Incorporation of CpG into a Liposomal Vaccine Formulation Increases the Maturation of Antigen-Loaded Dendritic Cells and Monocytes To Improve Local and Systemic Immunity

Melanie R. Neeland, Martin J. Elhay, Jackie Nathanielsz, Els N. T. Meeusen and Michael J. de Veer
J Immunol April 15, 2014, 192 (8) 3666-3675; DOI: https://doi.org/10.4049/jimmunol.1303014

Abstract

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine immunity. Following vaccination, innate cell populations respond to immune stimuli, phagocytose and process Ag, and migrate from the injection site, via the afferent lymphatic vessels, into the local lymph node. In this study, the signals received in the periphery promote and sculpt the adaptive immune response. Effector lymphocytes then leave the lymph node via the efferent lymphatic vessel to perform their systemic function. We have directly cannulated the ovine lymphatic vessels to detail the in vivo innate and adaptive immune responses occurring in the local draining lymphatic network following vaccination with a liposome-based delivery system incorporating CpG. We show that CpG induces the rapid recruitment of neutrophils, enhances dendritic cell–associated Ag transport, and influences the maturation of innate cells entering the afferent lymph. This translated into an extended period of lymph node shutdown, the induction of IFN-γ–positive T cells, and enhanced production of Ag-specific Abs. Taken together, the results of this study quantify the real-time in vivo kinetics of the immune response in a large animal model after vaccination of a dose comparable to that administered to humans. This study details enhancement of numerous immune mechanisms that provide an explanation for the immunogenic function of CpG when employed as an adjuvant within vaccines.

The generation of a successful immune response to vaccination relies on appropriate activation of the innate immune system. Vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine-induced immunity (1). A major class of innate simulators under investigation to serve as adjuvants are the TLR ligands. These ligands bind to and stimulate TLRs, triggering a cascade of events that leads to the initiation of an immune response at the site of injection, ultimately resulting in the generation of an adaptive immune response in the local draining lymph node and the induction of immunological memory (2).

CpG is a synthetic oligonucleotide that signals predominantly through TLR9 and mimics the immunostimulatory activity of bacterial DNA (3). When used as an adjuvant within vaccine formulations, CpG typically induces a strong type 1, proinflammatory immune response, generating long-lasting cellular and humoral Ag-specific immunity (3–8). The addition of CpG to the licensed human anthrax vaccine induced Ag-specific Ab titers in mice that were significantly higher and persisted for longer than those induced by the vaccine alone (9). Enhanced Ab titers generated by CpG have also been observed in large animal models (10, 11), nonhuman primates (12), and human clinical trials (13–15). Complexing CpG into liposomes has been shown to further augment the stimulatory capacity of the adjuvant and increase cell-mediated immunity (16–19). In addition to acting as a delivery vehicle, liposomes protect the components from degradation and facilitate Ag uptake by APCs, leading to enhanced Ag recognition and vaccine-specific immune responses (20–23). Several liposome-based vaccines are currently in clinical trials; however, the cellular targets of these formulations and mechanisms of immune induction are yet to be defined (24).

The early immune response to vaccination is characterized by activation of cells present at the injection site and their subsequent migration to the local lymph node via the afferent lymphatics. The immunological signals received by innate cells at the periphery are conveyed to lymphocytes in the local lymph node, leading to the generation of an adaptive immune response in which Ag-specific lymphocytes emigrate via the efferent lymphatics to perform their tailored effector function (25). Examination of the afferent and efferent lymphatic compartments during the innate and adaptive phases of an immune response allows investigation of the biological pathways at the injection site and within the draining lymph node responsible for vaccine immunity. We have directly cannulated these lymphatic vessels to explore the in vivo function of CpG when incorporated into a liposome vaccine formulation. We show that CpG significantly enhances multiple cellular responses at the site of injection that lead to increased Ag-specific immunity in the efferent lymph and peripheral blood compartments.

Materials and Methods

Immunizations

All injections consisted of 500 μl sterile PBS mixtures of liposomes (12% soy bean lecithin/cholesterol [9:1] obtained from Lipoid, Ludwigshafen, Germany) containing 400 μg OVA with or without the addition of 50 μg CpG, kindly provided by Zoetis (Parkville, VIC, Australia). The liposome preparations were injected s.c. in the area drained by the prefemoral lymph node at 500 μl/injection using a 25-gauge needle. For Ag labeling, 5 mg OVA was resuspended at 1 mg/ml in PBS, and a 15-M excess of A647 succinimidyl ester (Invitrogen) resuspended in DMSO at 5 mg/ml was added and incubated at room temperature for 1 h. Unconjugated fluorophore was removed from the solution using an Amicon Ultracel 3K centrifugal filter (Millipore) by centrifuging at 2000 × g for 30 min and washed with PBS five times.

Vaccination strategy and blood collection

One-year-old merino sheep were randomly assigned into two vaccination groups: liposome alone (n = 5) and liposome with the addition of CpG (n = 5). There were two cohorts of 10 animals, one for afferent lymphatic cannulation and the other for efferent lymphatic cannulation. Each animal received three injections of the respective formulation at both prefemoral drainage areas. Secondary injections were performed 4 wk following primary injections, and tertiary injections were performed 3 mo following secondary injections. Blood was collected prior to each injection, 7 and 10 d after secondary vaccination, and 3, 5, 7, 10, and 30 d after tertiary vaccination.

Pseudoafferent and efferent lymphatic cannulation surgery

Ovine prefemoral pseudoafferent and efferent lymphatic cannulations were performed as previously described (26, 27). For pseudoafferent lymphatic cannulation, the prefemoral lymph nodes of merino sheep were removed at 1 y of age, and at least 2 mo was allowed for reanastomosis of the afferent lymphatics with the larger efferent lymphatic vessel. A secondary surgery was performed to insert a 0.96 × 0.58-mm heparin-coated polyvinyl chloride cannula into the pseudoafferent lymphatic vessel. For efferent lymphatic cannulation, a 0.96 × 0.58-mm heparin-coated polyvinyl chloride cannula was inserted into the efferent lymphatic vessel of the prefemoral lymph node. For both models, the cannulae were exteriorized and placed in a sterile collection flask attached to the side of the sheep. Injections were given at least 7 d post–lymphatic surgery to allow for surgical recovery. Handling of animals and experimental procedures were approved by the Monash University Animal Ethics Committee in accordance with the relevant licensing agreement.

Lymph collection and flow cytometry analysis

Afferent and efferent lymph was collected in sterile 50-ml tubes containing 0.05 IU heparin (Pfizer) and 20 μl 100× cell-culture penicillin-streptomycin (Invitrogen) for a period of 1 h. Afferent lymph was collected prior to primary injection and 4, 24, 48, and 72 h postinjection, generating the innate vaccination time course. Efferent lymph was collected prior to secondary injection, 4 h, and 1, 2, 3, 6, 8, 10, and 13 d postinjection, generating the adaptive vaccination time course. Immediately after collection, afferent/efferent lymph cells and supernatant were separated by centrifugation at 400 × g at 4°C. The cell pellet was washed with 10 ml ice-cold PBS + 2 mM EDTA and resuspended in 1 ml 0.093 mM ammonium chloride with 0.1 M Tris (pH 7.2) to lyse RBCs. After 3 min incubation on ice, 9 ml ice-cold FACS buffer (5% horse serum and 2 mM EDTA in PBS) was added and the cells centrifuged, washed with FACS buffer, and used for flow cytometry analysis. The surface-marker Abs used were anti–MHC class II (MHC II)–Pacific Blue (clone 49.1; locally produced), anti-CD14-A700 (AbD Serotec), anti-CD172a (SIRPα) (clone DH59B; Veterinary Medical Research & Development, Pullman, WA), anti-CD80 (AbD Serotec), anti-CD86 (AbD Serotec), anti–CD4-FITC (clone 44.38; AbD Serotec), anti–CD8-PE (clone 38.65; AbD Serotec), anti–γδ TCR-A647 and -FITC (clone 86D; locally produced), anti-CD45R (clone 20.96; locally produced), and anti-mouse IgG1 coupled to PE (Caltag Laboratories). All samples for flow cytometry were prepared by resuspending 3 × 106 efferent lymph cells in 25 μl Fc-block (2% BSA, 2 mM EDTA, 0.05% azide, and 5% sheep serum in PBS) and then adding 25 μl surface-marker Ab mixes. For intracellular cytokine staining, efferent lymph cells were resuspended in 150 μl 1% paraformaldehyde for 20 min on ice, centrifuged at 400 × g, and washed twice in 500 μl saponin buffer (1% horse serum, 0.05% azide, and 0.1% saponin in PBS). The intracellular Ab mixes were then added (anti–IFN-γ–A647 and anti–IL-4–FITC; AbD Serotec). All samples for flow cytometry were performed on an LSRII machine (BD Biosciences) and analyzed using the FlowJo software (Tree Star). For all flow cytometry gating strategies, see Supplemental Figs. 1 and 2.

ELISA for the identification of OVA-specific Abs

An indirect ELISA was used to measure relative OVA-specific Ab levels in each efferent lymph and serum sample. Briefly, 100 μl OVA (4 μg/ml in PBS) was added to a flat-bottom 96-well plate (Immunosorb; Nunc) and left to bind overnight at 4°C. The plate was washed with 0.05% PBS plus Tween-20, blocked with 1% horse serum in PBS at 200 μl/well for 1 h at room temperature, and sera samples (diluted 1/500) and efferent lymph samples (diluted 1/2000) added at 100 μl/well for 1 h. The wells were washed before 100 μl rabbit anti-sheep Igs/HRP Ab (P0163; DakoCytomation) (diluted 1/1000 in PBS) was added and allowed to incubate for 1 h. Reactions were developed using TMB substrate (Invitrogen) at 100 μl/well, stopped by the addition of 50 μl/well 2 M H2SO4, and read at 450 nm on a spectrophotometer (Spectramax Plus; Molecular Devices).

Statistical analysis

Results are presented as mean ± SEM, with n = 5 in each treatment group. Differences between groups and within each group were calculated with a two-way repeated-measures ANOVA using a Sidak posttest to correct for multiple comparisons. Significance was determined as the confidence interval being >95% (p < 0.05). The statistical software used was GraphPad Prism, version 6.01 (GraphPad).

Results

Addition of CpG to liposomal Ag formulations alters the kinetics of innate cellular recruitment into afferent lymph

Although no statistically significant change in afferent lymph volume and total cell number was observed in either group after primary vaccination (Fig. 1A), the addition of CpG to liposomal OVA formulations elicited a dramatic and transient increase in the recruitment of neutrophils in afferent lymph, increasing 11-fold 4 h after injection and returning to baseline by 24 h (Fig. 1B). Although not significant, CpG induced a unique trend of monocyte recruitment, with a primary peak at 4 h and decline to baseline at 24 h, followed by a secondary peak at 48–72 h (Fig. 1B). This trend is distinct from that observed when other TLR ligands have been used as adjuvants in this model (28). Although total dendritic cell (DC) percentages were relatively consistent over the time course in both groups, CpG increased the percentage of the SIRPαhigh DC subset at 48 and 72 h after injection, increasing to 65% of DCs at 72 h (Fig. 1C). Lymphocyte populations in afferent lymph were also investigated in which CD4 T cells, CD8 T cells, γδ T cells, and B cells were all identified; however, these did not change significantly over time in either group (data not shown).

FIGURE 1.
FIGURE 1.

Vaccination induces temporal changes in the volume and cellular composition of afferent lymph. Changes over time in total volume of afferent lymph and total cell number in afferent lymph (A), the percentage of lymphocytes, neutrophils, monocytes, and DCs (B), and the percentage of DC subsets SIRPαhigh and SIRPαlow (C). Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicates differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001.

Liposomal CpG increases the total number of Ag-carrying cells in afferent lymph but not the capacity of individual cells to ingest Ag

To investigate the cell types within afferent lymph that transport Ag to the lymph node, the liposome injections contained A647-labeled OVA. CpG induced the greatest number of A647-OVA+ neutrophils at 4 h (Fig. 2C) and A647-OVA+ DC at 24 h (Fig. 2A) when compared with liposome alone. Both injections induced A647-OVA uptake by monocytes, with CpG inducing a two-wave trend of A647-OVA+ monocytes, first at 4 h, followed by a secondary peak between 48 and 72 h (Fig. 2B), a similar trend to that observed in the total CpG monocytes in Fig. 1B. When investigating the mean fluorescence intensity (MFI) of A647-OVA+ cells, it was observed that although CpG increased the number of DCs and neutrophils carrying Ag, this was not associated with an increase in the amount of A647-OVA fluorescence within individual cells above the liposome-alone control (Fig. 2).

FIGURE 2.
FIGURE 2.

Vaccination induces temporal changes in the number of Ag-positive cell populations and the level of Ag uptake. The number of A647-OVA+ DCs (A), A647-OVA+ monocytes (B), and A647-OVA+ neutrophils (C) per 10,000 cells in afferent lymph and MFI of these A647-OVA+ cells over time. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicates differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

SIRPαhigh DCs traffic more Ag into afferent lymph than SIRPαlow DCs

Although SIRPαhigh DCs are only 2-fold more abundant in afferent lymph (Fig. 1C), there were between 5- and 10-fold more A647-OVA+ SIRPαhigh DCs when compared with A647-OVA+ SIRPαlow DCs in both groups (Fig. 3B). The number of A647-OVA+ SIRPαhigh DCs was highest 4 h postinjection of the liposome-alone formulation; however, the amount of fluorescence within each SIRPαhigh DC peaked 24 h after injection (Fig. 3A). Conversely, the addition of CpG to the liposome formulation caused the number of A647-OVA+ SIRPαhigh DCs to peak at 24 h and the level of A647-OVA fluorescence to peak between 4 and 24 h after injection (Fig. 3A). CpG induced a 2-fold increase in the A647-OVA fluorescence of SIRPαhigh DCs at 4 h when compared with liposome alone (Fig. 3A). CpG did not increase the number of A647-OVA+ SIRPαlow DCs above the liposome alone injection; however, CpG increased the amount of A647-OVA fluorescence within individual SIRPαlow DCs at 24 h (Fig. 3B).

FIGURE 3.
FIGURE 3.

Vaccination induces temporal changes in the number of Ag-positive DC subsets and the level of Ag uptake. The number of A647-OVA+ SIRPαhigh (A) and A647-OVA+ SIRPαlow DCs (B) per 10,000 DCs in afferent lymph and the change in MFI of these A647-OVA+ DCs over time. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicate differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001.

Liposomal CpG induces the maturation of Ag-carrying monocytes and DCs in afferent lymph 72 h postvaccination

Injection with CpG induced a significant increase in the MFI of MHC II on A647-OVA+ monocytes, with a mean fold change of 2.0 at 72 h (Fig. 4A). The MFI of MHC II did not increase on any other A647-OVA+ cell population in afferent lymph (data not shown). All afferent lymph DCs expressed both costimulatory molecules CD80 and CD86 prior to injection. Significant increases in the MFI of CD80/CD86 on A647-OVA+ DCs were observed after injection with liposomal CpG, with a mean fold change of 2.32 and 6.33, respectively, at 72 h (Fig. 4B, 4C). This maturation effect was unique to A647-OVA+ monocytes and DCs and was not observed in any A647-OVA− cell population in either group (data not shown). Afferent lymph B cells were also shown to express CD80; however, expression did not change significantly over time or between groups (data not shown).

FIGURE 4.
FIGURE 4.

Vaccination induces temporal changes in the MHC II expression on Ag-positive monocytes and CD80/CD86 expression on Ag-positive DCs. Fold change over time in the MFI of MHC II on A647-OVA+ monocytes (A), CD80 on A647-OVA+ DCs (B), CD86 on A647-OVA+ DCs (C) and the respective flow cytometry plots of the MFI of A647-OVA+ cells compared with A647-OVA− cells. All flow cytometry plots are of one representative animal from each treatment group. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicates differences between groups, and # indicates differences from 4 h within each group. *p < 0.05, **p < 0.01, #p < 0.05.

The adaptive response in efferent lymph: CpG extends lymph node cell shutdown and increases lymphocyte traffic

Secondary vaccination with the liposomal formulations induced temporal changes in the total volume and number of cells within efferent lymph flowing from the local lymph node. Both injections caused a marked reduction in cell output, but not volume, 4 h after injection, reducing from ∼5 × 107 cells/h prior to injection to 1 × 107 cells/h 4 h after injection (Fig. 5A). This phenomenon is a process known as lymph node cell shutdown (29). CpG extended the duration of lymph node cell shutdown by a further 24 h, with cell numbers remaining at ∼1 × 107 cells/h. This was followed by a dramatic and unique increase in the number of cells and lymph volume leaving the lymph node 2 d postinjection (Fig. 5A). All lymphocyte populations were equally reduced during the period of lymph node cell shutdown induced by both injections (Fig. 5B). The increase in cell number 2 d after injection with CpG was due to an increase in the number of conventional α-β CD4 and CD8 T cells (Fig. 5B). Interestingly, CpG also increased the number of CD8 T cells expressing the γδ TCR at 2 d postinjection (Fig. 5B).

FIGURE 5.
FIGURE 5.

Vaccination induces temporal changes in the volume and cellular composition of efferent lymph. (A) Changes in total volume and total cell number of efferent lymph over time. (B) Changes in the cell number per hour of CD4, CD8, γδ, and γδ CD8 T cells and B cells in efferent lymph over time. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups, * indicates differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, ###p < 0.001.

Vaccination with liposomal CpG increases IFN-γ production by CD8 T cells in efferent lymph

Prior to secondary injection of both liposomal formulations, there were a greater percentage of IFN-γ+ lymphocytes circulating in efferent lymph when compared with IL-4+ lymphocytes, 0.5 and 0.09%, respectively (Fig. 6A). Following injection, the percentage of IFN-γ+ CD4 and CD8 T cells remained higher than IL-4+ cells, whereas equal percentages of γδ T cells were observed with both cytokines (Fig. 6B–D). Injection of CpG induced a significant 3-fold increase in the number of IFN-γ+ lymphocytes 3 d after injection (Fig. 6A). This effect was due to an increase in the percentage of both IFN-γ+ CD8 T cells, increasing from 1.6% prior to injection to 3.6% 3 d after injection, and IFN-γ+ CD4 T cells, increasing from 0.5% prior to injection to 1.3% 3 d after injection (Fig. 6B, 6C). Additionally, there were significantly more IFN-γ+ CD8 T cells at both 2 and 3 d after injection with CpG when compared with liposome alone (Fig. 6B). CpG did not significantly increase the percentage of γδ T cells producing IFN-γ or IL-4.

FIGURE 6.
FIGURE 6.

Vaccination induces temporal changes in IFN-γ and IL-4 production by lymphocyte populations in efferent lymph. IFN-γ– and IL-4–positive total lymphocytes (A) and CD8 (B), CD4 (C), and γδ T cells (D) in efferent lymph over time. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicates differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Liposomal CpG increases Ag-specific Abs in efferent lymph and sera following vaccination

OVA-specific Ab levels were measured in the efferent lymph and sera of vaccinated sheep. In the efferent lymph, secondary vaccination with CpG induced greater OVA-specific Ab between 3 and 13 d after vaccination when compared with liposome alone (Fig. 7A). In fact, mean end point Ab titers from efferent lymph were ∼35-fold greater at day 5 in the CpG group (Fig. 7A). This effect was also observed in the sera, where secondary vaccination with CpG induced greater OVA-specific Ab 7 and 10 d postvaccination when compared with liposome alone (Fig. 7B). Tertiary vaccination with both liposomal formulations further increased OVA-specific Ab levels in the sera (Fig. 7C). CpG induced greater Ag-specific Ab levels than liposome alone from day 6 that continued to remain elevated for 30 d after vaccination (Fig. 7C). Additionally, Ag-specific Abs were vastly more concentrated in the efferent lymph than the sera, highlighting efferent lymphatic cannulation as an ideal model to assess both cellular and humoral immunity induced by vaccination.

FIGURE 7.
FIGURE 7.

Vaccination increases Ag-specific Ab concentration in efferent lymph and sera. (A) OVA-specific Ab concentration in efferent lymph after secondary injection expressed as OD (450 nm) and mean end point titer at baseline (−2 h) and at different times after injection. OVA-specific Ab concentration in sera after secondary (B) or tertiary (C) injection expressed as OD (450 nm) and mean end point titer prior to primary injection (naive), prior to secondary or tertiary injection (−2 h), and at different days after injection. Data expressed as mean ± SEM of five individually analyzed animals at each time point in both treatment groups. * indicates differences between groups, and # indicates differences from baseline (−2 h) within each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

Discussion

The immunogenicity of liposomal vaccine formulations can be improved by the addition of immunostimulatory components (24, 30). Several recent studies have demonstrated that incorporating innate immune agonists into liposomes improves both cell-mediated and humoral Ag-specific immunity in mice (18, 19). However, little is known about the effect of liposomal adjuvant formulations on migratory cell populations obtained in vivo from nonmanipulated tissues. By directly cannulating ovine lymphatic vessels, we were able to intercept and characterize the cells migrating from the skin and lymph nodes into the draining lymphatic network.

The majority of inflammatory stimuli, including sterile cell damage, microorganisms, and vaccine adjuvants, induce an early inflammatory environment at the site of injection, characterized by an influx of neutrophils from the blood (31). Typically, the more potent the stimuli, the longer this inflammatory environment persists and the stronger the ensuing immune response. This is desirable when clearing a persistent infection; however, a mild inflammatory response with a strong immune outcome is preferable for human vaccination, in which excess inflammation can lead to enhanced reactogenicity (32). Our studies have shown that injection with most vaccine adjuvants and even sterile saline results in the rapid recruitment of neutrophils and monocytes within afferent lymph following injection (26, 28, 33). Consistent with this, injection of sterile liposomes alone or containing CpG induced neutrophils and monocytes to rapidly enter the afferent lymph within 4 h. Interestingly, the addition of CpG, a bacterial pathogen-associated molecular pattern, increased the total number of neutrophils leaving the injection site but did not significantly prolong the duration of a nonspecific neutrophil-associated inflammatory response at the site of injection.

Consistent with previous studies using this model (26, 28), the number and percentage of total DCs in afferent lymph is remarkably unaffected by vaccine adjuvants, including liposomes and CpG. This is in contrast to some murine studies, in which DCs accumulate within the lymph node following injection with CpG (34). This phenomenon may be the result of DC recruitment from the blood into the lymph node or increased damage at the site of injection from relatively higher vaccine doses that may cause aberrant migration. However, when we calculated the relative contribution of Ag transport by each afferent lymph cell type, DCs were the major cell type responsible, and this was significantly enhanced by CpG 24 h after injection.

Several innate immune agonists have been shown to activate DC maturation in vitro, characterized by upregulation of MHC II and the costimulatory molecules CD80 and CD86 (35–38). There are limited studies that show migratory DCs, which display a highly mature phenotype, can further mature in response to inflammatory stimuli (39). In the current study, CpG induced an increase in expression of CD80 and CD86 on Ag-positive migratory DCs 72 h after vaccination. No increase in MHC II expression on afferent lymph DCs was observed throughout the time course. CpG has also been shown to induce the maturation of naive human blood monocytes into functionally mature DCs over 2–4 d in vitro, demonstrated by an increase in MHC II expression (40, 41). From our data, it appears that this is occurring in vivo within the peripheral tissues prior to emigration to the lymph node, but only with monocytes that have taken up the Ag, presumably in concert with CpG within the liposomes. Of the vaccine adjuvants we have tested within ovine lymphatics (26, 28, 33), CpG uniquely triggers Ag-carrying monocytes and DCs to increase their T cell–activating machinery at the injection site before migrating to the local lymph node. Taken together with increased DC Ag uptake over the vaccination time course, these results may provide an explanation for the increased Ag-specific adaptive immunity induced by CpG (18, 19, 42).

Although CpG increased the number of afferent lymph neutrophils and monocytes carrying Ag, this was generally not associated with an increase in the amount of fluorescence within each cell above the liposome-alone control. This suggests that CpG does not increase the phagocytic ability of these cell types, but rather induces greater recruitment of these cells to the injection site where Ag is present. Interestingly, monocytes and DCs contained comparable levels of Ag fluorescence, ∼2-fold greater than the mean fluorescence of neutrophils across the time course. A significant increase in the amount of Ag fluorescence within each cell was observed in both the SIRPαhigh and SIRPαlow DC subsets after injection with CpG. A recent comparative genomics study revealed that the ovine afferent lymph SIRPαhigh DCs are genetically and functionally equivalent to the mouse CD11b+ DCs and human blood DC Ag 1+ DC, whereas the SIRPαlow DCs represent the mouse CD8α+ DCs and human blood DC Ag 3+ DCs (43). Consistent with other studies (33, 43–45), the SIRPαhigh DC subset transported the majority of DC-associated Ag after injection with both formulations. The fact that these cells have been shown to be more efficient at CD4 T cell activation via classical MHC II presentation (46) and the observation that CpG increased the ability of these cells to take up Ag suggest that this may lead to increased CD4 T cell stimulation in the local lymph node.

Recent studies have shown that a subset of the afferent lymph SIRPαlow DCs are efficient at activating CD8 T cells via the cross-presentation pathway (44). Similarly, studies in mice have also demonstrated that skin-derived DC populations are able to cross present viral Ags to CD8 T cells in the lymph node (47). In our study, despite existing at low numbers in afferent lymph, Ag-positive SIRPαlow DCs demonstrated an improved ability to ingest Ag after injection with CpG when compared with liposome alone. Whether this subset is responsible for the significant increase in the number of CD8 T cells expressing IFN-γ exiting the lymph node will require further investigation. CpG-induced increases in IFN-γ production by CD8 T cells have been observed previously (48, 49), and incorporating CpG into liposomes was shown to enhance Ag-specific cell-mediated immune responses (50).

Unlike the conventional α-β TCR CD4- and CD8-expressing T cells, the γδ T cell population in efferent lymph produced approximately equal amounts of IFN-γ and IL-4 that did not change significantly over the vaccination time course or between treatments, suggesting that CpG does not skew these cells to induce a specific immune phenotype following vaccination. Interestingly, the γδ T cell population was responsible for almost all of the IL-4–positive cells present over the time course. The production of both IFN-γ and IL-4 by γδ T cells has been observed previously where γδ T cells exhibit both proinflammatory and regulatory functions, depending on the type or severity of the infection (51, 52).

Our study shows that secondary injection with both formulations induced a rapid reduction in the number of cells leaving the lymph node immediately after vaccination. This phenomenon, known as lymph node cell shutdown, has been observed in several studies using the ovine efferent lymphatic cannulation model (29, 53, 54). In our study, this process was extended by an additional 24 h after vaccination with CpG and observed in all lymphocyte populations. A similar effect has been shown previously where the addition of the adjuvant ISCOMATRIX to the influenza Ag induced an extended lymph node cell shutdown period (53). It was hypothesized that this cell shutdown occurred to increase the time for Ag-carrying APCs and Ag-specific lymphocytes to interact in the lymph node, promoting the generation of an adaptive immune response (29). The acute increase in the number of IFN-γ–producing CD4 and CD8 T cells leaving the lymph node immediately following CpG-induced cell shutdown and the significant increase in the concentration of Ag-specific Abs observed in our study support this hypothesis.

Injection of CpG induced a significant increase in Ag-specific Ab in both the efferent lymph and peripheral blood of vaccinated animals, suggesting that the liposomal CpG vaccine formulation has both local and systemic effects on adaptive immunity. In fact, tertiary injection of CpG increased Ag-specific Abs that remained elevated in the circulation for 30 d after vaccination. This has been shown previously where liposome vaccine formulations containing CpG were up to 30 times more effective than unencapsulated CpG at inducing Ag-specific Abs to influenza and hepatitis B Ags (42). The elevation of Ag-specific Ab induced by CpG occurred after secondary vaccination; however, tertiary vaccination was required for the liposome-alone injections to demonstrate any significant increase in Ag-specific Ab. This reinforces previous observations that liposomal formulations without the addition of immune-stimulatory components often show limited immunogenicity and require multiple boost injections to elicit strong immunity (24).

This study characterizes the action of liposomal vaccine formulations incorporating CpG in the local draining lymphatic network of vaccinated animals. We have shown that the addition of CpG to these formulations significantly enhances Ag uptake by afferent lymph cell populations and induces their maturation prior to their migration to the local lymph node. This ultimately leads to an extended lymph node cell shutdown period, promoting the generation of an adaptive immune response, characterized by increased CD8 T cell IFN-γ production and the persistence of local and systemic Ag-specific Abs. Taken together, the results of this study establish an in vivo explanation for the increased immune response induced by CpG and enhance our understanding of the immunogenic function of liposomal vaccine formulations.

Disclosures

J.N. was and still is an employee of Zoetis and M.J.E. was an employee of Zoetis at the time of this study.

Acknowledgments

We thank the Large Animal Facility (Department of Physiology, Monash University) for excellent care of animals and Mr. Gary Nguyen for technical assistance.

Footnotes

  • ↵1 Current address: UoM Commercial Ltd., University of Melbourne, Carlton, VIC, Australia.

  • This work was supported by an Australian Research Council linkage grant with Pfizer Veterinary Medicine Research and Development (now Zoetis Veterinary Medicine Research and Development).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    DC
    dendritic cell
    MFI
    mean fluorescence intensity
    MHC II
    MHC class II.

  • Received November 22, 2013.
  • Accepted February 12, 2014.

References

  1. ↵
    1. Coffman R. L.,
    2. A. Sher,
    3. R. A. Seder
    . 2010. Vaccine adjuvants: putting innate immunity to work. Immunity 33: 492–503.
    OpenUrlCrossRefPubMed
  2. ↵
    1. de Veer M.,
    2. E. Meeusen
    . 2011. New developments in vaccine research—unveiling the secret of vaccine adjuvants. Discov. Med. 12: 195–204.
    OpenUrlPubMed
  3. ↵
    1. Bode C.,
    2. G. Zhao,
    3. F. Steinhagen,
    4. T. Kinjo,
    5. D. M. Klinman
    . 2011. CpG DNA as a vaccine adjuvant. Expert Rev. Vaccines 10: 499–511.
    OpenUrlCrossRefPubMed
    1. Overstreet M. G.,
    2. H. Freyberger,
    3. I. A. Cockburn,
    4. Y. C. Chen,
    5. S. W. Tse,
    6. F. Zavala
    . 2010. CpG-enhanced CD8+ T-cell responses to peptide immunization are severely inhibited by B cells. Eur. J. Immunol. 40: 124–133.
    OpenUrlPubMed
    1. Jung J.,
    2. A. K. Yi,
    3. X. Zhang,
    4. J. Choe,
    5. L. Li,
    6. Y. S. Choi
    . 2002. Distinct response of human B cell subpopulations in recognition of an innate immune signal, CpG DNA. J. Immunol. 169: 2368–2373.
    OpenUrlAbstract/FREE Full Text
    1. Davis H. L.,
    2. R. Weeratna,
    3. T. J. Waldschmidt,
    4. L. Tygrett,
    5. J. Schorr,
    6. A. M. Krieg
    . 1998. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. [Published erratum appears in 1999 J. Immunol. 162: 3103.] J. Immunol. 160: 870–876.
    OpenUrlAbstract/FREE Full Text
    1. Lipford G. B.,
    2. M. Bauer,
    3. C. Blank,
    4. R. Reiter,
    5. H. Wagner,
    6. K. Heeg
    . 1997. CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur. J. Immunol. 27: 2340–2344.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Chu R. S.,
    2. O. S. Targoni,
    3. A. M. Krieg,
    4. P. V. Lehmann,
    5. C. V. Harding
    . 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186: 1623–1631.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Tross D.,
    2. D. M. Klinman
    . 2008. Effect of CpG oligonucleotides on vaccine-induced B cell memory. J. Immunol. 181: 5785–5790.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Mapletoft J. W.,
    2. M. Oumouna,
    3. H. G. Townsend,
    4. S. Gomis,
    5. L. A. Babiuk,
    6. S. van Drunen Littel-van den Hurk
    . 2006. Formulation with CpG oligodeoxynucleotides increases cellular immunity and protection induced by vaccination of calves with formalin-inactivated bovine respiratory syncytial virus. Virology 353: 316–323.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Linghua Z.,
    2. T. Xingshan,
    3. Z. Fengzhen
    . 2008. In vivo oral administration effects of various oligodeoxynucleotides containing synthetic immunostimulatory motifs in the immune response to pseudorabies attenuated virus vaccine in newborn piglets. Vaccine 26: 224–233.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Klinman D. M.,
    2. H. Xie,
    3. S. F. Little,
    4. D. Currie,
    5. B. E. Ivins
    . 2004. CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of rhesus macaques. Vaccine 22: 2881–2886.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Cooper C. L.,
    2. H. L. Davis,
    3. J. B. Angel,
    4. M. L. Morris,
    5. S. M. Elfer,
    6. I. Seguin,
    7. A. M. Krieg,
    8. D. W. Cameron
    . 2005. CPG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. AIDS 19: 1473–1479.
    OpenUrlCrossRefPubMed
    1. Cooper C. L.,
    2. H. L. Davis,
    3. M. L. Morris,
    4. S. M. Efler,
    5. M. A. Adhami,
    6. A. M. Krieg,
    7. D. W. Cameron,
    8. J. Heathcote
    . 2004. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J. Clin. Immunol. 24: 693–701.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Halperin S. A.,
    2. G. Van Nest,
    3. B. Smith,
    4. S. Abtahi,
    5. H. Whiley,
    6. J. J. Eiden
    . 2003. A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant. Vaccine 21: 2461–2467.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Gursel I.,
    2. M. Gursel,
    3. K. J. Ishii,
    4. D. M. Klinman
    . 2001. Sterically stabilized cationic liposomes improve the uptake and immunostimulatory activity of CpG oligonucleotides. J. Immunol. 167: 3324–3328.
    OpenUrlAbstract/FREE Full Text
    1. Shargh V. H.,
    2. M. R. Jaafari,
    3. A. Khamesipour,
    4. I. Jaafari,
    5. S. A. Jalali,
    6. A. Abbasi,
    7. A. Badiee
    . 2012. Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis. Vaccine 30: 3957–3964.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Zaks K.,
    2. M. Jordan,
    3. A. Guth,
    4. K. Sellins,
    5. R. Kedl,
    6. A. Izzo,
    7. C. Bosio,
    8. S. Dow
    . 2006. Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes. J. Immunol. 176: 7335–7345.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Jaafari M. R.,
    2. A. Badiee,
    3. A. Khamesipour,
    4. A. Samiei,
    5. D. Soroush,
    6. M. T. Kheiri,
    7. F. Barkhordari,
    8. W. R. McMaster,
    9. F. Mahboudi
    . 2007. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. Vaccine 25: 6107–6117.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Lonez C.,
    2. M. Vandenbranden,
    3. J. M. Ruysschaert
    . 2008. Cationic liposomal lipids: from gene carriers to cell signaling. Prog. Lipid Res. 47: 340–347.
    OpenUrlCrossRefPubMed
    1. Ma Y. F.,
    2. Y. Zhuang,
    3. X. F. Xie,
    4. C. Wang,
    5. F. Wang,
    6. D. M. Zhou,
    7. J. Q. Zeng,
    8. L. T. Cai
    . 2011. The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses. Nanoscale 3: 2307–2314.
    OpenUrlCrossRefPubMed
    1. Zhang Y. M.,
    2. M. Rusckowski,
    3. N. Liu,
    4. C. Liu,
    5. D. J. Hnatowich
    . 2001. Cationic liposomes enhance cellular/nuclear localization of 99mTc-antisense oligonucleotides in target tumor cells. Cancer Biother. Radiopharm. 16: 411–419.
    OpenUrlCrossRefPubMed
  23. ↵
    1. De Oliveira M. C.,
    2. V. Boutet,
    3. E. Fattal,
    4. D. Boquet,
    5. J. M. Grognet,
    6. P. Couvreur,
    7. J. R. Deverre
    . 2000. Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice. Life Sci. 67: 1625–1637.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Nordly P.,
    2. H. B. Madsen,
    3. H. M. Nielsen,
    4. C. Foged
    . 2009. Status and future prospects of lipid-based particulate delivery systems as vaccine adjuvants and their combination with immunostimulators. Expert Opin. Drug Deliv. 6: 657–672.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Neeland M. R.,
    2. E. N. Meeusen,
    3. M. J. de Veer
    . 2013. Afferent lymphatic cannulation as a model system to study innate immune responses to infection and vaccination. Vet. Immunol. Immunopathol. 158: 86–97.
    OpenUrlPubMed
  26. ↵
    1. de Veer M.,
    2. J. Kemp,
    3. J. Chatelier,
    4. M. J. Elhay,
    5. E. N. Meeusen
    . 2010. The kinetics of soluble and particulate antigen trafficking in the afferent lymph, and its modulation by aluminum-based adjuvant. Vaccine 28: 6597–6602.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Hall J. G.
    1967. A method for collecting lymph from the prefemoral lymph node of unanaesthetised sheep. Q. J. Exp. Physiol. Cogn. Med. Sci. 52: 200–205.
    OpenUrl
  28. ↵
    1. de Veer M.,
    2. J. Kemp,
    3. J. Chatelier,
    4. M. J. Elhay,
    5. E. N. Meeusen
    . 2012. Modulation of soluble and particulate antigen transport in afferent lymph by monophosphoryl lipid A. Immunol. Cell Biol. 90: 404–410.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Wee J. L.,
    2. D. L. Greenwood,
    3. X. Han,
    4. J. P. Scheerlinck
    . 2011. Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node. Vet. Immunol. Immunopathol. 144: 95–103.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Jiang Z. H.,
    2. R. R. Koganty
    . 2003. Synthetic vaccines: the role of adjuvants in immune targeting. Curr. Med. Chem. 10: 1423–1439.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Egan P. J.,
    2. W. Kimpton,
    3. H. F. Seow,
    4. V. M. Bowles,
    5. M. R. Brandon,
    6. A. D. Nash
    . 1996. Inflammation-induced changes in the phenotype and cytokine profile of cells migrating through skin and afferent lymph. Immunology 89: 539–546.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Caskey M.,
    2. F. Lefebvre,
    3. A. Filali-Mouhim,
    4. M. J. Cameron,
    5. J. P. Goulet,
    6. E. K. Haddad,
    7. G. Breton,
    8. C. Trumpfheller,
    9. S. Pollak,
    10. I. Shimeliovich,
    11. et al
    . 2011. Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J. Exp. Med. 208: 2357–2366.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. de Veer M.,
    2. M. Neeland,
    3. M. Burke,
    4. J. Pleasance,
    5. J. Nathanielsz,
    6. M. Elhay,
    7. E. Meeusen
    . 2013. Cell recruitment and antigen trafficking in afferent lymph after injection of antigen and poly(I:C) containing liposomes, in aqueous or oil-based formulations. Vaccine 31: 1012–1018.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Yang G. X.,
    2. Z. X. Lian,
    3. K. Kikuchi,
    4. Y. J. Liu,
    5. A. A. Ansari,
    6. S. Ikehara,
    7. M. E. Gershwin
    . 2005. CD4- plasmacytoid dendritic cells (pDCs) migrate in lymph nodes by CpG inoculation and represent a potent functional subset of pDCs. J. Immunol. 174: 3197–3203.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Sparwasser T.,
    2. E. S. Koch,
    3. R. M. Vabulas,
    4. K. Heeg,
    5. G. B. Lipford,
    6. J. W. Ellwart,
    7. H. Wagner
    . 1998. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur. J. Immunol. 28: 2045–2054.
    OpenUrlCrossRefPubMed
    1. Carrasco C. P.,
    2. R. C. Rigden,
    3. I. E. Vincent,
    4. C. Balmelli,
    5. M. Ceppi,
    6. O. Bauhofer,
    7. V. Tâche,
    8. B. Hjertner,
    9. F. McNeilly,
    10. H. G. van Gennip,
    11. et al
    . 2004. Interaction of classical swine fever virus with dendritic cells. J. Gen. Virol. 85: 1633–1641.
    OpenUrlCrossRefPubMed
    1. Behboudi S.,
    2. D. Chao,
    3. P. Klenerman,
    4. J. Austyn
    . 2000. The effects of DNA containing CpG motif on dendritic cells. Immunology 99: 361–366.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Hackstein H.,
    2. A. Knoche,
    3. A. Nockher,
    4. J. Poeling,
    5. T. Kubin,
    6. M. Jurk,
    7. J. Vollmer,
    8. G. Bein
    . 2011. The TLR7/8 ligand resiquimod targets monocyte-derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation. Cell. Immunol. 271: 401–412.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Bertho N.,
    2. F. Marquet,
    3. F. Pascale,
    4. C. Kang,
    5. M. Bonneau,
    6. I. Schwartz-Cornil
    . 2011. Steady state pig dendritic cells migrating in skin draining pseudo-afferent lymph are semi-mature. Vet. Immunol. Immunopathol. 144: 430–436.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Gursel M.,
    2. D. Verthelyi,
    3. D. M. Klinman
    . 2002. CpG oligodeoxynucleotides induce human monocytes to mature into functional dendritic cells. Eur. J. Immunol. 32: 2617–2622.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Ziegler-Heitbrock L.,
    2. P. Ancuta,
    3. S. Crowe,
    4. M. Dalod,
    5. V. Grau,
    6. D. N. Hart,
    7. P. J. Leenen,
    8. Y. J. Liu,
    9. G. MacPherson,
    10. G. J. Randolph,
    11. et al
    . 2010. Nomenclature of monocytes and dendritic cells in blood. Blood 116: e74–e80.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Joseph A.,
    2. I. Louria-Hayon,
    3. A. Plis-Finarov,
    4. E. Zeira,
    5. Z. Zakay-Rones,
    6. E. Raz,
    7. T. Hayashi,
    8. K. Takabayashi,
    9. Y. Barenholz,
    10. E. Kedar
    . 2002. Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines. Vaccine 20: 3342–3354.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Contreras V.,
    2. C. Urien,
    3. R. Guiton,
    4. Y. Alexandre,
    5. T. P. Vu Manh,
    6. T. Andrieu,
    7. K. Crozat,
    8. L. Jouneau,
    9. N. Bertho,
    10. M. Epardaud,
    11. et al
    . 2010. Existence of CD8α-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species. J. Immunol. 185: 3313–3325.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Hope J. C.,
    2. E. Guzman,
    3. C. Cubillos-Zapata,
    4. S. A. Stephens,
    5. S. C. Gilbert,
    6. H. Prentice,
    7. P. Sopp,
    8. C. J. Howard,
    9. B. Charleston
    . 2012. Migratory sub-populations of afferent lymphatic dendritic cells differ in their interactions with Mycobacterium bovis Bacille Calmette Guerin. Vaccine 30: 2357–2367.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Stephens S. A.,
    2. J. Brownlie,
    3. B. Charleston,
    4. C. J. Howard
    . 2003. Differences in cytokine synthesis by the sub-populations of dendritic cells from afferent lymph. Immunology 110: 48–57.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Milling S. W. F.,
    2. C. D. Jenkins,
    3. U. Yrlid,
    4. V. Cerovic,
    5. H. Edmond,
    6. V. McDonald,
    7. M. Nassar,
    8. G. Macpherson
    . 2009. Steady-state migrating intestinal dendritic cells induce potent inflammatory responses in naive CD4+ T cells. Mucosal Immunol. 2: 156–165.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Bedoui S.,
    2. P. G. Whitney,
    3. J. Waithman,
    4. L. Eidsmo,
    5. L. Wakim,
    6. I. Caminschi,
    7. R. S. Allan,
    8. M. Wojtasiak,
    9. K. Shortman,
    10. F. R. Carbone,
    11. et al
    . 2009. Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nat. Immunol. 10: 488–495.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Cobb D.,
    2. S. Guo,
    3. R. B. Smeltz
    . 2013. CpG and interleukin-15 synergize to enhance IFN-gamma production by activated CD8+ T cells. BioMed Res. Int. 2013: 924023.
    OpenUrlPubMed
  49. ↵
    1. Zhao K.,
    2. H. Wang,
    3. C. Y. Wu
    . 2011. The immune responses of HLA-A*0201 restricted SARS-CoV S peptide-specific CD8⁺ T cells are augmented in varying degrees by CpG ODN, PolyI:C and R848. Vaccine 29: 6670–6678.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Andrews C. D.,
    2. M. S. Huh,
    3. K. Patton,
    4. D. Higgins,
    5. G. Van Nest,
    6. G. Ott,
    7. K. D. Lee
    . 2012. Encapsulating immunostimulatory CpG oligonucleotides in listeriolysin O-liposomes promotes a Th1-type response and CTL activity. Mol. Pharm. 9: 1118–1125.
    OpenUrlPubMed
  51. ↵
    1. Ferrick D. A.,
    2. M. D. Schrenzel,
    3. T. Mulvania,
    4. B. Hsieh,
    5. W. G. Ferlin,
    6. H. Lepper
    . 1995. Differential production of interferon-gamma and interleukin-4 in response to Th1- and Th2-stimulating pathogens by gamma delta T cells in vivo. Nature 373: 255–257.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Pinheiro M. B.,
    2. L. R. Antonelli,
    3. R. Sathler-Avelar,
    4. D. M. Vitelli-Avelar,
    5. S. Spindola-de-Miranda,
    6. T. M. P. D. Guimaraes,
    7. A. Teixeira-Carvalho,
    8. O. A. Martins,
    9. V. P. C. P. Toledo
    . 2012. CD4-CD8-alpha beta and gamma delta T Cells Display Inflammatory and Regulatory Potentials during Human Tuberculosis. PLoS ONE 7: e50923.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Windon R. G.,
    2. P. J. Chaplin,
    3. P. McWaters,
    4. M. Tavarnesi,
    5. M. Tzatzaris,
    6. W. G. Kimpton,
    7. R. N. Cahill,
    8. L. Beezum,
    9. A. Coulter,
    10. D. Drane,
    11. et al
    . 2001. Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX. Vaccine 20: 490–497.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Young A. J.,
    2. T. J. Seabrook,
    3. W. L. Marston,
    4. L. Dudler,
    5. J. B. Hay
    . 2000. A role for lymphatic endothelium in the sequestration of recirculating gamma delta T cells in TNF-alpha-stimulated lymph nodes. Eur. J. Immunol. 30: 327–334.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section