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(Journal of Leukocyte Biology. 2002;71:813-820.)
© 2002 by Society for Leukocyte Biology

Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide

Section of Retroviral Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

Correspondence: Dennis M. Klinman, CBER/FDA, Bldg. 29A Rm. 3 D 10, 8800 Rockville Pike, Bethesda, MD 20892. E-mail: Klinman{at}CBER.FDA.GOV

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synthetic oligodeoxynucleotides (ODN) expressing "CpG motifs" show promise as immune adjuvants, antiallergens, anticancer, and immunoprotective agents. Two structurally distinct classes of CpG ODN have been identified that stimulate human PBMC. This work establishes that both types of ODN bind to and are internalized by the same individual B cells, NK cells, and monocytes. However, the intracellular localization of "D" and "K" ODN differs, as does their functional activity: "K" type ODN trigger monocytes and B cells to proliferate and secrete IL-6 and IgM, whereas "D" type ODN induce NK cells to produce IFN- and monocytes to differentiate into CD83⁺/CD86⁺ dendritic cells. In monocytes, these two types of ODN (which differ in backbone composition and CpG motif) cross-inhibit one another’s activity. Thus, different types of CpG ODN have distinct and in some cases incompatible effects on the same cells, a finding with important implications for the therapeutic use of these agents.

Key Words: CpG DNA • B cells • NK cells • monocytes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial DNA contains unmethylated "CpG motifs" that strongly activate the mammalian immune system [^{1 2 3 4 5} ]. Synthetic oligodeoxynucleotides (ODN) containing such CpG motifs stimulate B cells [² , ⁶ ], natural killer (NK) cells [⁷ , ⁸ ], and professional antigen-presenting cells (APCs) [^{9 10 11 12} ] to proliferate and/or secrete a variety of cytokines, chemokines, and immunoglobulins (Ig). Animal studies suggest that CpG ODN may be therapeutically useful as vaccine adjuvants, antiallergens, chemotherapeutic, and immunoprotective agents [^{13 14 15 16 17} ].

Previously, we demonstrated that two structurally distinct classes of CpG ODN are capable of activating human peripheral blood mononuclear cells (PBMC), a finding since confirmed by others [⁸ , ¹⁸ , ¹⁹ ]. "K" type phosphorothioate ODN expressing multiple TCGTT and/or TCGTA motifs stimulate human immune cells to proliferate and secrete interleukin (IL)-6 and IgM (referred to as CpG-B by other groups) [^{20 21 22 23} ]. "D" type ODN, which contain a phosphodiester purine/pyrimidine/CG/purine/pyrimidine motif, capped at each end by a phosphorothioate poly G tail, stimulate NK cells to produce interferon- (IFN-; referred to as CpG-A by other groups) [⁸ , ¹⁸ , ¹⁹ ]. Whether these differences in activity reflect variation in cellular binding, uptake, or signaling by "K" versus "D" ODN and whether these two types of ODN interact synergistically or competitively are unknown.

This work compares the ability of "K" and "D" ODN to activate B cells, NK cells, and monocytes. Results indicate that both types of ODN bind to and enter the same immune cells. However, their intracellular localization differs, as does their functional activity. Moreover, these two types of ODN cross-inhibit one another’s ability to activate human monocytes. Thus, ODN, differing in CpG motif and backbone composition, have distinct and in some cases incompatible effects on the same immune cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Oligonucleotides and antibodies
ODN were synthesized at the Center for Biologics Evaluation and Research (Bethesda, MD) core facility. Sequences of the CpG ODN used in this study are 5'-TCGAGCGTTCTC-3' (K23) and 5'-GgtgcatcgatgcaggggGG-3' (D35) [⁸ ]. The control for "K" ODN was 5'-TCAAGTGTTCTC-3' and for "D" ODN, 5'-GgtgcatctatgcaggggGG-3'. Bases shown in capital letters are phosphorothioate, and those in lower case are phosphodiester. CpG dinucleotides are underlined. All fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and cychrome-labeled monoclonal antibodies (mAb) were purchased from Pharmingen (San Jose, CA). All ODNs used in this study contained <0.1 U/mg endotoxin.

Cell cultures
PBMC from normal donors (provided by the NIH Department of Transfusion Medicine, Bethesda, MD) were isolated by Ficoll-Hypaque density-gradient centrifugation [⁸ ]. Countercurrent centrifugal elutriation was used to isolate monocytes that were >95% pure. Cells (0.5–4x10⁶/ml) were cultured in RPMI 1640 containing 5% fetal calf serum (FCS), 50 U/ml penicillin, 50 µg/ml streptomycin, 0.3 mg/mL L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 10^-5 M 2-mercaptoethanol. Cells were stimulated with ODN for 8–72 h with 1–3 µM ODN depending on the assay.

Analysis of cell proliferation
PBMC were cultured in complete medium plus 3 µM ODN for 72 h. To study B-cell proliferation, cells were loaded with 10 nM carboxy fluorescein succinimidyl ester energy (Molecular Probes, Eugene, OR) as described before [²⁴ ]. Proliferation of CD11c+ monocytes was monitored by adding 10 µM BrdU (Pharmingen) for the last 18 h of culture. Staining for BrdU was performed as recommended by the manufacturers.

Enzyme-linked immunosorbent assays (ELISAs)
Ninety-six-well microtiter plates (Millipore, Bedford, MA) were coated with anticytokine or anti-IgM Ab and blocked with phosphate-buffered saline (PBS)-5% bovine serum albumin [⁸ ]. The plates were incubated for 2 h with culture supernatants from PBMC (5x10⁵/ml) that had been stimulated for 8–24 h with ODN as described above. IL-6, IFN-, and IgM were detected colorimetrically using biotin-labeled antibodies followed by phosphatase-conjugated avidin and a phosphatase-specific colorimetric substrate [⁸ ]. The detection limit of the assays was 6 pg/ml IFN-, 20 pg/ml IL-6, and 10 ng/ml IgM. All assays were performed in triplicate.

Staining for cell-surface markers and intracellular cytokine
Cultured cells were washed in cold PBS, fixed, and stained with fluorescent-labeled anti-CD69 (24 h), anti-CD25 (72 h), anti-CD83 (72 h), or anti-CD86 (72 h). To detect intracytoplasmic cytokine, cells incubated with ODN plus 10 µg/ml Brefeldin A for 8 h were washed, fixed, permeabilized (as per the manufacturer’s instructions; Caltag, S. San Francisco, CA), and stained with 4 µg/ml PE-conjugated anti-IL-6 or 2 µg/ml PE-conjugated anti-IFN- (Pharmingen) plus various FITC and Cy-Chrome-labeled surface markers for 30 min at room temperature. Samples were washed and analyzed (20,000–40,000 events) on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) after gating on live cells with proper electronic compensation. The data were analyzed using CELLQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Analysis of cell-surface binding and internalization of ODN
PBMC (4x10⁶/ml) were incubated with biotinylated ODN (1–3 µM) for 10 min at 4°C (binding experiments) or 37°C for 1 h (uptake experiments). To detect internalized ODN, surface-bound ODN were blocked with 100 µg/ml "cold" streptavidin. After washing, these cells were permeabilized, fixed, and stained with PE-conjugated streptavidin (1 µg/ml) plus FITC or Cy-Chrome-conjugated cell-surface markers.

Confocal microscopy
Elutriated monocytes (4x10⁶/ml) were incubated with Cy-3 or FITC-labeled "K" and/or "D" ODN at 37°C for 1 h. The cells were washed and mounted using the Prolong antifade kit (Molecular Probes) Subcellular localization of Cy3 and FITC-labeled ODN was determined by confocal microscopy under 1000x magnification (LSM5 PASCAL; Carl Zeiss, Thornwood, NY).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Binding and internalization of CpG ODN
The ability of human PBMC to bind and internalize CpG ODN was examined using biotin-labeled K23 and D35 ODN. Both types of ODN bound rapidly to the surface of virtually all human monocytes at 4°C (Fig. 1 , upper panel). A significant fraction of B lymphocytes (20–45%) and NK cells (10–20%) also bound these ODN. Simultaneous staining with "K" and "D" ODN showed that the same cells were binding both types of ODN. In contrast, interaction with T cells barely exceeded background levels (Fig. 1) .

View larger version (20K): [in this window] [in a new window]   Figure 1. Binding and uptake of K23 and D35 ODN. Binding studies: Freshly isolated PBMC were incubated with 3 µM biotinylated ODN for 10 min at 4°C, washed, and counter-stained with FITC-avidin and Cy3-labeled phenotype-specific Ab. Uptake studies: PBMC were incubated with 3 µM biotinylated ODN for 60 min at 37°C. Surface-bound ODN was blocked with excess streptavidin, and internalized ODN was detected by FITC-avidin staining of permeabilized cells. Cells incubated with ODN at 4°C showed no internalization of ODN. Data represent the mean percent ± SEM of CD3+ T cells, CD16+ NK cells, CD19+ B cells, and CD14+ monocytes from six independent experiments.

The ratio of membrane bound:internalized ODN was compared. Based on differences in mean fluorescence intensity (MFI), we calculate that target cells internalized approximately half of the ODN that had bound to their cell surface (unpublished results). For all cell types, the absolute magnitude of "D" ODN uptake exceeded that of "K" ODN. For example, the amount of labeled "D" ODN that bound to and was taken up by monocytes exceeded that of equimolar "K" ODN by about twofold throughout the functional concentration range of these agents (P<0.001; Fig. 2A ). To achieve equivalent levels of binding and uptake required that "D" ODN be used at a fourfold lower concentration than "K" (e.g., 0.75 vs. 3.0 µM; Fig. 2 ).

View larger version (26K): [in this window] [in a new window]   Figure 2. (A) Internalization and localization of K23 versus D35 ODN. PBMC were incubated with increasing concentrations of biotinylated "K" () or "D" ODN (•). A tenfold excess of unlabeled "D" ODN did not inhibit the uptake of labeled "K" ODN () nor did unlabeled "K" ODN block the uptake of biotinylated "D" (). Note that 3 µM "K" and 0.75 µM "D" ODN yield equivalent levels of uptake. Results represent the mean fold increase in MFI over background ± SE from four independent experiments. (B) Subcellular distribution of K23 and D35 ODN in elutriated monocytes. Purified monocytes were incubated with 3 µM Cy-3-labeled "K" (red) and FITC-labeled "D" (green) ODN for 2 h at 37°C. The intracellular localization of these ODN was determined by confocal microscopy. Sections are at 1000x original magnification.

Differential effect of "K" versus "D" ODN on B-cell function
Whole PBMC were treated with optimal concentrations of K23 and D35 ODN. "K" ODN activated CD19⁺ B cells rapidly, reflected by a significant increase in the expression of the CD69 early activation marker and the CD25 late-activation marker (P<0.001; Table 1 and Fig. 3 ). "K" ODN also triggered a greater-than tenfold increase in B-cell proliferation (P<0.05), a greater-than tenfold increase in IgM production (P<01), and a fivefold increase in the number of B cells secreting IL-6 (P<0.001). The effect of K23 exceeded that of D35 (and of a control for the "K" type ODN of the same structure but lacking the critical CpG motif) by more than tenfold in each of these functional assays. However, "D" ODN were not entirely inactive, because they induced a modest increase in CD25 and CD69 expression by CD19⁺ B cells (Table 1 and Fig. 3 ).

View larger version (59K): [in this window] [in a new window]   Figure 3. B-cell activation by K23 versus D35 ODN. PBMC were incubated with 3 µM ODN for 8–72 h. The number of CD19+ B cells induced to up-regulate expression of CD25 and CD69, produce IL-6, and proliferate was monitored by cell-surface and intracytoplasmic staining (see Materials and Methods). Note that in this representative example, the stimulation induced by "K" ODN exceeded that of "D" ODN by every parameter measured.

View larger version (51K): [in this window] [in a new window]   Figure 4. NK cell activation by K23 versus D35 ODN. PBMC were incubated with 3 µM ODN for 24–72 h. The number of CD16+ NK cells induced to up-regulate expression of CD25 and CD69 and produce IFN was monitored by cell-surface and intracytoplasmic staining. Note that in this representative example, the stimulation induced by "D" ODN exceeded that of "K" ODN in every parameter measured.

View larger version (78K): [in this window] [in a new window]   Figure 5. Monocyte activation by K23 versus D35 ODN. Elutriated monocytes were incubated with 3 µM ODN for 8–72 h. The number of CD11c+ monocytes induced to up-regulate expression of CD25 and CD69 and proliferate was monitored by cell-surface and intracytoplasmic staining. Production of IL-6 by CD14+ monocytes was monitored by intracytoplasmic staining. Note that in this representative example, "K" ODN triggered monocyte proliferation and production of IL-6, and "D" ODN induced monocytes to mature into CD83+/CD86+ dendritic cells.

To clarify this situation, monocytes were treated simultaneously with D35 plus K23. At optimally stimulatory concentrations, these ODN did not cross-compete for uptake or binding (Fig. 2A) . Yet, when their function was analyzed, coadministration of "K" ODN reduced the ability of "D" ODN to trigger monocyte differentiation by 70% (P<0.001; Table 3 ). The inhibitory effect of "K" ODN on the activity of "D" ODN was sequence-specific and concentration-dependent, because control, non-CpG ODN did not interfere significantly with the activity of "D" ODN (unpublished results). Conversely, "D" ODN significantly reduced the ability of "K" ODN to induce monocytes to proliferate (P<0.05; Table 3 ). As above, the inhibitory effect of "D" on the activity of "K" ODN was sequence-specific and concentration-dependent.

A very different pattern emerged when B and NK cells were studied. In these cells, the coadministration of "D" with "K" ODN was not inhibitory. Rather, the ability of "K" ODN to stimulate B cells to proliferate and secrete IL-6 and IgM was unaffected by the presence of "D" ODN, and the ability of "D" ODN to stimulate NK cells to secrete IFN- was not reduced by inclusion of "K" ODN.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CpG ODN show promise as immune adjuvants and antiallergens and for the treatment of infectious disease and cancer [^{13 14 15} , ^{25 26 27 28 29} ]. Previously, we demonstrated that "K" and "D" ODN might support distinct therapeutic activities. The current work monitored the uptake, distribution, and functional characteristics of "D" versus "K" ODN in multiple cell types. "K" ODN uniquely stimulated B cells to proliferate and secrete, and "D" ODN uniquely activated NK cells to produce IFN-. The response of monocytes to these two types of ODN was particularly informative. "K" ODN trigger elutriated monocytes to proliferate and secrete IL-6, and "D" ODN stimulate them to differentiate into dendritic APCs. When mixed, "K" and "D" ODN cross-inhibited one another’s effects on monocytes.

The ability of CpG ODN to trigger immune cells to proliferate and secrete Ig and cytokines was documented first in mice [¹⁴ ]. Because of evolutionary divergence in CpG recognition between species, ODN that are highly active in rodents are poorly immunostimulatory in primates [⁸ , ²³ , ³⁰ ]. This prompted efforts to identify CpG ODN that stimulated human PBMC, culminating in the discovery of "K" and "D" type ODN. Optimally active "K" ODN are composed of multiple TCGxTT and/or TCGxTA motifs on a phosphorothioate backbone [⁷ , ⁸ , ²³ , ^{30 31 32} ]. In contrast, "D" ODN consist of a phosphodiester purine-pyrimidine-CpG-purine-pyrimidine hexamer flanked by self-complementary bases that form a stem-loop structure capped at the 3' end by a phosphorothioate poly G tail [⁸ ]. The ability of "K" ODN to stimulate monocytes and B cells is well-documented, and "K" ODN have entered phase I clinical trials [⁷ , ²³ , ^{30 31 32} ]. By comparison, much less is known of the functional activity of "D" ODN. The current work not only confirms that "D" ODN induce NK cells to secrete IFN- but demonstrates that they also trigger monocytes to mature into CD83⁺/CD86⁺ dendritic cells.

This work is the first to establish that these two different types of CpG ODN can block one another’s immunostimulatory activity. We find that "D" ODN were unable to trigger monocytes to differentiate into DC when "K" ODN were present and that the proliferation induced by "K" ODN was disrupted when "D" ODN were present (Table 3) . These observations suggest that "D" and "K" ODN trigger competing signaling pathways or that the activation induced by one type of ODN precludes monocytes from responding to the other. Consistent with the latter possibility, "K" ODN stimulate monocytes to proliferate, secrete IL-6, and differentiate into macrophages—effects known to inhibit the maturation of monocytes into DC [^{33 34 35} ].

Of interest, "D" and "K" ODN did not interfere with one another’s ability to activate B or NK cells. Both cell types bound and internalized these ODN, yet "K" ODN uniquely triggered B cells to proliferate and secrete, and "D" ODN uniquely stimulated NK cells to produce IFN-. Thus, it appears that competition does not occur when "D" and "K" ODN trigger convergent activation pathways.

We and others [³⁶ , ³⁷ ] have demonstrated that "K" ODN interact with Toll-like receptor 9 (TLR-9). Studies show that the cellular activation mediated by "K" ODN involves a signaling cascade in which the serine kinase interleukin-1 receptor-associated kinase interacts with the adaptor protein tumor necrosis receptor-associated factor 6, which in turn links to the mitogen-activated protein-3 kinase transforming growth factor ß activated kinase-1. TAK-1 induces the activation of the transcription factor nuclear factor-B (NF-B) as well as activated protein-1 transcription family members Jun and Fos, which contribute to the transcription of immune response genes [⁶ , ³⁸ , ³⁹ ]. Unlike "K" ODN, there is no evidence that "D" ODN interact with TLR-9. Rather, ongoing research suggests that the poly G tail of "D" ODN may interact with scavenger receptors on immune cells [⁴⁰ ], and studies in our lab indicate that "K" but not "D" ODN stimulate cells transfected with TLR-9 (unpublished results). Consistent with differences in recognition and subsequent uptake, "D" ODN do not compete with "K" ODN for binding or cell entry (Fig. 1) . Moreover, "D" and "K" ODN primarily occupy discrete locations within a single cell (although some colocalization is observed; Fig. 2 ).

K23 and D35 were used as representative "K" and "D" ODN in the current study. However, other "K" and "D" ODN were examined with similar results. As controls, ODN with the same structure as K23 and D35 but lacking the critical CpG motif were used. These controls demonstrate that CpG motifs were responsible for the immune activation observed. Moreover, when the poly G tail of D35 was replaced by a poly T tail, enhanced cellular uptake and immunostimulatory activity were abolished totally, indicating that the poly G component was indispensable for "D" ODN activity. Addition of a poly G tail to K23 altered the uptake and intracellular distribution of this ODN, as a result, decreasing the level of proliferation and IL-6 secretion elicited. Because some lots of FCS (and lipopolysaccharide contamination of any cell-culture reagent) can synergistically enhance the immune activation induced by CpG ODN, all materials used in these studies were prescreened and shown to be immunologically inert in our assays.

"D" and "K" motifs are present in biological products currently undergoing clinical testing (ranging from DNA vaccines to gene-therapy vectors). Preliminary studies suggest that the number and location of CpG motifs can influence the nature and magnitude of the host’s immune response to these products. In this context, ongoing studies in our lab suggest that "D" ODN are significantly better than "K" ODN as immune adjuvants in vivo. Knowledge of the specific cell types and functions triggered by these two types of ODN and of their competitive activities should facilitate the rational design of novel and effective therapeutic agents.

	ACKNOWLEDGEMENTS

 
This work was supported in part by Military Interdepartmental Purchase Request MM8926. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration at large.

Received November 26, 2001; revised January 11, 2002; accepted January 16, 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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