Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable.
Pathogen associated molecular patterns (PAMPs) activate antigen presenting cells (APCs) via pattern recognition receptors (PRRs) and this process is required for the development of an efficient immune response against the pathogens. Bacterial DNA and synthetic oligonucleotides like CpG oligonucleotides [
Therefore, it would be very important to understand how T cells and B cells costimulated with non-CpG ODN contribute to the development of an adaptive immune response. Our aim was to investigate how non-CpG ODN modulates antibody production following cognate interaction of T cells with B cells presenting antigen. To this end we examined early and late activation events leading to isotype switching in B cells, a process that enables a more effective host defense against pathogens.
All the treatments of animals (mice) in this research followed the guidelines of the Institutional Animal Care and Ethics Committee at Eötvös Loránd University that operated in accordance with permissions 22.1/828/003/2007 issued by the Central Agricultural Office, Hungary, and all animal work was approved by the appropriate committee.
BALB/c mice were purchased from Charles River Laboratories; DO11.10 mice (on the BALB/c background) were derived from The Jackson Laboratory. Both strains were bred and maintained under specific pathogen free conditions in the animal unit of the Eötvös Loránd University. Mice were used at 6–18 wk of age. Spleen or lymph node cell suspensions were cultured in RPMI 1640 medium (GIBCO, Invitrogen, Carlsbad, CA, US) supplemented with 5% heat-inactivated FCS (GIBCO), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, US), 100 U/mL penicillin (Sigma-Aldrich), 100
All oligodeoxynucleotides (ODN) had phosphorothioate (PS) linkages between the nucleobases (marked with capital letters), except the one preceding the last two 3′ base (marked with lowercase letters). CpG (ODN 1668; TCCATGACGTTCCTGATGCt), Control (ODN 1720; TCCATGAGCTTCCTGATGCt), and Inhibitor (ODN 2088; TCCTGGCGGGGAAGt) were purchased from Sigma-Aldrich. The ovalbumin derived peptide (OVA) with the following sequence Biotin-KISQAVHAAHAEINEAGR was synthesized by the CASLO Laboratory ApS (Lyngby, Denmark).
Freshly isolated spleen or lymph node cells (pooled from the subiliac, popliteal, proper and accessory axillary, superficial parotid, mandibular, and sciatic lymph nodes) were plated onto 96-well plates in 2 × 105 cells/well density. Inhibitor, Control, and CpG ODNs were added in low (0.25
Fluorescently labeled mAbs obtained from eBioscience (San Diego, CA, US) were the following: anti-mouse CD45R-PerCP-Cy5.5 (clone: RA3-6B2), rat IgG2a-PerCP-Cy5.5 isotype control (clone: eBR2a); BD Biosciences (Franklin Lakes, New Jersey, US): anti-mouse CD4-PE (clone: (L3T4) (RM4-5)), rat anti-mouse I-A/I-E-PE (clone: M5/114.15.2), rat
16-pad nitrocellulose-covered glass slides (UniSart, Sartorius Stedim Biotech, Goettingen, Germany) were put into slide modules (ProPlate, Grace Bio-Labs, Bend, OR, USA) and rinsed with PBS for 5 min before use and coated overnight with 2.5
Statistical difference was calculated using two-tailed Wilcoxon signed rank test in case of flow cytometric data and two-tailed permutation test was applied for fluorescent ELISPOT results. The permutation test was performed as follows: Values from the two groups to be compared were randomly reassigned to two groups and the difference between the group means was calculated. Distribution of 5000 randomizations was drawn and the two-tailed
In order to extend earlier observations showing costimulation of T cells by non-CpG ODN [
Non-CpG ODN enhances CD69 expression in T and B cells engaging in cognate interaction. DO11.10 splenocytes were incubated with different combinations of ODNs and OVA; after 24 hours CD69 expression was measured on T and B cell populations by flow cytometry ((a)-(b)). Medians and interquartile ranges of 13 independent experiments are shown. Asterisks indicate significant difference from the group receiving no ODN at all, within the respective OVA treatment group. MFI: mean fluorescence intensity;
After 24 hours of incubation about 40% of T cells expressed the early activation marker CD69 in the presence of OVA (Figure
After receiving appropriate activation stimuli lymphocytes express various surface markers and start to differentiate and divide. To better understand the enhancing effects of non-CpG ODN exerted on T and B cells upon cognate interaction, we looked at two late activation events: expression of MHC class II on B cells and the synthesis of novel DNA as a preparation for cell division in both B and T cells.
Interestingly, the expression of MHCII did not reflect our observations with CD69. Non-CpG ODN triggered the increase of MHCII in the absence of OVA (both low and high concentrations of Control and high concentrations of Inhibitor) but had no effects in the presence of OVA (Figure
Effects of non-CpG ODN on late activation events. Spleen cells were incubated with the different combinations of ODNs and OVA as indicated. For MHCII expression measurements cells were harvested and stained for flow cytometry after 4 days (a). Medians and interquartile ranges of 9 independent experiments are shown. Asterisks indicate significant difference from the group receiving no ODN at all, within the respective OVA treatment group. MFI: mean fluorescence intensity;
B cells differentiate into antibody secreting plasmablasts and plasma cells when they receive proper stimuli. This stimulus may include T cell help, involving MHCII-TCR interactions along with the engagement of coreceptors but may also take place without T cell help. We examined the effects of non-CpG ODN on B cell differentiation into ASCs by measuring the number of cells producing antibodies of various isotypes. Using the very same experimental setup of coincubation of splenocytes with or without OVA and with the addition of ODN, after 4 days of culture the cells were transferred into wells coated with antibodies capturing light chains.
The number of both IgM and IgG-producing ASCs was higher in the presence of Inhibitor or Control ODNs, as well as in the presence of CpG ODN (Figures
Enhancement of ASC formation by non-CpG ODN. Cells were incubated with the different combinations of ODNs and OVA as indicated; then after 4 days the cultures were transferred onto nitrocellulose-covered slides coated with light chain capture antibodies. After 10 hours of incubation, slides were washed and IgM (a) and IgG (b) spots produced by ASCs were detected using labeled antibodies and a fluorescent scanner. A representative fluorescent spot experiment is shown in (c). Statistical significance was calculated using two-tailed permutation test. Treatment pairs with significant differences (
The same trends were observed in the case of IgG1- and IgG2a-producing cells, but differences were not statistically significant (a representative measurement is shown in Figure
To further confirm that non-CpG ODN synergize with T cell derived stimuli in enhancing antibody production and isotype switching in B cells, we repeated experiments using wild type (wt) mice. While some OVA specific T cells may be present in naïve wt mice, their numbers are expected to be so low as to be negligible (1 : 10 000 or lower). Indeed, the percentage of CD69 positive T cells was unaffected by the addition of OVA in wt mice (Figure
No synergism between OVA and ODN treatments when cognate T cells are not present. Wild type (wt) or TCR transgenic (tg) splenic cells were incubated with different combinations of ODN and OVA for 1 day ((a), (b)) or 4 days (c). After 1 day CD69 expression was measured on B and T cell populations by flow cytometry ((a), (b)). Cells from the 4-day cultures were washed and then transferred onto nitrocellulose-covered slides that were previously coated with light chain capture antibodies. After 10 h of incubation, slides were washed and spots indicating IgG producing ASCs were detected using fluorescently labeled antibodies and a fluorescent scanner (c). Results represent a single experiment.
Efficient vaccination presents two challenges to the immunologist: one is finding the appropriate antigen that will stimulate the ideal B and T lymphocyte clones for achieving pathogen recognition, neutralization, and removal; the other is the formulation and administration of the antigen, in a way that the optimal costimulation is provided for those specific lymphocyte clones. Adjuvants promote these latter events, where a delicate balance should be reached between overstimulation, causing unacceptable side effects, and understimulation, leading to poor vaccination response. Immunostimulatory oligodeoxynucleotides are potent adjuvants [
The exact mechanism by which different ODNs exert their costimulatory effect on T cells is not fully elucidated yet, as there is incongruity between reports regarding the dependence of ODN effects on the presence of TLR9 and MyD88 [
Our approach of examining non-CpG ODN with moderate adjuvant effects [
As expected based on previous observations, T cells were not activated when treated by different ODN alone, even when B cells were activated by CpG ODN treatment (Figure
ODNs that do not or only moderately activate B cells could be used in combination with antigen to achieve B cell activation, differentiation and isotype switching via cognate interaction with antigen specific T cells. From our experiments we cannot categorically conclude whether non-CpG ODN showed antigen presentation enhancing effects via B cells, T cells, or both. The non-CpG ODN dosage we utilized showed modest effects on early B cell activation in itself and enhanced MHCII expression and ASC formation. The contribution of this effect on B cells, therefore, should be taken into account. The experiments of Landrigan et al. clearly showed a costimulatory effect of non-CpG ODN on T cells [
Class switching in murine B cells follows a pattern that is the function of the number of cell divisions [
Although there is evidence concerning the requirement of CpG DNA-mediated PI-3 kinase activation via T cell-expressed MyD88 in a CD4+ T cell-dependent response
Taking into account that CpG ODN induce cell activation irrespective of the coadministration of antigen, we propose that nonstimulatory ODNs are promising candidates as possible vaccine adjuvants, because they costimulate only T cells responding to the administered antigen and in turn provide help for appropriate B cell clones presenting the antigen, a feature that could provide a more restricted and focused B cell activation and differentiation profile.
Antigen-presenting cell
Antibody-secreting cell
Cytosine guanine dinucleotide-containing unmethylated DNA motif (in this study: ODN 1668)
Control oligonucleotide (in this study: ODN 1720)
Incomplete Freund’s adjuvant
TLR9 antagonist oligonucleotide (in this study: ODN 2088)
Lymph node
Mean fluorescence intensity
Oligodeoxynucleotide
Ovalbumin peptide (in this study: Biotin-KISQAVHAAHAEINEAGR)
Phosphorothioate.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Árpád Mikesy for his invaluable assistance in animal care and Márta Pásztor and Gősi Sándorné (Gizike) for technical assistance. These studies were funded by the NKTH-OTKA K109683 grant to József Prechl. The European Union and the European Social Fund have provided financial support to the project under the Grant agreement no. TÁMOP 4.2.1./B-09/1/KMR-2010-0003. The financial support of the Hungarian Academy of Sciences is gratefully acknowledged. Krisztián Papp is supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The research was supported by the Hungarian Scientific Research Fund (OTKA-NK 104846).