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Immunology. Oct 2003; 110(2): 250–257.
PMCID: PMC1783041

Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cells


Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine–phosphate–guanosine (CpG)-DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG-DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG-ODN-induced innate immune responses, including natural-killer-like activity [non-major histocompatibility complex (MHC)-restricted cytotoxicity], interferon-α secretion and 2′-5′A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non-MHC-restricted killing was not associated with interferon-γ production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG-ODN-induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses.


The first indication that cytosine–phosphate–guanosine (CpG) -DNA possessed immunostimulatory effects was reported in 1893. A sustained clinical remission was observed in 45% of 849 patients, with biopsy-proven carcinomas, when treated with a bacterial lysate known as Coley's toxin.1,2 In 1984 Tokunaga and colleagues demonstrated that bacterial DNA may be the active component responsible for the immunostimulatory effects previously observed by Coley, as purified bacterial DNA was shown to induce tumour regression and could prevent metastasis in mice and guinea-pigs.3 Despite these promising results it was not until 1995 that Krieg and colleagues demonstrated that unmethylated CpG dinucleotides in a specific base pair sequence conferred immunostimulatory activity to bacterial DNA, and that synthetic oligodeoxynucleotide (ODN), containing these same sequences, also had immunostimulatory activity.4

There is now evidence in the literature that CpG-DNA treatment can protect mice against challenge by intracellular pathogens.511 Although the effector mechanisms that mediate this disease protection remain to be fully determined, the recognition of specific CpG motifs is known to involve Toll-like receptors (TLR) and TLR9 appears to be critical in this signalling pathway.1214 Studies using human- and mouse-derived cells have demonstrated that CpG immunostimulatory ODN can induce a broad range of innate immune responses. Macrophages, monocytes and dendritic cells, when directly stimulated by CpG-ODN, increase their expression of interleukin-1 (IL-1), IL-6, IL-12, tumour necrosis factor-α (TNF-α), interferon-α (IFN-α), IFN-β, B7-1 and B7-2 costimulatory molecules, and major histocompatibility complex (MHC) class II molecules.1521 Furthermore, cytokine production by these myeloid cells has been reported to activate natural killer (NK) cell activity, including IFN-γ production.22,23 B cells, which can also be directly activated by CpG-DNA, can increase their expression of IL-6, IL-10, MHC class II and the B7-1 and B7-2 costimulatory molecules, as well as increasing cellular proliferation and resistance to apoptosis.2430 It is hypothesized that such a broad range of cellular responses can contribute to enhanced disease protection and reflects an integrated activation of both the innate and adaptive immune systems. Although a broad range of cellular responses is observed following CpG stimulation, it is hypothesized that induction of IFN production and NK activity may contribute significantly to protection against viral infection.5

The antiviral effects that IFNs can exert are impressive. Upon secretion and binding to specific cell surface receptors, IFNs induce a complex pattern of physiological changes that include the up- and down-regulation of gene expression, which can significantly limit viral replication.31,32 One gene that is up-regulated following IFN stimulation encodes for 2′-5′-oligoadenylate (2′-5′A) synthetase.32 This enzyme is activated upon contact with double-stranded (ds) RNA, which is commonly produced during the life cycle of a range of viruses including single-stranded (ss) RNA, dsRNA, and dsDNA viruses.33 Interaction with dsRNA results in a conformational change in the structure of 2′-5′A synthetase which results in its capacity to polymerize ATP and other nucleotides in novel 2′,5′ linkages.34 These 2′-5′As proceed to activate RNase L which then cleaves ssRNA, thus hindering gene expression.3540 Thus, activation of 2′-5′A synthetase confers significant protection against viral infection. Type 1 IFNs (IFN-α and IFN-β) are also potent inducers of NK cytotoxicity; they up-regulate MHC class I expression on antigen-presenting cells and activate a variety of innate immune responses that can effectively limit viral infection.41,42

Two classes of ODN, namely class A and class B, with distinct structure, currently exist. Class A ODNs consist of phosphorothioate poly(G) sequences on both the 3′ and 5′ end of a phosphodiester core containing CpG motifs.43 This class of ODN is characterized by its ability to induce better NK cytotoxicity and higher IFN-α secretion in mice than class B ODNs, which have a complete phosphorothioate backbone. However, unlike class B ODNs, class A ODNs do not induce significant lymphocyte proliferative responses (LPR).44,45 The objective of the present investigation was to evaluate, in sheep, the immunostimulatory effects of a class A and class B CpG-ODN. Peripheral blood mononuclear cells (PBMC) were stimulated with CpG-ODNs and several immune responses were measured. These responses included LPR and innate immune responses, including IFN-α and IFN-γ expression, non-MHC-restricted cytotoxicity and 2′-5′A synthetase activity. These immune responses were analysed for each CpG-ODN to determine if the structure of CpG-ODNs significantly influenced the activation of the innate immune system.

Materials and methods


Ten 2–4-month-old Suffolk sheep of either sex were obtained from the Department of Animal and Poultry Science (University of Saskatchewan; Saskatoon, Sask., Canada). Animals were fed a ration of rolled barley and alfalfa hay. All experiments were carried out according to the guidelines in the Guide to the Care and Use of Experimental Animals, provided by the Canadian Council on Animal Care.


Oligodeoxynucleotides were purchased from OPERON (Alameda, CA) and the sequences of ODN used are shown in Table 1.

Table 1
Class A and B ODN sequences and backbone structure

Tissue culture conditions

PBMC were cultured in Aim-V medium (GibcoBRL, Burlington, Ont., Canada) supplemented with 2% fetal bovine serum (FBS, GibcoBRL), 50 μg/ml streptomycin sulphate, 10 μg/ml gentamicin sulphate, 2 mm l-glutamine (Sigma-Aldrich, St Louis, MO) 50 μm 2-mercaptoethanol and 10 μg/ml polymyxin B sulphate (Sigma-Aldrich) (culture medium). K562 cells were grown in 75 cm2 flasks (Nunc, Naperville, IL), in minimum essential medium (MEM; GibcoBRL) supplemented with 10% FBS (GibcoBRL), 2 mm l-glutamine (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich) and 100 μg/ml streptomycin sulphate (Sigma-Aldrich). All other tissue culture was performed in 96-well, round-bottom plates (Nunc) using culture medium. Cells were incubated at 37° in an atmosphere of 5% CO2 and 95% humidity. PBMC stimulation with concanavalin A (Con A; Sigma-Aldrich) was used as a positive control for lymphocyte proliferation assays and PBMC stimulation with phorbol 12-myristate 13-acetate (PMA; 100 nm) plus calcium ionophore A2387 (CI; 400 nm) (Sigma-Aldrich) was used as a positive control for the detection of IFN-γ secretion.

PBMC isolation

Blood was collected from the jugular vein in ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) solution to a final concentration of 0·2% EDTA. PBMC were isolated following the protocol described by Dudler and colleagues46 with the modification that a 54% Percoll™ (Pharmacia Biotech AB, Uppsala, Sweden) gradient was used. Purified PBMC were counted using a cell counter (Dual Diluter III, Coulter electronics Ltd, Luton, UK) and resuspended in the culture medium.

IFN-α and IFN-γ assay by enzyme-linked immunosorbent assay (ELISA)

Ovine IFN-α and IFN-γ were detected by a capture ELISA as described previously.47,48 Supernatants from CpG-stimulated cells, plated at 0·5 × 106 cells per well, were collected at 24 and 48 hr post stimulation. IFN concentrations were calculated using Bio-Rad Microplate Manager version 5.0.1 software (Bio-Rad Laboratories, Hercules, CA).

Lymphocyte proliferative responses (LPR)

PBMC were resuspended in culture medium at 3 × 105 cells per well and cultured in a final volume of 200 μl. Triplicate cultures were stimulated with either ODN (0·2, 1, 2, 5, or 10 μg/ml) or Con A (5 μg/ml). During the final 6 hr of a 72-hr incubation, cells were pulsed with 0·4 μCi [5-3H]uridine (Amersham Pharmacia, Piscataway, NJ) per well. [5-3H]Uridine was used to avoid the possibility of competitive labelling by cold thymidine, which may be heightened when stimulating cells with ODN containing a phosohodiester backbone.49 Cells were harvested using standard liquid scintillation protocols and uptake of 3H was assessed in a beta-counter (Topcount, Packard Instrument Company, Meriden, CT). The LPR were calculated as the mean counts per minute (c.p.m.) of triplicate cultures and expressed as a stimulation index (c.p.m. in the presence of stimulus/c.p.m. in the absence of stimulus).

2′-5′A synthetase assay

The activity of 2′-5′A synthetase in culture supernates was measured using a commercial radioimmunoassay kit according to the manufacturer's instructions (Eiken Chemical Company, Tokyo, Japan). Briefly, culture supernatants were added to a poly(I)poly(C) agarose suspension to adsorb and activate 2′-5′A synthetase. The activated enzyme converted available adenosine-5′-triphospahte (ATP) substrate to 2′-5′-oligoadenyl-5′-triphosphate. 125I-labelled 2′-5′A and a mixed suspension of rabbit anti-2′-5′A serum and goat anti-rabbit immunoglobulin G (IgG) serum were then added. The tubes were incubated at 37° for 1 hr. The free enzyme was separated by centrifugation and radioactivity of the bound enzyme in the precipitate was measured using a gamma-counter (Model 5500, Beckman Instruments Inc., Fullerton, CA). The binding rate with respect to the initially added 125I-labelled 2′-5′A was calculated, and the quantity of 2′-5′-oligoadenyl-5′-triphosphate produced by the 2′-5′A synthetase in the test sample was obtained from the standard curve derived from the samples of known 2′-5′A synthetase concentration provided with the kit.

Cytotoxicity assay

K562 cells, a known ovine NK-sensitive target cell50,51 were prepared by labelling 1 × 106 cells with 100 μCi Na251CrO4 (Amersham Pharmacia Biotech, Baie de Urfe, PQ, Canada) for 2 hr in 200 μl MEM/10% FBS. Target cells were washed three times in MEM/10% FBS and then resuspended in culture media and 1 × 104 cells were added to each well in a round-bottom, 96-well plate. All assays were performed in triplicate and six wells per plate were used to determine the mean spontaneous release (release of 51Cr from K562 cells in media alone) and total release (lysis of K562 cells with 5% Triton X-100). One million effector cells (PBMC) were added per well for an effector to target ratio of 100 : 1. PBMC and K562 cells were cocultured for 24 hr at 37°, either in medium alone, stimulated with 15 ng/ml of recombinant bovine IL-2 (bIL-2; Ciba Giegy, Basel, Switzerland), or incubated with 2 μg/ml of either 2216, 2216-GC, 2007 or 2007-GC ODN in a final volume of 200 μl. Supernatants were harvested using a supernatant collection system (Skatron, Sterling, VA) and c.p.m. were determined using a gamma-counter (Model 5500, Beckman Instruments). Spontaneous release was always less than 25% of total release. The percentage cytotoxicity was calculated using the formula [(experimental c.p.m.) − (spontaneous c.p.m.)]/[(total c.p.m.) − (spontaneous c.p.m.)] × 100.


Data were analysed using the statistical software program spss 10·0 for windows (Chicago, IL). Data that were not normally distributed were transformed by ranking. Means of the ranks were compared using Tukey's multiple comparison test. A Spearman two-tailed non-parametric correlation analysis was also used to assess the degree of correlation between IFN-α and 2′-5′A synthetase.


LPR induced by class A (2216) and class B (2007) CpG-ODNs

LPR have previously been used to screen the biological activity of CpG-ODNs with PBMC from many species including sheep.52 To assess the activity of ODN 2216 and 2007 we first determined if both ODNs induced LPR. Ovine PBMC were stimulated with a range of ODN concentrations (0·2, 1, 2, 5, or 10 μg/ml) and [3H]uridine incorporation was measured. ODN 2007, at a final concentration of 1 μg/ml induced maximum LPR (data not shown) but no significant LPR was detected with ODN 2216 (data not shown). Figure 1 demonstrates that at 1 μg/ml only ODN 2007 induced a significant LPR relative to the 2007-GC control ODN (P < 0·05). No significant differences were observed between ODN 2216 and either the media or 2216-GC control ODN (P > 0·05). Additionally, the LPR induced by the 2007-GC control ODN were significantly greater than either media or 2216-GC control ODN-stimulated cells (P ≤ 0·001). These data demonstrate that the class B ODN (2007) but not the class A ODN (2216) was a potent inducer of ovine PBMC LPR. Furthermore, the backbone chemistry may contribute marginally (24·8 ± 10·6%) to the mitogenic activity of the class B ODN.

Figure 1
Proliferative responses of ODN-stimulated ovine PBMC. PBMC were stimulated with either medium alone, or 1 μg/ml ODN or 5 μg/ml Con A. Data for individual animals are presented with the median value indicated by a horizontal bar for each ...

Induction of IFN-α and IFN-γ secretion by class A (2216) and class B (2007) CpG-ODNs

Interferons are potent activators of the innate immune system and class A and class B ODNs are reported to differ in their capacity to induce IFN secretion.45,53 In preliminary experiments, various concentrations of both class A (2216) and class B (2007) ODNs (0·3–10·0 μg/ml) were used to stimulate IFN secretion by ovine PBMC. A final concentration of 2 μg/ml was observed to induce maximum IFN-α secretion (data not shown). Therefore, this ODN concentration was used throughout subsequent experiments. Figure 2(a) demonstrates that ovine PBMC stimulation for 48 hr with either class A (2216) or class B (2007) CpG-ODN did not induce a significant increase in IFN-γ secretion relative to either medium alone or the respective GpC control ODNs (P > 0·05). PBMC supernatants harvested 24 hr after ODN stimulation were also negative for IFN-γ secretion (data not shown). A significant increase in IFN-γ secretion was, however, detected following stimulation with PMA/CI (P < 0·001). This control confirmed that high levels of IFN-γ could be produced by ovine PBMC and could be detected by ELISA. In contrast, Fig. 2(b) demonstrates that both the class A (2216) and class B (2007) CpG-ODN induced significantly increased levels of IFN-α secretion relative to the respective GpC control ODNs (P < 0·001 and p < 0·05, respectively). Furthermore, the class A CpG-ODN (2216) induced a significantly greater (P < 0·001) level of IFN-α secretion than did class B ODN (2007).

Figure 2
Analysis of IFN secretion by ovine PBMC following stimulation with ODN. Interferon levels in culture medium were determined by capture ELISA. (a) and (b) demonstrate IFN-γ and IFN-α secretion, respectively, at 48 hr following stimulation ...

The kinetics of IFN-α production was also analysed following PBMC stimulation with ODN 2216. Culture supernatants were collected at 3, 6, 12, 18, 24, 36 and 48 hr post-stimulation and IFN-α levels were measured by ELISA. Figure 3 demonstrates that ODN 2216 stimulation required at least 18 hr to induce a detectable level of IFN-α secretion and IFN-α reached peak levels at 36 hr post-stimulation. No further increase in IFN-α secretion was detected at 48 hr post-stimulation. This plateau in cytokine secretion suggested that the response to CpG-ODN was transient.

Figure 3
Temporal kinetics of IFN-α secretion following ovine PBMC stimulation with ODN 2216. PBMC were stimulated with 2 μg/ml ODN 2216 and culture supernatants were collected at 3, 6, 12, 18, 24, 36 and 48 hr post-stimulation. IFN-α secretion ...

In summary, these data indicate that neither class A nor B CpG-ODN induced a detectable level of IFN-γ secretion but both CpG-ODNs induced detectable levels of IFN-α secretion. There was, however, a significant difference in the level of IFN-α secretion induced by the class A ODN when compared to the class B ODN and this response was CpG specific.

Induction of 2′-5′A synthetase activity by class A (2216) and class B (2007) CpG-ODNs

Interferons are potent inducers of 2′-5′A synthetase and this enzyme is a known antiviral effector molecule.33,34 To determine if CpG-ODN stimulation of ovine PBMC was associated with 2′-5′A synthetase activity, culture supernatants collected at 48 hr for the analysis of IFN secretion were also used to assess 2′-5′A synthetase activity. Figure 4 demonstrates that both the class A and B CpG-ODN induced increased 2′-5′A synthetase activity relative to both media and respective GpC control ODNs (P < 0·05). Although both class A and B CpG-ODNs induced 2′-5′A synthetase activity, PBMC stimulated with ODN 2216 secreted significantly higher levels of enzyme than PBMC stimulated with ODN 2007 (P < 0·05).

Figure 4
Induction of 2′-5′A synthetase activity following CpG-ODN stimulation of ovine PBMC. Culture supernatants were collected 48 hr after stimulation with either medium alone, or 2 μg/ml ODN 2007, ODN 2216, or the respective GpC ODNs. ...

The higher level of IFN-α secretion and 2′-5′A synthetase activity following class A CpG-ODN (2216) stimulation was consistent with previous reports that IFN-α is a potent inducer of 2′-5′A synthetase.32 A Spearman two-tailed non-parametric correlation analysis was used to determine if there was a correlation between IFN-α secretion and 2′-5′A synthetase activity. The results of this analysis indicated that a significant correlation (r = 0·62) existed between IFN-α secretion and 2′-5′A expression (P < 0·01).

Induction of NK-like activity following stimulation of ovine PBMC with class A (2216) and class B (2007) CpG-ODNs

CpG-ODNs are potent activators of both human and murine NK cells.22,54 Similarly, stimulation of ovine PBMC with either class A (2216) or class B (2007) CpG-ODN resulted in increased cytolysis of the NK-sensitive K562 cells (Fig. 5). The non-MHC-restricted cytotoxicity was CpG-specific because both class A and B CpG-ODNs induced a significant increase in the percentage of specific 51Cr-release relative to respective GpC control ODNs (P ≤ 0·001). Furthermore, no significant difference in the NK-like cytotoxicity was observed following incubation of PBMC with either the class A or class B ODN (P > 0·05).

Figure 5
NK-like cytotoxicity following CpG-ODN stimulation of ovine PBMC. Data presented for individual animals (n = 10) are the mean values of triplicate cultures and the horizontal lines represent the median value for each treatment group. The effector (PBMC) ...


In addition to differing in structure, evidence is emerging that class A and B ODNs also have distinct biological activities. In this study we have established, for the first time, that a range of innate immune responses is induced in sheep PBMC upon stimulation with CpG-ODN. This direct comparison of a class A and B ODN demonstrated both qualitative and quantitative differences in these innate immune responses that were dependent upon the class of CpG-ODN used. Although both classes of ODN induced non-MHC-restricted NK-like activity, ODN 2007 was the only ODN able to induce proliferative responses and ODN 2216 induced significantly greater IFN-α secretion and 2′-5′A synthetase activity than ODN 2007. Interestingly, although both ODNs were able to induce NK-like activity, neither ODN induced detectable IFN-γ secretion, which has been reported for both human and mouse NK cells.20,5558 Finally, the present investigation with an outbred species revealed substantial individual animal variation for CpG-ODN-induced innate immune responses.

LPR have been the primary assay used to screen CpG-ODNs for biological activity.52,59 To assess the ability of class A and B CpG-ODNs to induce LPR, which is indicative of a broad non-specific immune activation, ovine PBMC were stimulated with ODN 2216 and 2007, respectively. The present results demonstrated that only class B CpG-ODN induced a significant LPR (P < 0·05). This observation is consistent with previous investigations that used PBMC from a variety of species to determine that only class B ODNs induced LPR.52,59 Furthermore, in the present investigations the amplitude of LPR induced by ODN 2007 and Con A, a potent mitogen, were similar. This comparison provides some perspective regarding the potency of ODN 2007 as a stimulator of lymphocyte activation. It is difficult however, to relate mitogenic activity directly to innate immunity. Immune protection against pathogens can only be inferred by a more detailed analysis of innate immune responses with known effector functions.

To characterize CpG-ODN activation of the ovine innate immune system, the secretion of the potent antiviral cytokine, IFN-α, was measured in PBMC culture supernatants. Consistent with previous reports in mice and humans21,60,61 sheep PBMC secreted significant levels of IFN-α in response to the class A ODN and much lower levels in response to the class B ODN. No DNA backbone effect was observed as neither ODN 2007-GC nor ODN 2216-GC induced detectable IFN-α secretion in ovine PBMC. Also consistent with previous observations22,43,62 the class A ODN (2216) induced significantly higher levels of IFN-α secretion than the class B ODN (2007). These observations support the conclusion that class A ODNs might be more effective than the class B ODNs for clinical applications which require antiviral activity. This conclusion was further supported by the observation that production of 2′-5′A synthetase, a potent antiviral effector molecule, was directly correlated with the level of IFN-α secretion. This 2′-5′A synthetase response was also CpG specific as the respective GpC-ODN did not induce detectable levels of 2′-5′A synthetase activity. Collectively, these observations support the conclusion that class A ODNs have the capacity to induce potent antiviral defences in sheep.

The ability of the class A (2216) and B (2007) ODNs to induce NK-like cytotoxicity was assessed as a more specific measure of innate immune activation. Results presented here demonstrate that both the class A and B CpG-ODNs induced significant levels of non-MHC-restricted NK-like cytotoxicity by sheep PBMC. Furthermore, the level of cytotoxicity induced by both the class A and B ODN was similar to that previously reported for ovine NK-like cells isolated from the endometrium.51 It has been reported that class A-type ODNs are more potent inducers of mouse and human NK-cell lytic activity22,63 but with sheep PBMC no statistically significant difference (P > 0·05) was observed for the NK-like cytotoxicity assayed following stimulation with the class A or B CpG-ODN. It might be argued that the use of alternative NK-sensitive target cells could reveal CpG-ODN class-specific differences in NK-cell activation. It was clearly apparent, however, that despite the induction of CpG-specific NK-like cytotoxicity, the ovine PBMC failed to secrete detectable levels of IFN-γ. These data are not consistent with previous reports that CpG-ODNs induce IFN-γ secretion by both mice and human NK cells.20,5558 Sheep NK cells have been identified by function but have not been phenotypically characterized.50,51 Therefore, it might be postulated that CpG-ODN-induced non-MHC-restricted killing was mediated by a PBMC subpopulation distinct from NK cells. The absence of IFN-γ secretion following CpG-ODN stimulation would then be explained by a failure to activate NK cells. Alternatively, CpG-ODN stimulation of ovine NK cells might be adequate to induce cytotoxic activity but not IFN-γ secretion. Ovine NK-like cytotoxicity can be induced by IFN-α.50 However in the present experiments, both class A and B ODN-stimulation of PBMC induced similar levels of NK-like cytotoxicity, but the class A ODN stimulated significantly higher levels of IFN-α secretion. Thus, a distinct pathway for CpG-ODN-induced activation of NK cells may be present in sheep.

In summary, this is the first demonstration that both class A and B CpG-ODN can induce a range of innate immune responses in sheep. These results clearly demonstrate that CpG-ODNs, differing in sequence and backbone chemistry, have distinct immunostimulatory properties and these properties are CpG specific. The fully phosphorothioate class B ODN (2007) induced strong LPR but significantly lower levels of IFN-α and 2′-5′A synthetase than the class A ODN (2216). Both class A and B ODN induced non-MHC-restricted NK-like cytotoxicity but we were unable to detect increased IFN-γ secretion. These observations support the conclusion that screening CpG-ODN for immune protection should include a variety of assays that reflect relevant innate immune defences.


We thank Don Wilson, Brock Evans, Carolyn McCormack, Jan Erickson and Sherry Tetland for the care and handling of animals. We are also grateful to Xenia P. Ioannou for her support with the statistical analysis used during this study. Financial support was provided by grants from QIAGEN Inc, National Science and Engineering Research Council (NSERC), Canadian Adaption and Rural Development (CARD), Alberta Agriculture Research Institute (AARI) and Health Services Utilization and Research Commission (HSURC). Lorne A. Babiuk holds the Canada Research Chair in Vaccinology.


calcium ionophore A2387
counts per minute
ethylenediamine tetraacetic acid
enzyme-linked immunosorbent assay
fetal bovine serum
lymphocyte proliferative responses
major histocompatibility complex
natural killer
peripheral blood mononuclear cells
phorbol myristate acetate
Toll-like receptor
tumour necrosis factor


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