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. 2010 Jul;130(3):374–387. doi: 10.1111/j.1365-2567.2009.03237.x

Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model

Yifan Ma 1,2, Louis Poisson 1, Guzman Sanchez-Schmitz 1, Santosh Pawar 1, Chunfeng Qu 3, Gwendalyn J Randolph 3, William L Warren 1, Eric M Mishkin 1, Russell G Higbee 1
PMCID: PMC2913217  PMID: 20331478

Abstract

The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimensional tissue-engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1α/β, 6, 8 and 10 and tumour necrosis factor-α. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100-fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up-regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE-derived DC treated with Gardiquimod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33+) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen-presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.

Keywords: cytokine, dendritic cell, endothelial cells, human, in vitro tissue engineered immunological model, three-dimensional, Toll-like receptor

Introduction

Dendritic cells (DC) are the most potent antigen-presenting cells (APC), and they play an essential role in both innate and adaptive immunity. They normally develop from circulating bone-marrow-derived DC precursors that distribute into the peripheral tissues and give rise to immature DC (iDC).1 The tissue-residing iDC capture antigens from the local environment and release cytokines/chemokines, thereby participating in innate immunity. Moreover, antigen capture also triggers DC maturation and migration into draining lymph nodes. In the T-cell region of lymph nodes, mature DC (mDC) present antigens to naive T cells via major histocompatibility complex molecules, triggering the adaptive immune response.2 Hence, DC are an important link between innate and adaptive immunity.

In vitro DC production is an important strategy for generating large numbers of DC. To date, the most commonly used method to generate human DC is to culture blood monocytes with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5–7 days.3 Although this method can produce a large population of DC, it remains questionable whether this method faithfully recapitulates DC development in vivo.4 Besides cytokine-derived DC, Randolph et al.5–8 have reported a tissue-engineered in vitro immunological model that allows for autonomous generation of DC. We termed this system the in vitro‘Peripheral Tissue Equivalent’ (PTE) module. The PTE module is a three-dimensional culture system consisting of a confluent monolayer of unstimulated endothelial cells grown on top of an endotoxin-free collagenous matrix (see Fig. 1). When peripheral blood mononuclear cells (PBMC) are applied onto the endothelial monolayer, monocytes preferentially migrate across the endothelium into the sub-endothelial collagen. This process probably mimics the constitutive entry of monocytes into tissues as observed in vivo.9 After 48 hr in culture, approximately half of the migrated monocytes differentiate into DC with varying degrees of maturity, and migrate across the intact endothelium in the abluminal-to-luminal direction (reverse transmigration),5 which may mimic the in vivo DC migration from peripheral tissue into the local lymphatics.1 In the unstimulated PTE module, the percentage of mature DC in the reverse-transmigrated (RT) cell fraction is < 10%, with the majority of RT cells resembling immature DC or monocytes. Incorporation of various stimuli, such as lipopolysaccharide (LPS), influenza virus or zymosan, significantly increases the percentage of mature DC in the RT cell fraction.6 Compared with conventional cytokine-derived DC, PTE-derived DC (PTE-DC) differentiate more rapidly and do not require application of exogenous cytokines. Moreover, the composition of PTE-DC is more heterogeneous than that of cytokine-cultured DC, and may more closely resemble the composition of the DC populations developed in vivo.5,10 In addition, the PTE module contains endothelial cells that can sense ‘danger signals’ in peripheral tissues and contribute to immune responses.11,12 Indeed, the presence of endothelial cells has a critical role in the generation of DC in the system,5 which probably increases the sensitivity of the PTE module in response to various stimuli.

Figure 1.

Figure 1

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The scheme of the in vitro peripheral tissue equivalent (PTE) module. The PTE module consists of a quiescent monolayer of human umbilical vein endothelial cells (HUVEC) grown on a collagen matrix. When peripheral blood mononuclear cells (PBMC) are applied onto HUVEC, monocytes migrate into the subendothelial collagen and form a PTE module (a). After an additional 48 hr of culture, about half of the monocytes reverse transmigrate across the endothelium and develop into dendritic cells (b).

The Toll-like receptor (TLR) family is a group of pattern-recognition receptors that play a crucial role in both innate and adaptive immunity. TLRs can recognize conserved microbial structures or products of microbial metabolism called ‘pathogen-associated molecular pattern’, which consequently triggers innate immunity. The TLR signalling also promotes the activation and maturation of APCs, thereby facilitating adaptive immunity. In addition, cytokines and chemokines elicited by TLR stimulation further regulate downstream T-cell and B-cell responses.13 Currently, several TLR agonists are being evaluated as potential adjuvants for vaccine development against infectious diseases and cancer. For example, polyinosine-polycytidylic acid (Poly I:C), a TLR3 agonist, has been shown to be a potent adjuvant to enhance vaccine-induced protective immune responses.14 Agonists of TLR7/8, such as Imiquimod and Resiquimod, have been used to treat skin neoplasms and viral infections in humans.15 CpG oligonucleotides (ODN), TLR9 agonists, have been reported to significantly enhance the antibody responses induced by hepatitis B and anthrax vaccines in human clinical trials.16,17 Previous studies have demonstrated that conventional and plasmacytoid DC (pDC) are the major cell types mediating the adjuvant effect of TLR agonists.18 Stimulation by TLR can promote the differentiation and maturation of freshly isolated DC or cytokine-cultured DC, triggering the release of cytokines.19–21 Moreover, there is evidence that TLR stimulation, such as stimulation of TLR8 by single-stranded RNA, triggers PTE-DC maturation.7 However, a generally broader survey as to how TLR stimulation will affect the phenotype and function of PTE-DC has not been performed, nor is it known how the response to TLR agonists in the PTE module compares with traditional PBMC culture and cytokine-cultured DC. In the present study, the immunopotency of TLR agonists, such as Poly I:C (TLR3), LPS (TLR4), Gardiquimod (TLR7/8) or CpG 2006 (TLR9), was tested in the PTE module. These TLR agonists evoked cytokine and chemokine production, and promoted DC generation and maturation in the PTE module. When compared with traditional PBMC culture methods, the PTE module was more sensitive and so was a promising predictor of immune response. These studies support the use of the PTE module as a reliable immunological test bed for adjuvant and vaccine screening and development.

Materials and methods

Human PBMC preparation

Apheresis blood products were obtained from Florida’s Blood Centers (Orlando, FL). The study was reviewed and approved by Chesapeake Research Review Inc. (Columbia, MD). All donors were in good health and all blood products were negative for blood-borne pathogens as detected by standard blood bank assays. The PBMC were enriched by Ficoll density gradient separation according to standard laboratory procedures. After washing, PBMC were either resuspended in culture media or cryopreserved in dimethylsulphoxide-containing freezing media for extended storage in liquid nitrogen vapour phase for future assays.

In vitroPTE module preparation

The PTE module was fabricated as described previously,10 as shown in Fig. 1. Briefly, second-passage human umbilical vein endothelial cells (HUVEC; Lonza, Walkersville, MD) were cultured on a polymerized type I collagen matrix (Inamed Biomaterials, Fremont, CA) and maintained in M199 media (Lonza) supplemented with 20% fetal bovine serum until confluent. The PBMC or purified monocytes were suspended in M199 media containing 0·1% (weight/volume) bovine serum albumin, and applied onto the HUVEC monolayer at 500 000 cells/well and 200 000 cells/well, respectively. In some experiments, pDC were depleted from whole PBMC by using CD304 (BDCA-4/Neuropilin-1) MicroBeads (Miltenyi Biotec Inc., Auburn, CA). After a 90-min incubation, non-migrated cells were removed, and PTE modules were incubated in culture medium (M199 medium with 20% heat-inactivated human autologous serum) with or without the following TLR agonists: 5 μg/ml CpG 2006, 25 μg/ml Poly I:C, 5 μg/ml Gardiquimod (all from Invivo-Gen, San Diego, CA), or 10 ng/ml LPS (Sigma, St Louis, MO). The PTE culture supernatants were collected at 24 hr or 48 hr for cytokine analysis, and the RT cells were carefully harvested at 48 hr for fluorescence-activated cell sorting analysis.

PBMC culture

The PBMC were resuspended at 2·5 × 106 cells/ml in RPMI-1640 supplemented with 10% heat-inactivated autologous human serum, and stimulated with or without the following TLR agonists: 5 μg/ml CpG 2006, 25 μg/ml Poly I:C, 5 μg/ml Gardiquimod (all from InvivoGen), or 10 ng/ml LPS (Sigma). PTE culture supernatants were collected at 24 hr for cytokine analysis.

Cell isolation

The T and B cells were negatively selected using pan-T-cell isolation kit II or B-cell isolation kit II from Miltenyi Biotec, Inc. For isolation of CD33+ myeloid cells, PBMC were stained with phycoerythrin (PE) -labelled anti-human CD33 antibody (BD Biosciences, San Jose, CA). Labelled cell populations were then enriched to > 99% purity by selection over LS separation columns after labelling with anti-PE microbeads (Miltenyi Biotec Inc). For pDC reconstitution experiments, the CD33/pDC fraction was positively selected using CD33 and BDCA-4 microbeads, and the percentage of pDC in the CD33/pDC-enriched fraction was 1·4–2%.

Cytokine-cultured DC preparation and stimulation

CD14+ monocytes were positively selected using CD14+ microbeads (Miltenyi Biotec), and cultured with GM-CSF (100 ng/ml) and IL-4 (25 ng/ml) for 5 days to generate iDC. The iDC were then stimulated with equivalent doses of TLR agonists as previously described. After 48 hr culture, cells were collected for DC phenotypic analysis.

Flow cytometry

The phenotype of DC was determined using seven-colour staining. The RT cells or iDC were first incubated with mouse immunoglobulin G1 (IgG1; 1 μg/sample; Sigma) at room temperature to block Fc receptor along with the vital dye Annexin V (2·5 μl/sample; Invitrogen Corporation, Carlsbad, CA) to identify apoptotic cells. After 15 min, the cells were incubated for 40 min on ice with the cocktail of fluorochrome-labelled monoclonal antibodies. The antibody cocktail contained non-myeloid lineage markers, allophycocyanin-conjugated CD3, CD19 and CD56, and the relevant DC surface markers, allophycocyanin-Cychrome 7-conjugated CD14, peridinin chlorophyll protein-Cychrome 5.5-conjugated human leucocyte antigen (HLA) -DR, CCR7-PE-Cy7, fluorescein isothiocyanate-conjugated CD83 and CD86-PE (BD Biosciences). After incubation, the cells were washed twice with Hanks’ buffered salt solution (HBSS, +Ca/+Mg) followed by fixation in 2% (volume/volume) paraformaldehyde, also in HBSS. Viable cellular events were accrued on a BD LSRII flow cytometer equipped with BD FACSDiva software (BD Biosciences). Data analysis was with FlowJo software (Tree Star Inc., Ashland, OR).

Cytokine and chemokine analysis

Production of cytokines and chemokines was analysed using a Beadlyte human 22-plex multi-cytokine detection system (Millipore, Billerica, MA). Briefly, 50 μl appropriately diluted cytokine standard or culture supernatants was incubated with 25 μl of multi-cytokine beads in a 96-well filter plate at room temperature for 2 hr. The beads were then washed and incubated with 25 μl of multi-cytokine biotin for 90 min, followed by mixing with 25 μl of diluted Beadlyte strepavidin-PE. After 30 min, liquid was removed, and the beads were resuspended in 125 μl of assay buffer and analysed using the Bio-Plex 200 system (Bio-Rad, Hercules, CA). Cytokine and chemokine concentrations were calculated based on relevant standard curves using the bio-plex manager software (Bio-Rad).

Antigen presentation

The PTE modules were prepared as previously described. After removing the non-migrated cells, the PTE modules were incubated at 37° with either culture medium alone, tetanus toxoid (TT, 1 μg/ml; Calbiochem, La Jolla, CA), Gardiquimod (5 μg/ml), or TT plus Gardiquimod. The RT cells were carefully collected after 48 hr and co-cultured with 2·5 × 105 autologous T cells and 2·5 × 105 autologous B cells in a 24-well plate at an APC : lymphocyte ratio of 1 : 30. The anti-TT IgG response was evaluated on day 7 using enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) detection.

Enumeration of anti-TT antibody-secreting cells

Numbers of TT-specific antibody-secreting cells were assessed by ELISPOT as previously described, with minor modifications.22 Briefly, a 96-well Multiscreen HA filtration plate (Millipore Corporation) was coated with 1 μg/ml TT at 4° overnight, followed by blocking with RPMI-1640 containing 1% bovine serum albumin at 37° for 2 hr. Serially diluted lymphocyte suspensions were added to the plate and incubated at 37° for 4–5 hr. After incubation, cells were discarded and the plate was incubated with 1 μg/ml goat anti-human IgG-biotin (Jackson ImmunoLab, West Grove, PA) at 4°, overnight. On the next day, the plate was washed and incubated with 5 μg/ml horseradish peroxidase-conjugated avidin (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature. The plate was washed and incubated with 3-amino-9-ethylcarbazole substrate solution until spots were well developed. The plate was rinsed with deionized water to stop the reaction and spots were counted using the AID ELISPOT reader system (Cell Technology, Columbia, MD).

Results

TLR agonists induce more cytokines in the PTE module than in PBMC culture

The TLR-induced cytokine production in the PTE module was measured first to evaluate the immunopotency of the TLR agonists. Overall, TLR agonists induced higher levels of cytokines in the PTE module than in conventional PBMC cultures (Fig. 2). For example, both Poly I:C and CpG 2006 induced the pro-inflammatory cytokines IL-1α/β in the PTE module, which were not observed in PBMC cultures (Fig. 2a,c). Poly I:C also triggered tumour necrosis factor-α (TNF-α) production solely in the PTE culture (Fig. 2a). Moreover, the production of IL-6 and IL-8 induced by Poly I:C and CpG 2006 treatments in the PTE module was significantly higher than in PBMC culture (Fig. 2b,d). Although Gardiquimod and LPS dramatically induced IL-1β, IL-6, IL-8, IL-10 and TNF-α, both in the PTE module and in the PBMC culture, the PTE module produced approximately three- to sixfold more IL-1β and IL-10, and 10- to 50-fold more IL-6 and IL-8, than PBMC culture (Fig. 2e–h). Consequently, the PTE module was more sensitive than conventional PBMC culture in response to TLR stimulation.

Figure 2.

Figure 2

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Toll-like receptor (TLR) agonists induce cytokine production in the peripheral tissue equivalent (PTE) module. Peripheral blood mononuclear cells (PBMCs) were suspended in M199-0·1% bovine serum albumin and applied onto the endothelium at 500 000 cells/well to generate PTE modules. After a 90-min incubation period, non-migrating cells were removed and the PTE was incubated in M199 with 10% autologous serum in the presence or absence of CpG 2006 (5 μg/ml), Poly I : C (50 μg/ml), Gardiquimod (Gar, 2·5 μg/ml), or lipopolysaccharide (LPS; 10 ng/ml). For PBMC cultures, PBMC were suspended in RPMI-1640 with 10% autologous serum (2·5 × 106 cells/ml), and stimulated with the same concentration of Toll-like receptor (TLR) agonists. The culture supernatants were collected after 24 hr incubation, the cytokine levels in supernatants were analysed by Q-plex cytokine array (IL, interleukin; TNF, tumour necrosis factor). The numbers shown in the bar graphs represent the mean ± SE of four or five different donors.

The PTE module contains a confluent HUVEC monolayer, so it was important to determine if the higher sensitivity of the PTE module to TLR agonists was attributable to the presence of HUVEC. There was a basal production of IL-6 (286 ± 26 pg/ml) and IL-8 (3822 ± 347 pg/ml) in unstimulated HUVEC cultures. Although treatment with Poly I:C or LPS significantly increased the production of IL-6 and IL-8 by about twofold to fourfold, Gardiquimod and CpG 2006 did not trigger cytokine production in the HUVEC culture (Fig. 3). These data suggest that enhanced TLR-induced cytokine production in the PTE module is attributed to a synergistic interaction between migrated cells and the endothelium.

Figure 3.

Figure 3

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Toll-like receptor (TLR) agonists induce cytokine production in the human umbilical vein endothelial cell (HUVEC) culture. HUVEC were grown on a collagen matrix and maintained in M199-10% fetal bovine serum. Once the HUVEC monolayer became confluent, treatment with medium, CpG 2006 (5 μg/ml), Poly I : C (50 μg/ml), Gardiquimod (Gar, 2·5 μg/ml), or lipopolysaccharide (LPS; 10 ng/ml) was carried out for 48 hr. Interleukin-6 (IL-6) and IL-8 in culture supernatants were analysed by Q-Plex cytokine array. Values shown in the bar graphs represent the mean ± SE of four or five different donors. Differences between unstimulated and stimulated groups were analysed using the paired Student’s t-test. *P < 0·05.

TLR agonists promote differentiation and maturation of DC in the PTE module

Previous studies have shown that TLR stimulation can induce DC maturation and enhance antigen presentation.23 The PTE module has been reported as a novel tissue-engineered system to generate DC autonomously, so we determined if TLR stimulation could also regulate the phenotype of the PTE-DC. For this purpose, RT cells were carefully harvested from unstimulated PTE modules and labelled with an antibody cocktail. Antigen-presenting cells generated in the PTE module (PTE-APC) were defined as Annexin− Lineage− DR+ cells, which could be further divided into two subpopulations: DR+ CD14+ cells and DR+ CD14− cells (Fig. 4). DR+ CD14+ cells expressed a low level of CD86 and lacked CD83 and CCR7. In contrast, DR+ CD14− cells expressed CD83 and CCR7, with elevated levels of CD86 and HLA-DR compared with DR+ CD14+cells (Fig. 4), suggesting that these cells were DC generated autonomously in the PTE module (PTE-DC). Although PTE-DC (DR+ CD14−) accounted for 20–30% of total PTE-APs in untreated PTE modules, Gardiquimod and LPS approximately doubled the percentage of DC recovered, a change that was statistically significant (Fig. 5a). Notably, the application of TLR agonists did not significantly impact the absolute number of live RT cells (data not shown), which is consistent with a previous report.5 Hence, selected TLR stimulation enhanced the differentiation of monocytes into DC in the PTE module. Moreover, TLR agonists promoted the maturation of PTE-DC. Unstimulated PTE-DC expressed CD86 but lacked CD83 and CCR7, a chemokine receptor essential for DC migration into the lymphatic vessels.24 Gardiquimod and LPS significantly increased the expression of CCR7, CD83 and CD86 on PTE-DC, suggesting that they drive the maturation of DC (Fig. 5b). Poly I:C and CpG 2006 promoted DC maturation to a lesser extent, with augmented expression of CD86 and CCR7, respectively (Fig. 5b).

Figure 4.

Figure 4

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Phenotypic analysis of unstimulated peripheral tissue equivalent antigen-presenting cells (PTE-APC). The PTE module was prepared as described in Fig. 1, and then incubated in culture medium for 48 hr. The reverse transmigrated (RT) cells were collected and labelled with a mixture of antibodies, including the lineage markers allophycocyanin-CD3, -CD19, -CD56 and the DC relevant markers, allophycocyanin-Cychrome 7-CD14, peridinin chlorophyll protein-Cychrome 5.5-HLA-DR, phycoerythrin-Cychrome 7-CCR7, fluorescein isothiocyanate-CD83 and phycoerythrin-CD86. Viable cells were assessed by Trypan Blue exclusion and Annexin V-Pacific Blue negativity. No significant difference between absolute numbers of viable cells counted between different conditions was observed. PTE-APC are defined as Annexin V− Lin− DR+, and can be further divided into two subpopulations: DR+ CD14+ and DR+ CD14−. The expressions of CD83, CD86, CCR7 and HLA-DR on these two subpopulations are shown in histograms, and the grey areas represent the staining with isotype-matched control antibodies.

Figure 5.

Figure 5

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Toll-like receptor (TLR) agonists promote the generation and maturation of peripheral tissue equivalent dendritic cells (PTE-DC). The PTE modules were prepared and treated as described in Fig. 1. The reverse transmigrated (RT) cells were carefully harvested at 48 hr and stained with fluorochrome labelled antibodies as described in Fig. 2. PTE-antigen-presenting cells (APC) are defined as Annexin V− Lin− DR+ shown in (a), and can be further divided into two subpopulations: PTE-DC (DR+ CD14−) and CD14+ APC (DR+ CD14+). The percentage of PTE-DC is shown in (a), and the expression of CD83, CD86 and CCR7 on PTE-DC and CD14+ APC are shown in (b) and (c), respectively. CD14+ cells positively selected from peripheral blood mononuclear cells (PBMC) were cultured with granulocyte–macrophage colony-stimulating factor (100 ng/ml) and interleukin-4 (25 ng/ml) for 5 days to generate immature cytokine-cultured DCs (iDC). The iDC were then cultured in the presence or absence of CpG 2006 (5 μg/ml), Poly I : C (50 μg/ml), Gardiquimod (Gar, 2·5 μg/ml), or lipopolysaccharide (LPS; 10 ng/ml). The cells were harvested at 48 hr and stained with fluorochrome labelled antibodies as previously described, and the expression of CD83, CD86 and CCR7 on DC are shown in (d). The numbers shown in the bar graphs represent the mean ± SE of four or five different donors, and differences between the stimulated and unstimulated PTE modules were analysed using the Student’s t-test. *P < 0·05; **P < 0·01.

In addition to promoting DC generation and maturation, TLR agonists also triggered the activation of Lineage− DR+ CD14+ APC in the PTE module. Under unstimulated conditions, CD14+ APC expressed low levels of CD83, CD86 and CCR7. However, administration of Gardiquimod and LPS induced the expression of CD83 and CD86 by threefold to sixfold, and CCR7 over 10-fold in these cells (Fig. 5c). Just as for CD14− DC, CpG 2006 and Poly I:C were less potent than LPS and Gardiquimod in activating CD14+ APC in the PTE module (Fig. 5c).

The PTE-DC were further characterized by comparing their phenotype with conventional cytokine-cultured DC. In general, unstimulated PTE-DCs expressed similar levels of CCR7, CD83 and CD86 as cytokine-cultured iDC (Fig. 5d). Lipopolysaccharide significantly promoted maturation of cytokine-cultured DC by inducing CCR7, CD83 and CD86, which was consistent with observations made in the PTE module. However, Gardiquimod significantly promoted generation and maturation of PTE-DC, but failed to regulate cytokine-cultured DC maturation. Conversely, Poly I : C moderately enhanced the maturation of PTE-DC, yet dramatically induced expression of CCR7 and CD86 on cytokine-cultured DC (Fig. 5d). Hence, cytokine-cultured DC and PTE-DC respond differently to TLR stimulations.

Non-monocytic cell populations contribute to TLR-induced cytokine production in the PTE module

Previous studies6–8 and our unpublished observations have indicated that the unstimulated RT fraction contained about 10–15% non-monocytic cells, including lymphocytes and pDC. The PTE-DC and cytokine-derived DC showed differential responsiveness to TLR agonists, especially Gardiquimod and Poly I:C, so it was of interest to determine if a small population of non-monocytic cells was involved in the response of the PTE module to TLR stimulation. For this purpose, we established the PTE module with PBMC (PTE-PBMC) or monocytes (PTE-Mo) by applying PBMC or purified CD33+ monocytes onto the HUVEC monolayer, respectively. Although the PBMC populations used in these studies were typically comprised of ∼ 20% monocytes, the enriched myeloid cell populations for the PTE-Mo module were devoid of non-monocytic cell populations and consistently exhibited purities in excess of 99%.

Cytokine analysis indicated that TLR agonists evoked different cytokine profiles in the PTE-PBMC and PTE-Mo modules. The production of IL-6, IL-8 and IL-10 was not significantly different between the PTE-PBMC and PTE-Mo modules (data not shown), yet several cytokines were significantly reduced only in the PTE-Mo module. For example, levels of IL-12 p70 and TNF-α were significantly increased by TLR stimulation in the PTE-PBMC module. However, in the PTE-Mo module, the production of IL-12 p70 and TNF-α was reduced by between five- and 20-fold (Fig. 6a,b). Similarly, production of interferon (IFN) also declined in the PTE-Mo module. Interferon-α, a type I IFN, is a potent stimulus for DC maturation.3,25 In the PTE-PBMC module, IFN-α was induced by Poly I:C and Gardiquimod, whereas in the PTE-Mo module its production was reduced or completely abolished (Fig. 6c). In addition to the cytokines, the production of regulated on activation, normal T-cell expressed and secreted (RANTES) and interferon-inducible protein-10 (IP-10) was reduced in the PTE-Mo module. Although TLR agonists induced RANTES about 10- to 200-fold in the PTE-PBMC module, RANTES production was partially or completely ablated in the PTE-Mo module (Fig. 6d). Another chemokine, IP-10, was dramatically reduced in the PTE-Mo, particularly in those treated with LPS, Gardiquimod and CpG 2006 (Fig. 6e). These data suggest that the relatively few non-monocytic cells in the PTE module may play a complementary role in TLR-triggered cytokine and chemokine production.

Figure 6.

Figure 6

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Purified monocytes diminish the effect of Toll-like receptor (TLR) agonists on cytokine/chemokine production in the peripheral tissue equivalent (PTE) module. Peripheral blood mononuclear cells (PBMC) or purified monocytes were applied onto the human umbilical vein endothelial cell (HUVEC) monolayer to establish the PTE-PBMC module and the PTE-monocyte (Mo) module, respectively. After removal of the non-migrated cell fraction, the PTE modules were incubated in the presence or absence of CpG 2006 (5 μg/ml), Poly I : C (50 μg/ml), Gardiquimod (Gar, 5 μg/ml), or lipopolysaccharide (LPS; 10 ng/ml) for 48 hr. The cytokine levels in culture supernatants were analysed by the Bio-plex cytokine array. The numbers shown in the bar graphs (a–e) represent the mean ± SE of four or five different donors.

Non-monocytic cell populations contribute to TLR-induced DC generation and non-DC APC activation in the PTE module

As TLR agonists were observed to evoke different cytokine profiles in the PTE-PBMC and PTE-Mo modules, we next wished to determine if myeloid APC and DC derived from PTE-Mo and PTE-PBMC modules would also respond differently to TLR stimulation. The percentage of DC derived from the PTE-Mo module was less than half that in the PTE-PBMC module in unstimulated conditions. The TLR-induced DC generation was significantly reduced by 30–60% in the PTE-Mo module compared with the PTE-PBMC module (Fig. 7a). Surprisingly, despite decreased DC generation, the induction of CD83, CD86 and CCR7 on DC by TLR stimulation remained similar in the PTE-Mo module (Fig. 7b). These data suggest that the small population of non-monocytic cells in the PTE module participate in DC generation but may not be essential for DC maturation.

Figure 7.

Figure 7

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Purified monocytes diminish the Toll-like receptor (TLR) agonist-induced dendritic cell (DC) generation and antigen-presenting cell (APC) activation in the peripheral tissue equivalent (PTE) module. PTE-peripheral blood mononuclear cells (PBMC) and PTE-monocytes (Mo) were established and treated as described in Fig. 4. After 48 hr, the reverse transmigrated (RT) cells were carefully harvested and stained with fluorochrome labelled antibodies. PTE-DC and CD14+ APC are defined as described in Fig. 3. The percentage of PTE-DC in total PTE-APC are shown in (a), and the expression of CCR7, CD83 and CD86 on PTE-DC and CD14+ APC are shown in (b) and (c), respectively. The numbers shown in the bar graphs represent the mean ± SE of four or five different donors, and differences between PTE-Mo and PTE-PBMC were analysed using the paired Student’s t-test. *P < 0·05; **P < 0·01.

Furthermore, the PTE-Mo module dampened the activation of CD14+ APC. The expression of CD83, CD86 and CCR7 on CD14+ APC in unstimulated PTE-Mo modules was approximately half that observed in PTE-PBMC modules. Although Gardiquimod was the most potent inducer of CD14+ APC activation, the enhancement of CD83, CD86 and CCR7 on APC was reduced by over 50% in PTE-Mo modules compared with PTE-PBMC modules (Fig. 7c). In contrast, LPS-induced expression of activation markers on CD14+ APC remained similar in both PTE-PBMC and PTE-Mo modules except for CD83, which was significantly reduced in the PTE-Mo module (Fig. 7c). CpG 2006 and Poly I:C both moderately activated non-DC APC by inducing CD86 in the PTE-PBMC module; however, their effect was partially or completely abolished in the PTE-Mo module. These data indicated that the relatively few non-monocytic cells in the PTE module may contribute to TLR-induced DC generation and APC activation.

Plasmacytoid DC recovers the immunoregulatory effect of Gardiquimod in the PTE module

Plasmacytoid DC are the key cell population for producing type I IFN and mediating TLR7/TLR9-triggered immune responses.26 Considering that Gardiquimod is a particularly potently TLR7 agonist,27 we wanted to determine if the impaired effects of Gardiquimod on DC development and cytokine production in the PTE-Mo module were the result of the lack of a pDC population, which are always depleted by CD33-positive selection. We first tested whether the removal of pDC alone was sufficient to change Gardiquimod-induced DC maturation in the PTE module. The up-regulation of CCR7 and CD86 on PTE-APC induced by Gardiquimod in the PTE-PBMC module were markedly diminished when pDC were selectively depleted from the PBMC fraction (Fig. 8a). However, when pDC were added back into the PTE-Mo module, Gardiquimod-induced DC generation and the expression of CD86 and CCR7 were effectively rescued (Fig. 8b,c). These data indicated that the small number of pDC remaining in the PTE module crucially contribute to Gardiquimod-induced PTE-DC differentiation and APC activation.

Figure 8.

Figure 8

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Plasmacytoid dendritic cells (pDC) are involved in Gariquimod-induced DC development and antigen-presenting cell (APC) activation in peripheral tissue equivalent (PTE) modules. Different PTE modules were established either using peripheral blood mononuclear cells (PBMC) versus PTE. PBMC depleted with pDC (a), or using purified monocytes (CD33+ cells) versus purified monocytes with pDC (CD33/pDC) (b, c), as described in the Materials and methods. The PTE modules were treated with medium only or Gardiquimod for 48 hr. The reverse transmigrated (RT) cells were carefully harvested and labelled with a mixture of antibodies, including the lineage markers allophycocyanin-CD3, -CD19, -CD56 and the DC relevant markers, allophycocyanin-Cychrome 7-CD14, peridinin chlorophyll protein-Cychrome 5.5-HLA-DR, phycoerythrin-Cychrome 7-CCR7, fluorescein isothiocyanate-CD83 and phycoerythrin-CD86. The expression of CCR7, CD83 and CD86 on PTE-APC are shown in (a) and (c) (a representative of three independent experiments, using different donors) and the percentage of PTE-DC is shown in (b). Filled area: unstained control; dash line: isotype control; thin line: medium-treated group; thick line: Gardiquimod-treated group.

Gardiquimod enhances TT-specific IgG responses in vitro

The major function of APC is to present antigen to T cells and initiate adaptive immunity. As TLR agonists could enhance the generation and/or maturation of APC and DC, we next investigated if adjuvant-treated PTE-APC could facilitate antigen-specific immune responses. For these studies, we used Gardiquimod because it is a novel TLR7 ligand and has been shown to potently enhance the maturation of PTE-APC. The RT cells were collected from the PTE module treated with either culture medium alone or with TT ± Gardiquimod for 48 hr. Consistent with previous results, Gardiquimod alone significantly enhanced DC generation and maturation; TT alone also markedly increased the DC generation and promoted the expression of CD86 and CCR7 on DC. The combination of TT and Gardiquimod resulted in the highest percentage of CD86+ CCR7+ DC (Fig. 9a), indicating that TT synergized with Gardiquimod in the generation and maturation of PTE-DC. When co-cultured with autologous T cells and B cells, PTE-DC treated with Gardiquimod and/or TT did not impact TT-specific T-cell and B-cell proliferation (data not shown). However, TT-primed PTE-DC significantly induced the anti-TT IgG response, suggesting that these DC were competent APC. Notably, the PTE-DC treated with TT + Gardiquimod elicited about a twofold increase in the number of anti-TT IgG-secreting cells (Fig. 9b). Clearly, administration of TLR agonists in the PTE module not only promoted the generation and maturation of PTE-DCs, but also facilitated a more pronounced antigen-specific B-cell response.

Figure 9.

Figure 9

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Co-administration of Gardiquimod with tetanus toxoid (TT) in the peripheral tissue equivalent (PTE) module enhances the anti-TT immunoglobulin G (IgG) response. The PTE module was cultured in medium alone or in the presence of TT (1 μg/ml) ± Gardiquimod (Gar, 5 μg/ml). After 48 hr, the reverse transmigrated (RT) cells were carefully harvested and stained with fluorochrome labelled antibodies. The percentage of PTE-dendritic cells (DC) in the total PTE-antigen-presenting cell (APC) fraction and the expression of CCR7 and CD86 on DC are shown in (a). RT cells were co-cultured with 2·5 × 105 autologous CD3 T cells and 2·5 × 105 autologous B cells at an APC : T cell ratio of 1 : 30 in a 96-well plate. After 7 days, the cells were harvested and the anti-TT IgG response was evaluated using an enzyme-linked immunosorbent spot-forming cell assay. The numbers shown in (b) represents the mean ± SE of three or four different donors, and differences between the unstimulated and stimulated groups were analysed using the Student’s t-test. *P < 0·05.

Discussion

Vaccination is currently the most cost-effective and successful approach to prevent infectious disease in the world. Adjuvants are an important part of vaccines and are frequently used to enhance vaccine immunogenicity. Over the past few decades, numerous adjuvants have been discovered, but only a few are licensed for human use.28 The limited application of new vaccine adjuvants can be attributable to the paucity of reliable evaluation systems. Although human clinical trials are the most reliable way to evaluate vaccine/adjuvant safety and efficiency, the cost and sample sizes required for validation are daunting.29 Some in vitro assessments, such as PBMC culture assays and/or cytokine-derived DC culture assays, are used for screening and validating adjuvants. Unfortunately, results from these in vitro studies do not provide a complete immunological picture and as such have been poor predictors of response because of functional limitations. Therefore, it is desirable to have a new reliable test bed to evaluate adjuvants. In the present study, the immunopotency of TLR agonists using the PTE module, a tissue engineered in vitro immunological model, was tested. The PTE module has been reported as a novel autonomous DC-generating system that more closely resembles in vivo physiological conditions5,10 than other models. Results reported herein show that the PTE module is highly sensitive to TLR stimulation and may be a reliable test bed for adjuvant evaluation.

Compared with conventional PBMC cultures, the PTE module produced significantly higher levels of cytokines in response to TLR stimulation, suggesting that it has a better sensitivity. The increased sensitivity of the PTE module may be partly the result of the enrichment of APC (monocytes and DC), which are the major cell population expressing TLR. Muller et al.30 demonstrated that over 97% of migrated cells in a similar PTE module were monocytes. We report that about 80% of live RT cells were DR+ APC. Enriched APC in the PTE module can amplify the stimulatory signals of TLR, and therefore will help to assess the immunoregulatory potency of TLR agonists and other adjuvant candidates. Moreover, endothelial cells (HUVEC) play a key role in TLR-triggered DC differentiation and cytokine production in the PTE module. Although endothelial cells are classically regarded as non-immune cells, they can produce a variety of inflammatory mediators (chemokines and adhesion molecules) upon stimulation, and mediate local recruitment and migration of leucocytes. Previous studies have shown that endothelial cells express certain levels of TLR2, -3, -4, -5 and -9, and can be stimulated by TLR agonists to release cytokines and chemokines.10,30 More importantly, the presence of endothelial cells synergized with monocytes to produce cytokines, and enhanced monocyte activation in a co-culture system.31 In addition, the cell–cell contact between endothelial cells and monocytes was found to be critical for the rapid DC differentiation and antigen-induced DC maturation in vitro.5,6 In the present study, Poly I:C (TLR3) and LPS (TLR4) induced production of IL-6 and IL-8 about two- to threefold in the HUVEC cultures, indicating a direct stimulation of HUVEC by TLR agonists. More importantly, compared with the conventional PBMC culture, the PTE module produces significantly higher levels of cytokines after TLR stimulation, confirming that HUVEC and migrated monocytic cells synergistically contributed to TLR-induced cytokine production in the PTE module.

Furthermore, the PTE module is capable of elucidating the effect of TLR agonists on both DC differentiation and maturation. Both LPS and Poly I : C have been shown to fully induce the maturation and the activation of cytokine-cultured DC.32,33 However, their effect on DC generation remains unclear. The present study shows that LPS up-regulates the expression of CD83, CD86 and CCR7 on PTE-DC, consistent with observations in cytokine-cultured DC. Moreover, LPS significantly increases the percentage of DC in the PTE module. Hence, LPS promotes both DC generation and maturation in the PTE module. On the other hand, although Poly I : C induces CD86 expression on PTE-DC, it does not significantly regulate the percentage of DC produced. These data suggest that Poly I:C can moderately induce PTE-DC maturation without impacting DC generation. Surprisingly, our results showed a weaker effect of Poly I : C on PTE-DC maturation in comparison with that shown in cytokine-cultured DC, which may be to the result of the heterogeneity of the PTE-APC population, consisting of ∼ 20% PTE-DC and 80% monocyte-like APC. As human TLR3 has been shown to be restricted to freshly isolated mature DC and cytokine-derived DC,34 the presence of monocyte-like APCs may diminish the stimulatory effect of Poly I:C in the PTE module. In fact, in vivo DC have been shown as a heterogeneous population. Morelli et al.10 reported that migrating DC from skin tissue were heterogeneous and contained both dermal DC and Langerhans cells (LC) as well as a subpopulation (∼ 20%) of CD14+ LC precursors. Additionally, cells in healthy human superficial lymph are known to contain three- to fourfold more monocytes than DC.10 Therefore, although the heterogeneity of PTE-APC diminished the extent of Poly I:C-induced DC maturation, it may more closely resemble in vivo physiological conditions.

The CpG ODNs are synthetic oligonucleotides containing unmethylated CpG motifs, which can mimic bacterial DNA and stimulate both innate and adaptive immunity.16 Latest clinical studies also indicated that co-administration of CpG ODN with vaccine was safe and effectively increased vaccine-specific immune responses in humans.35 The immunostimulatory effects of CpG ODN are mediated by intracellular TLR9 expression predominantly in pDC and B cells.36 The recognition of CpG ODN by TLR9 can directly activate B cells and pDC, up-regulating type I IFN production.37 Moreover, CpG ODN are known to induce the differentiation of monocytes into DC via pDC-derived type I IFN.38 Interestingly, although cytokine-cultured DC did not respond to CpG ODN, CpG significantly induces differentiation and maturation of freshly isolated peripheral blood DC.37 The different responses of cytokine-cultured DC compared with in vivo DC implies that there are also functional differences. In the present study, the application of CpG 2006 increased PTE-DC generation by about 50%, and moderately enhanced PTE-DC maturation by inducing CCR7 expression. These data are consistent with observations made in fresh-blood DC.37 On the other hand, CpG 2006 failed to augment DC generation in the PTE-Mo modules, suggesting that CpG-induced PTE-DC generation should require the involvement of pDC, which account for 1–2% of live migrated cells in the PTE-PBMC module but were absent in the PTE-Mo module. Compared with conventional PBMC cultures and cytokine-derived DC, the PTE module shows better sensitivity in response to CpG 2006, and therefore could serve as a promising test bed for evaluating the immunopotency of different CpG motifs.

Gardiquimod, as a novel TLR7 ligand, has been shown to enhance vaccine-induced immune responses in animal models.39 However, the role of TLR7 in DC differentiation and maturation remains controversial. Ito et al.40 reported that TLR7 is expressed on freshly isolated mDC and pDC, and the treatments with TLR7 ligands, such as Imiquimod and Resiquimod, significantly enhances expression of CD80 and CD86 on freshly isolated DC. In contrast, cytokine-induced iDC neither express TLR7 nor respond to TLR7 agonists.41,42 Furthermore, co-administration of TLR7 agonists with GM-CSF suppressed differentiation and maturation of cytokine-derived DC.43 These conflicting observations between cytokine-cultured DC and freshly isolated DC further supports the theory that functional differences exist between cytokine-derived and in vivo DC. Interestingly, although cytokine-induced iDC did not respond to TLR7 agonist, priming iDC with type I IFN induces TLR7 expression and sensitizes iDC to TLR7 stimulation.42

In the present study, Gardiquimod did not affect the maturation of traditional cytokine-cultured DC; however, it did enhance PTE-DC generation and maturation as well as cytokine/chemokine production. Notably, the immunoregulatory effects of Gardiquimod were significantly reduced when PBMC were replaced with purified monocytes in the PTE module. Analysis of whole PBMC selectively depleted of pDC indicates that reduced sensitivity of the PTE module in response to Gardiquimod could be the result of the absence of pDC in the PTE module. Furthermore, the reconstitution of pDC effectively rescued the immunoregulatory effect of Gardiquimod in the PTE module. Taken together, our studies indicate that pDC are indispensable for Gardiquimod-triggered immune responses in the PTE module.

In addition to promoting PTE-DC development and inducing cytokine production, Gardiquimod co-administered with TT in the PTE module also significantly increases the production of anti-TT IgG antibodies, which is consistent with the results in animal models.39 The correlation between the PTE-DC development and antibody production suggests that adjuvant-triggered immune responses in the PTE module could be used to predict their modulatory effects on the downstream antigen-specific immune response.

Overall, the PTE module shows marked sensitivity to TLR stimulation. Application of TLR agonists into the PTE module not only induces cytokine and chemokine production, but also promotes DC differentiation and maturation. Moreover, the PTE module mimics the generation of DC that functionally resembles in vivo DC. Hence, the tissue-engineered PTE module may serve as a sensitive and reliable in vitro immunological model for adjuvant assessment.

Glossary

Abbreviations

APC

antigen-presenting cells

DC

dendritic cell

ELISPOT

enzyme-linked immunosorbent spot-forming cell assay

GM-CSF

granulocyte–macrophage colony-stimulating factor

HBSS

Hanks’ buffered salt solution

HUVEC

human umbilical vein endothelial cell

iDC

immature DC

IFN

interferon

IgG

immunoglobulin G

IL-4

interleukin-4

IP-10

interferon inducible protein-10

LC

Langerhans cells

LPS

lipopolysaccharide

mDC

mature DC

ODN

oligonucleotides

PBMC

peripheral blood mononuclear cell

pDC

plasmacytoid DC

PE

phycoerythrin

Poly I:C

polyinosine-polycytidylic acid

PTE module

in vitro peripheral tissue equivalent module

PTE-DC

PTE-derived DC

RANTES

regulated on activation, normal T-cell expressed and secreted

RT

reverse transmigrated

TLR

Toll-like receptor

TNF

tumour necrosis factor

TT

tetanus toxoid

Disclosures

G.J.R. has received stock options from VaxDesign. G.J.R. and Mount Sinai School of Medicine (MSSM) have applied for a patent with VaxDesign for technology in which vascular and connective tissue is reconstructed from human cells for the purposes of vaccine testing and selection. If this technology were licensed to a commercial entity, then G.J.R. and MSSM would benefit financially.

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