were cultured for 24 h with medium, CD40L, MV, AT-2 SIV, C661, or C274 before being stained with FITC-conjugated anti-CD20 and PE-conjugated anti-CD40, -CD80, or -CD86 (Fig. 2) . The MFIs of CD40, CD80, and CD86 expressed by all CD20+ B cells within the differently stimulated cultures were determined for each and are presented as mean MFIs (±SEM; left y-axes, black, solid symbols). The MFIs of CD40, CD80, and CD86 on the double-positive populations in each culture (i.e., CD20+ cells expressing CD40, CD80, or CD86 levels above the cut-off set based on the PE-conjugated Ig controls, typically at ~1 log) are also shown in black, open symbols (left y-axes). The percentage of double-positive cells was calculated, and the mean values (±SEM, right y-axes, blue, open symbols) are shown for each condition.

 
C274 typically induced the highest levels (MFIs) of CD40, CD80, and CD86 expression on CD20+ B cells (total B cells and double-positive cells) from SHIV-infected and naïve monkeys (although CD86 levels on the double-positive B cells from naïve animals were comparable or slightly higher following AT-2 SIV or CD40L stimulation; Fig. 3 ). Kruskal-Wallis analyses by ranks revealed that CD40 and CD80 were increased significantly in response to C274 on all B cells (P values <0.05) as well as on the double-positive populations (P values <0.006). Overall, CD86 levels were not significantly increased on the double-positive cells or total B cells in response to C274, but C274 did induce significantly higher levels on the total B cell population compared with AT-2 SIV (P=0.01). Similar increases were seen in the percentages of double-positive B cells after C274 stimulation, but these increases were only significant for the CD86+CD20+ subset (P<0.002). Any nonspecific effects of C661 were less evident at the level of the marker expression.

The initially observed, similar responses to C274 by purified B cells and B cells within PBMC mixtures (Fig. 2) were confirmed using cells from two naïve and two SHIV-infected macaques (Fig. 4A ). Much like the activated B cells in the PBMC cultures (Fig. 3) , up-regulation of CD40, CD80, and CD86 expression was detected upon C274 stimulation of the purified B cells from all animals. The individual datasets shown in Figure 4A also highlight the variations in the levels of costimulatory molecule expression between different animals, but the consistent up-regulation of these markers by C274 is appreciated. As seen in the PBMCs, there was no discernable difference between the changes in B cell surface immunophenotypic seen for B cells from infected and uninfected animals after overnight exposure to C274. In contrast, the small effect of AT-2 SIV on B cell phenotype in PBMC cultures (Figs. 2 and 3) was not observed when purified B cells were cultured with AT-2 SIV. Specifically, control B cell cultures exhibited MFIs (±SEM) of CD40, CD80, and CD86 of 5.53 ± 1.61, 6.63 ± 1.9, and 11.77 ± 3.75, and AT-2 SIV-stimulated B cells expressed MFIs of 5.96 ± 1.79, 8.44 ± 2.76, and 10.93 ± 4.94, respectively (averaged data from two naïve and three SHIV-infected animals).


Figure 4
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  		Figure 4. Comparable B cell activation in short- and long-term cultures of cells from SHIV-infected and uninfected macaques. (A) CD20+ B cells were isolated from the blood of two SHIV-infected (T373 and T528) and two naïve (CN82 and T613) macaques and cultured for 24 h with 5 µg/ml control ODN C661 (–) or C274 (+). After antibody staining and flow cytometric analysis, the MFIs of the indicated markers on the double-positive cells were determined as described in Figures 2 and 3 . (B) PBMCs from four SHIV-infected (solid symbols) or four naïve (open symbols) monkeys were cultured with C661 (–) or C274 (+) for 7 days. The MFIs of CD40, CD80, and CD86 on the double-positive cells were determined (mean±SEM).

 
Knowing that C274 maintained B cell viability, especially after longer culture durations (Fig. 1D) , we examined the B cell surface immunophenotype at later time-points to ascertain how long the activated phenotype persisted. Coincident with the augmented B cell numbers, CD20+ B cells within the C274-activated PBMC cultures maintained their activated phenotype for up to 7 days (Fig. 4B) . This was true for cells from naïve and SHIV-infected macaques. Cells were typically less healthy (cell numbers, phenotype) after 13 days of culture with the individual stimuli (not shown). As plasma cell differentiation is characterized by CD20 down-regulation with increased CD38 expression, we measured the CD38 levels in the C274-stimulated cultures. CD38 expression was never detected on the cultured cells, even after up to 13 days of culture with the individual stimuli (not shown).

Cytokine and chemokine responses stimulated by C274
C274 induces IFN-{alpha} and IL-12 responses by macaque PBMCs and DC-enriched mixtures, paralleling increases in DC-driven, SIV-specific IFN-{gamma} responses [63 ]. Knowing this, along with the current finding that C274 also activates macaque B cells, we set out to ascertain what other cytokines and chemokines are induced by C274, which might be important in boosting B cell, DC, and other immune functions. Cell-free tissue-culture supernatants were collected from the differently activated PBMC versus B cell cultures, and the presence of a variety of factors was measured using the fluorescent-based bead assay.

Overnight exposure to C274 induced PBMCs and B cells (from infected and uninfected macaques) to release IL-3, IL-6, CXCL8, CCL3, and CCL2 (Fig. 5A and 5B ). C274-induced responses were two- to 30-fold above those detected in the control ODN-treated cells. CD40L also induced similar responses; however, the CCL2, IL-3, and IL-6 responses were lower than those induced by C274. In fact, IL-3 production was negligible after CD40L activation. AT-2 SIV stimulated solid IL-6, CXCL8, CCL2, and CCL3 secretion but low levels of IL-3 by cells from infected and uninfected animals. Although variable between donors, IFN-{alpha} release was detected in PBMCs and B cell-enriched cultures upon exposure to AT-2 SIV (407±183 and 534±396 pg/ml, respectively; mean±SEM from five animals). For PBMCs and purified B cells, C274 stimulated significantly larger amounts of IL-3 and IL-6 compared with CD40L (P<0.002). The IL-3 response induced by C274 was only significantly greater than that induced by AT-2 SIV in the PBMCs (P<0.002), and compared with AT-2 SIV, C274 induced significantly more IL-6 release from the B cells (P<0.04) but not the PBMCs. Compared with the CD40L-induced response, C274 also induced significantly more CCL2 release by the PBMCs (P<0.002). These responses were maintained or reduced when monitored after 7–13 days of culture (often coincident with elevated, nonspecific chemokine production in the longer term cultures; not shown). Although the Luminex assay used includes antibodies cross-reactive with macaque-soluble factors, no increased production of IL-1ß, IL-4, IL-5, IL-12 (p40), IFN-{gamma}, GM-CSF, TNF-{alpha}, or CCL5 was detected in these cultures. At least in some instances, this likely reflects the sensitivity of the reagents in the kit, which are human-specific but monkey-cross-reactive (since the completion of this study, new kits have become available, which have greater reactivity for macaque factors). For instance, we previously showed that C274 induces IL-12 production, but this was detected using the Biosource anti-monkey IL-12 enzyme-linked immunosorbent assay (ELISA) [63 ], which was not used herein. Further analysis with sensitive monkey-reactive reagents is needed to elucidate whether other factors are produced in response to C274.


Figure 5
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  		Figure 5. C274 induces cytokine and chemokine release by macaque B cells. Cell-free supernatants were collected from 24 h-cultured PBMCs or B cells, stimulated with the indicated reagents (vs. controls). The levels of cytokine and chemokine production were measured using the fluorescent bead assay. (A) The mean secretion of the factors (mean pg/ml±SEM) induced under each condition is shown for the PBMCs (solid symbols) and B cells (open symbols) of up to 10 SHIV-infected macaques (10 PBMCs, three B cells) and seven naïve macaques (seven PBMCs, three B cells). (B) The levels of stimulus-specific production of each factor by PBMCs (solid bars; 17 animals) and B cells (shaded bars; six animals) from all animals are summarized (responses induced by the respective controls have been subtracted from each stimulus).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
By virtue of their ability to activate B cells and DCs, the C-type ISS-ODNs (e.g., C274) represent a particularly promising avenue through which vaccine immunogenicity may be enhanced. Cellular and humoral responses could be increased by C-type ISS-ODNs as a result of boosting the antigen-presenting functions of DCs and B cells and through direct activation of B cells. Nonhuman primate studies represent a uniquely valuable means of evaluating anti-HIV vaccine strategies, and there is accumulating experience with the use of ISS-ODNs in nonhuman primates. CpG-B ISS-ODNs boost antibody responses against hepatitis B [31 , 38 , 72 ], anthrax [31 , 73 ], or HIV [74 ] vaccines in macaques and reduce lesion size and parasite load in Leishmania-challenged, vaccinated macaques [75 ]. CpG-B ISS-ODN effects were even apparent in SIV-infected animals [72 , 75 ]. CpG-C ISS-ODNs were recently shown to induce IFN-{alpha}-inducible genes as well as several cytokines in baboons [76 ]; however, there have been no reports about the use of CpG-C ISS-ODNs in macaques to date.

To commence research on the application of the more recently identified CpG-C ISS-ODNs in the macaque model, we first demonstrated that the CpG-C ISS-ODN C274 effectively stimulates macaque PDCs as well as eliciting bystander MDC activation and enhancing in vitro SIV-specific, IFN-{gamma} release [63 ]. Evaluating the activity of CpG-C ISS-ODNs on macaque B cells was an additional, critical step in assessing the potential use of this novel adjuvant approach in macaques as a prelude to incorporating it into vaccine studies.

This work provides the first direct proof that CpG-C ISS-ODN exposure effectively activates and expands macaque blood-derived B cells. B cell proliferation is important for the development and expansion of antigen-specific B cell responses [11 ]. Paralleling the observations made with human cells [37 ], C274 stimulated purified macaque B cells to proliferate in culture (Fig. 1) . Such expansion was evident for up to after 1 week of culture, suggesting a promising, persistent effect from a single dose of C274 on macaque B cell biology, which would encourage the maintenance of the activated population. Despite DC–B cell communication occurring in macaque PBMC cultures to favor B cell activation, the lack of CD38 expression by the C274-activated cells (even after 7–13 days of culture) suggests that the single C274 stimulus was insufficient to drive differentiation to the plasma cell stage (CD20–CD38+ cells). This is not surprising, as plasma cell differentiation reportedly requires B cell–PDC interactions in concert with antigenic stimulation (e.g., anti-Ig stimulation), and this is enhanced by CpG-C ISS-ODNs [61 ]. Concordant with this, ELISA analysis did not detect anti-SIV antibodies in the supernatants from cells of SHIV-infected animals, which had been exposed to the single C274 stimulus (not shown).

However, these C274-expanded B cell populations did exhibit elevated levels of costimulatory molecule expression (Figs. 2 3 4) . Compared with the standard B cell stimulus CD40L, C274 induced the highest levels of costimulatory molecule expression combined with the best B cell viability over time. This complements our recent observations made about the effects of C274 on macaque PDC biology [63 ] and directly parallels the findings for human DCs and B cells [37 ]. Not only are there direct effects of C274 on macaque PDCs and B cells, the IFN-{alpha} responses elicited by C274 combined with the bystander MDC activation would further foster increased B cell activity [51 52 53 54 55 , 57 , 58 , 61 ]. Increasing evidence also highlights how B cells can play an important role in driving DC activation [77 ]. Therefore, through such direct and indirect effects on macaque B cells and DCs along with increased DC–B cell interplay known to favor immunity, C274 embodies an especially promising candidate to advance HIV vaccine testing in macaques.

Although SIV/HIV can trigger innate and adaptive responses, immunodeficiency viruses subvert the immune system by eliciting mediocre or inappropriate responses, which allow and possibly even facilitate the establishment of persistent infection. This is particularly apparent at the level of DC biology (reviewed in refs. [78 , 79 ]). Coincident with this concept, the limited impact of AT-2 SIV on the CD40, CD80, and CD86 levels on B cells in the PBMC mixtures likely reflected little (if any) functional activation. There were also no detectable differences in the cell-surface immunophenotypes of B cells freshly isolated from the blood of healthy versus SHIV-infected animals, suggesting that SHIV infection had not significantly modified the characteristics of circulating B cells. Adding to this, purified macaque B cells exhibited no changes in surface immunophenotype in response to AT-2 SIV. The subtle AT-2 SIV-induced elevation of costimulatory molecule expression by B cells in the PBMC mixtures might be a result of indirect effects on the B cells within the mixed cultures directed by the strong IFN-{alpha} responses and (at least) partial DC activation mediated by AT-2 SIV [63 ], favoring communication between IFN-{alpha}-triggered DCs and B cells. However, this cannot be attributed solely to IFN-{alpha}, as IFN-{alpha} production was detected in some AT-2 SIV-exposed, B cell-enriched cultures. We cannot rule out the persistence of PDCs in the CD20+-selected populations. However, it is also possible that SIV-triggered responses by other leukocytes within the PBMCs contribute to the B cell activation, mediated by cell–cell contact and other soluble factors, which are diminished in the B cell-enriched fractions.

Regardless of AT-2 SIV not fully activating B cells, as assessed by up-regulation of costimulatory molecules, AT-2 SIV did induce innate IL-3, IL-6, and chemokine release by the PBMC and B cell cultures. In addition to virus-induced B cell responses [24 ], these might also reflect DC [80 ] and/or monocyte [81 ] responses (in the mixed populations) to further encourage communication between cells and actually favor the spread of infection in the absence of the effective antiviral immunity. AT-2 SIV did not interfere with the C274-induced phenotypic and functional changes in the B cells (not shown), further supporting the use of AT-2 SIV as a vaccine [82 ], the immunogenicity of which might be augmented by the type of C274-mediated activation of DCs [63 ] and B cells described here.

Combined with the DC-driven IFN-{alpha} and IL-12 release triggered in C274-exposed macaque cells [63 ], C274 reliably induced IL-3, IL-6, CXCL8, CCL3, and CCL2 production from the B cells of healthy and SHIV-infected animals (Fig. 5) , much like what has been reported with human cells (reviewed in ref. [30 ]). Together, these cytokines and chemokines would contribute to macaque B cell and DC activation as well as encouraging leukocyte migration to facilitate vital DC–B cell–T cell interactions involved in immunity [11 , 30 , 36 , 83 84 85 86 ].

IL-3 improves macaque PDC function [63 ], which combined with the IFN-{alpha}, would further promote effective DC–B cell interactions in the C274-stimulated cultures to drive even better B cell activation. Immunodeficiency virus-infected PDCs have been reported to exhibit enhanced viability [87 , 88 ] and the (slightly greater) AT-2 SIV-induced IL-3 response from the cells of SHIV-infected animals could explain the larger numbers of PDCs, which we detected in the peripheral blood of the SHIV-infected animals [63 ]. This might also contribute to the slightly better B cell viability (over time as well as relative to the control-cultured cells at the later time-points), exhibited by cells from SHIV-infected animals (Fig. 1D) . As earlier studies have suggested that HIV infection modifies B cell functions [12 13 14 15 16 17 18 19 ], the presence of more functional PDCs in the SHIV-infected animals [63 ] possibly helps counter any SHIV-induced B cell dysfunction, allowing comparable B cell responses to be observed herein.

Our recent in vivo studies in the monkey lend further support to potential application of C274 to boost vaccine efficacy [89 ]. Specifically, C274 was found to stimulate B cells and DCs within the lymphoid tissues and most importantly, to function in vivo after intranodal injection. Exposure to C274 in vivo elicited immediate responses and augmented the responsiveness of the cells to subsequent, additional stimuli in vitro. Therefore, by triggering potent innate and adaptive responses in B cells and DCs (as well as monocytes and natural killer cells not considered here), CpG-C ISS-ODNs, like C274, represent a promising approach to boost responses that may be helpful in preventing or containing SIV/HIV infection. Combined with our studies demonstrating activity of C274 in vivo in macaques [89 ], this work sets the stage for essential therapeutic and prophylactic anti-HIV vaccine studies, testing the capacity of C274 to improve mucosal and systemic immunity in macaques.


   ACKNOWLEDGEMENTS
 
This work was supported by the Elizabeth Glaser Pediatric AIDS Foundation, as well as by National Institutes of Health (NIH) Grants R21 AI060405, R01 AI040877, and DE016256 (to M. P.) and the TNPRC Base Grant RR00164. M. P. is an Elizabeth Glaser Scientist. This work was also funded in part with federal funds from the NCI, NIH, under Contract NO1-CO-12400. N. T. and J. K. contributed equally to this study.

Received February 11, 2005; revised August 16, 2005; accepted August 23, 2005.


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 ABSTRACT
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 DISCUSSION
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