A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo

Authors


Abstract

Protein-based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non-toxic analogue of lipopolysaccharide. In mice, in comparison with non-formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T-cell responses to HIV gag-p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC-205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen-specific T-cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL-12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T-cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.

Introduction

The engineering of subunit proteins to produce protective vaccines against infectious diseases and cancer represents an exciting new area of research. Such vaccines can be injected repeatedly yet offer safety and ease of production 1. However when given alone, protein vaccines often lack the necessary immunogenicity to induce a protective response 2–4. The addition of adjuvants provides a means to initiate, direct, and sustain the immune response 5. Despite the success of currently approved adjuvants for generating protective antibody responses to viral and bacterial infections, there is still no effective adjuvant to generate strong T-cell immunity. Many components that activate the innate immune system are being tested, particularly synthetic compounds that are meant to mimic the presence of a microbe, but the research has emphasized studies with in vitro systems or transgenic mouse models 6–12.

DCs are the main antigen presenting cells for initiating immunity. The engagement of innate signaling receptors on DCs leads to cytokine and chemokine secretion, one consequence being the upregulation of costimulator molecules like CD86, to drive T-cell priming 13. Cytokines secreted by DCs further polarize the T cell to produce protective or “effector” products like IFN-γ 14. Also microbial products trigger DC migration to the T-cell areas of lymphoid organs, an effective site to select rare clones of antigen-specific, naïve T cells from the recirculating repertoire 15, 16. This intricate differentiation process that allows DCs to initiate immunity is called maturation. Maturation has generally been defined by high expression of costimulatory molecules and production of inflammatory cytokines in vitro, but to understand adjuvant action, it is necessary to study their effects on DCs in intact animals and, in addition to monitoring changes in DC phenotype (“phenotypic maturation”), prove that the DCs have become immunogenic or “functionally mature” for primary immune responses in vivo.

DCs express a variety of innate receptors, including toll-like receptors (TLRs) that signal the presence of microbial and viral products and trigger DC maturation 14. Lipopolysaccharide (LPS), found in the outer membrane of Gram-negative bacteria, is a natural agonist for TLR4 signaling of DCs 17. However, the toxicity of LPS precludes its use as a vaccine adjuvant in humans 18, 19. Chemical derivatives of LPS, especially its hydrophobic lipid A, have been synthesized to maintain immunogenicity while reducing toxicity 5. The LPS derivative, monophosphoryl lipid A (MPLA), was created through chemical modifications to the lipid A portion of LPS from the Salmonella minnesota R595 strain 20. MPLA adsorbed to alum, named Adjuvant System 04 (AS04) and owned by GlaxoSmithKine, is currently used in both Fendrix for hepatitis B and Cervarix for human papilloma virus 3, 21 vaccines. These vaccines are well tolerated and safe for human use, and generate high titers of antibodies conferring seroprotection to infection 20, 22, 23. In addition, when added to DCs in vitro, MPLA increases cell surface expression of costimulatory molecules as well as migration to lymph nodes and production of inflammatory cytokines 24, 25. MPLA promotes a Th1-cell immune response in an ovalbumin-specific TCR transgenic system 6, 25. However, in contrast to Mata-Haro et al. 6, we have previously found that MPLA and LPS are relatively weak adjuvants for inducing CD4+ T-cell responses from the polyclonal repertoire of intact mice, while still able to induce strong antibody responses 4, 26.

Glucopyranosyl lipid A (GLA) is a new synthetic lipid A agonist that combines six acyl chains with a single phosphorylation site. GLA has been formulated as a proprietary stable oil-in-water emulsion (GLA-SE) as well as in an aqueous form 27. GLA has already exhibited a good safety profile when tested in combination with the Fluzone vaccine against influenza in monkeys and a recently completed phase I trial 28. In mice, GLA-SE in combination with Fluzone enhanced vaccine-specific antibody responses and hemagglutination-inhibition titers, compared with emulsion alone and GLA as an aqueous formulation with Fluzone. Furthermore, Fluzone plus GLA-SE induced a Th1 type cell-mediated response with IFN-γ and IL-2 production, whereas Fluzone plus the emulsion alone induced a predominant Type 2 response 27, 28. However, the effects of GLA-SE on DCs in vivo have not been examined.

To understand how the new chemically defined GLA-SE adjuvant works, we have studied T-cell and antibody responses to the HIV gag p24 protein delivered within a monoclonal antibody to the DC endocytic receptor (DEC)-205, an uptake receptor, on DCs versus non-targeted gag p24. Protein vaccines are inefficiently captured by antigen presenting cells 29 but targeting vaccine proteins to DEC-205 enhances antigen presentation greater than 100-fold 26, 30, 31. Here we will show that GLA-SE serves as an adjuvant for the induction of antibody and T-cell responses to a HIV gag p24 protein in mice, including Th1 type CD4+ T cells in the intestinal mucosa. We find that DCs are required for adjuvant action, and that the GLA-SE adjuvant quickly renders the DCs functionally mature or immunogenic in vivo.

Results

GLA-SE is an active adjuvant for a Th1 type CD4+ T-cell response to a protein vaccine

To test the efficacy of GLA-SE as an adjuvant, we immunized mice with anti-DEC-HIV gag p24 or non-targeted gag-p24 protein along with GLA-SE twice i.p. over 4 weeks. After 1 week, antigen-specific T-cell responses were evaluated by IFN-γ secretion in response to re-stimulation with gag p24 15-mer peptides by flow cytometry. GLA-SE was an efficient adjuvant for the generation of gag-specific CD4+ T-cell responses in spleen and lymph nodes (Fig. 1 A and B, respectively). We had previously shown that LPS and its analogue MPLA were weak adjuvants for inducing CD4+ T-cell responses to HIV gag p24 delivered within anti-DEC antibody when compared with poly IC as the adjuvant 4, 26. Similar results were obtained when we used GLA-SE as an adjuvant and injected the protein vaccine s.c. (Supporting Information Fig. 1).

Figure 1.

GLA-SE is an effective adjuvant for the generation of Th1-cell responses. Mice were primed and boosted 4 weeks apart with 5 μg anti-DEC-p24 or 20 μg of untargeted p24 protein and 20 μg GLA-SE. After 1 week, IFN-γ secretion in gated CD3+ CD4+ T cells from (A) spleen, (B) lymph nodes, (C) lungs, and (D) lamina propia in response to gag p24 peptide mix or control gag p17 peptide mix was measured. Gating strategy is shown in Supporting Information Fig. 4. (E) As in (A), bulk splenocytes were stimulated with gag p24 peptide mix or control peptide mix for 3 days and the concentrations of the indicated cytokines measured in culture supernatants by Luminex, using duplicate samples and ELISA. Data are shown as mean+SD of n=6 mice from two experiments.

To test if GLA-SE as an adjuvant could induce cell-mediated immune responses at a mucosal site, as is likely helpful to protect against certain diseases, we assessed the presence of antigen-specific T cells in the lungs and lamina propia of mice immunized by the s.c. route. Surprisingly, after injection of anti-DEC-HIV gag p24 or nontargeted gag-p24 protein along with GLA-SE, we could detect gag-specific CD4+ T cells in a magnitude similar to four times bigger than spleen and lymph nodes (Fig. 1C and D).

To evaluate the type of cellular response induced by GLA-SE to a protein vaccine, we measured the production of Th1, Th2, and Th17 cytokines in supernatants of splenocytes stimulated with p24-peptides. In agreement with a previous publication using GLA-SE to adjuvant Fluzone vaccine 27, we found that gag-specific T cells induced by GLA-SE produced IFN-γ but not IL-17 or Th2 cytokines, verifying that GLA-SE allows a protein vaccine to induce a polarized Th1 T-cell response (Fig. 1E).

Induction of robust antibody responses with GLA-SE

To determine if the new synthetic TLR4 agonist GLA-SE could also generate a robust antibody response to protein vaccines, the sera of mice immunized with GLA-SE and anti-DEC-HIV gag p24 vaccine or nontargeted gag-p24 were assayed for anti-HIV gag antibody by ELISA. As expected from prior work with Fluzone, GLA-SE but not SE alone, adjuvanted strong antibody responses (Fig. 1A). Specific IgG1, IgG2b, and IgG2c titers against p24 antigen were detected with the GLA-SE adjuvant but not with the control emulsion (Fig. 2B–D). It is known that LPS as well as its analogue, MPLA, are good adjuvants for antibody responses 4, 32, 33. Our results indicate that GLA-SE is also effective at inducing antibody responses.

Figure 2.

Antibody response to a protein vaccine with GLA-SE as adjuvant. Mice were primed and boosted 4 weeks apart with 5 μg anti-DEC-p24 or 20 μg of untargeted p24 protein and 20 μg GLA-SE. One week after boost, (A) total gag-specific antibodies in serum as well as (B) subclass IgG1, (C) IgG2b, and (D) IgG2c were measured by ELISA. Data are given as mean+SD OD of n=4 mice from two independent experiments.

Adaptive immune responses induced by GLA-SE are dependent on TLR4

To prove that TLR4 was required in vivo, we assessed GLA-SE function in WT and TLR4−/− mice and found that similar to LPS, HIV-gag-specific T-cell and antibody responses were abolished in TLR4-deficient mice (Fig. 3A–C). Thus GLA-SE, a nontoxic derivative of LPS that is known to signal through TLR4 in vitro 34, 35, also requires TLR4 to act as an adjuvant in vivo.

Figure 3.

TLR4 requirement for GLA-SE adjuvanticity WT and TLR4−/− mice were injected i.p. with 5 μg of anti-DEC-p24 and 20 μg of GLA-SE. Mice were boosted with the same conditions at 4 weeks. After 1 week, HIV gag-specific, CD3+ CD4+ T cells were analyzed for IFN-γ in (A) spleen and (B) LNs by flow cytometry, and (C) total anti-gag antibody response by ELISA.

DCs are required for T-cell priming

To begin to obtain evidence that DCs were required for the adjuvant action of GLA-SE, we compared the response of DEC-targeted HIV gag p24 with soluble HIV p24 protein. All concentrations of anti-DEC-HIV gag p24 tested, 0.5–5.0 μg of fusion antibody corresponding to about 0.15–1.5 μg of gag protein, induced a similar CD4+ T-cell response. In contrast, a comparable strong immune response could only be detected with a high concentration, 15 μg, of soluble gag p24 protein (Fig. 4A and B).

Figure 4.

Requirement of DCs for the adjuvant effect of GLA-SE. (A) Mice were primed and boosted 4 weeks apart with different concentrations of anti-DEC-p24 and untargeted p24 protein plus 20 μg GLA-SE. After 1 week, IFN-γ secretion in gated CD3+ CD4+ T cells was measured in (A) spleen and (B) lymph nodes. (C–E) Two days prior to immunization, DT was injected s.c. to deplete DCs. CD11c-DTR and WT mice were immunized and Gag-specific T-cell responses in (C) spleen and (D) lymph node and (E) the antibody response in serum were measured as previously described. Data are shown as mean+SD of n=6 and are representative of two experiments.

To probe the essential role of DCs in T-cell priming in the intact animal, we ablated CD11chi DCs by administration of diphtheria toxin (DT), to CD11c-DTR bone marrow chimeras 36. The use of chimeras limits the toxicity of DT in CD11c-DTR mice. The CD11c-DTR and WT mice were treated with 100 ng of DT s.c. and 2 days later, at the time of vaccination, no DCs could be detected in spleen and lymph nodes (Supporting Information Fig. 2). Following vaccination, CD4+ T-cell responses did not develop if DCs were depleted with DT treatment of CD11c-DTR chimeras, whereas DT injection had no effect on nontransgenic WT bone marrow chimeras (Fig. 4B and C). Interestingly, depletion of CD11c+ cells had no effect on antibody responses (Fig. 4D). Thus the new GLA-SE adjuvant requires DCs for adaptive T-cell responses to take place but is less dependent on DCs for inducing antibodies.

GLA-SE generates immunogenic DCs

To test whether GLA-SE induces DC maturation in vivo, mice were injected s.c. with 20 μg of GLA-SE, SE control emulsion or PBS. After 6 or 18 h, spleen and lymph nodes were harvested and expression of costimulatory molecules (CD40, CD80, and CD86) analyzed on CD11chi MHCII+ cells by flow cytometry as showed in Supporting Information Fig. 3. GLA-SE-treated splenic DCs upregulated the expression of costimulatory molecules, especially CD86, as early as 6 h after injection, while in lymph nodes upregulation of CD40, CD80, and CD86 was evident after 18 h (Fig. 5A). DC maturation was dependent on the GLA since injection of the emulsion alone (SE) did not upregulate costimulatory molecules (Fig. 5A). In parallel experiments, to evaluate the profile of cytokines produced by DCs 4 h after s.c. injection of 20 μg of GLA-SE or SE control emulsion, purified CD11chi MHCII+ cells were incubated for an additional 18 h in vitro. As expected, the stimulated splenic DCs produced many inflammatory cytokines, in particular IL-12p70 (Fig. 5B). Therefore GLA induces two cardinal features of DC maturation, changes in cell surface costimulatory molecules and production of IL-12p70 and other cytokines. Since the magnitude and the nature of the T-cell response depends, to a large extent, on the presence of costimulatory molecules, such as CD80, CD86, and CD40 37, 38 as well as the production of cytokines and chemokines by DCs 39, these findings indicate that GLA is stimulating the appropriate changes in DCs in vivo that should lead to immunization.

Figure 5.

GLA-SE generates functionally mature DCs. (A) Mice were injected with 20 μg of GLA-SE, SE alone, or PBS in the foot pads and 6 or 18 h later, cell surface expression of CD86, CD80, and CD40 was analyzed on CD11c+ MHCII+ DCs from spleen and lymph nodes. (B) As in (A), but 4 h after GLA administration, the CD11c+ MHCII+ DCs were isolated and incubated for an additional 18 h to detect inflammatory cytokines in the supernatant by multiplex ELISA. (A and B) Data are representative of two experiments. (C) Mice were stimulated with 20 μg of GLA-SE or SE and PBS as negative controls. Graded numbers of fixed-splenic, purified CD11c+ MHCII+ DCs from C57BL/6 mice were added for 5 days to 2×105 allogeneic BALB/c T cells and T-cell proliferation detected by CFSE dilution of CD3+ cells. Data are representative of three experiments. (D) DCs from WT or MHCII−/− mice that had been injected with GLA-SE or SE together with DEC-p24 antigen were isolated and adoptively transferred into naïve mice for prime and boost. Graph represents the percentage of CD3+ CD4+ T cells producing IFN-γ. Data are shown as means+SD of n=9 mice from three independent experiments. (A and C) Data are representative of three experiments. (B and D) Data are shown as mean+SD of n=9 and are representative of three experiments.

As a first direct proof that DCs were functionally mature, i.e. immunogenic and able to find and activate rare clones of antigen-specific T cells, we sorted CD11c+ MHCII+ DCs from the spleen and lymph nodes 4 h after injecting mice with GLA-SE or SE as control. We then fixed the cells with a short exposure to formaldehyde to block further maturation in culture, and added the DCs in graded doses to allogeneic T cells in a mixed leukocyte reaction 40. DCs from GLA-SE but not SE-treated mice became active stimulators of the allogeneic mixed leukocyte reaction, inducing robust proliferation of both CD4+ and CD8+ T cells (Fig. 5C).

To further evaluate the capacity of DCs to become immunogenic following antigen capture in vivo, mice were injected with anti-DEC-HIV gag and either GLA-SE or SE. After 4 h, splenic DCs were purified by cell sorting and injected into naïve mice i.v. In addition, to check that antigen presentation was performed by the transferred and not recipient DCs, MHCII−/− DCs were used as negative controls. Only WT DCs, after targeting with anti-DEC-gag and stimulated with GLA-SE in vivo, were capable of inducing gag-specific T-cell immunity (Fig. 5D). These data indicate that GLA induces the full maturation of spleen and lymph node DCs in vivo.

Discussion

The discovery of receptors responsible for stimulating innate immunity, such as the TLR and RIG-like receptor pattern recognition receptors, makes it possible to test chemically defined agonists as new adjuvants to trigger the DC link between innate and adaptive immunity. To understand adjuvant action, these agonists need to be characterized in vivo at the level of antigen presenting DCs. Our experiments at this direct level indicate that a synthetic TLR4 agonist, GLA-SE, serves as an effective adjuvant and enhances the capacity of DCs in vivo to immunize against protein antigens. The adjuvant role of GLA-SE was dependent on TLR4. Similar results have been reported by Baldwin et al. where GLA induced production of IL-6 by monocyte-derived DCs in culture, and this was blocked with anti-TLR4 but not TLR2 antibodies 27. Our results extend prior research by showing a complete dependency of TLR4 stimulation for the induction of adaptive responses in vivo by GLA-SE.

DCs are the major link between the innate and the adaptive immune system, and its appropriate activation and maturation by agonists for innate signaling receptors should allow for the induction of an adaptive response 41, 42. However, much of the evidence involves studies of DCs stimulated in cell culture with adjuvants 43. In the current study, we demonstrated that GLA-SE injection together with a protein antigen allows the antigen-capturing DCs to quickly become immunogenic in vivo. Enhanced T-cell responses were detected when antigen was targeted to DCs. We did not detect qualitative difference in adaptive responses between untargeted or targeted protein. However, lower doses of antigen were required using anti-DEC-HIV gag p24 to achieve detectable responses. This finding highlights the importance of DCs for initiating adaptive T-cell immunity. After showing that DCs were essential for the generation of T-cell responses in lymph nodes to an s.c. injection of protein, using selective ablation of DCs by DT treatment of CD11c-DTR mice, we found that GLA-SE leads to increased expression of surface MHC and costimulatory molecules on DCs, and high levels of IL-12p70 production by DCs, which is known to polarize CD4+ Th1 cells. Importantly, adoptive transfer of antigen-loaded DCs stimulated with GLA-SE in vivo was sufficient to induce specific Th1-cell responses in naïve mice. In contrast, DCs stimulated with emulsion alone were unable to prime T cells. Since the DCs also had to express MHCII, this indicates that their T-cell immunizing function required direct presentation of antigen in the mice primed by adoptively transferred DCs. To our surprise, antibody responses were unaltered after CD11c+ depletion. In this paper, we only analyzed total IgG responses. Maturation of DCs may still have a role in antibody affinity.

The type of immune response that eliminates an infection depends on the type of pathogen. Induction of CD4+ T-cell responses by vaccination was associated with diminished simian immunodeficiency virus (siv) replication after intrarectal challenge and decreased HIV acquisition in the Thai HIV vaccine trial 44, 45. The results presented here demonstrate that GLA-SE is an efficient adjuvant for the generation of HIV-gag-specific Th1-cell immune response. IFN-γ was produced in large amounts by antigen-specific T cells in both spleen and lymph nodes. HIV-1 vaccines will most likely need to induce mucosal immunity. Mucosal tissues are the major site of natural HIV transmission and the reservoir for HIV replication quickly leading to a rapid loss of T cells in the intestine 46, 47. In addition, Th1 type CD4+ T cells are known to improve the mobilization of the cognate antigen-specific CD8+ T cells to a site of infectious challenge 48, 49. Thus GLA-SE has the capacity to adjuvant a protein vaccine to induce mucosal immunity that potentially is valuable to limit viral replication and curtail systemic dissemination. Previous studies successfully showed that local immune responses were able to prevent virus spread from the gut mucosa into the systemic circulation 50–52. However, the general belief is that local but not systemic immunization is required to induce robust mucosal responses 53–55. Interestingly, we found that s.c. injection of the GLA-SE and anti-DEC-HIV gag p24 vaccine was able to induce strong mucosal T-cell responses.

Immunization with HIV-gag targeted or untargeted protein plus GLA-SE induced a broad range of different antibody isotypes and therefore a combination of Th1 and Th2-cell responses. This contrast, i.e. with polarized Th1 T-cell responses, may be explained by the different requirement for DC priming. This result is consistent with previous studies where addition of GLA-SE gives a mixed Th1/Th2-cell response but also increases the IgG2/IgG1 ratio to an existent M. Tuberculosis and Influenza vaccine 27, 56. Interestingly, HIV-specific IFN-γ-producing CD4+ Th1 cells combined with IgG2 antibodies have been associated with a better control of HIV replication and act as better predictors of long-term nonprogression in HIV-1 infection than virus parameters 57, 58. No specific immune response was detected with SE used to formulate the GLA in our studies. Oil-in-water emulsion is considered an adjuvant by itself (e.g. MF59) and is believed to form a depot at the injection sites protecting the antigen from clearance, allowing its slow long-term release into the surrounding tissues and prolonging the duration of the interaction between antigen and the responding cell 59, 60. Formulations are also believed to enhance solubility and stability of adjuvants. For example, unformulated MPLA is insoluble and forms aggregates 61. We could not detect any difference in cell recruitment and lymph node inflammation between MPLA and GLA-SE supporting the second notion. Under this context, it is possible that formulation of MPLA with SE may increase T-cell responses. However, our paper focuses on the immune response induced by GLA-SE, a clinical feasible adjuvant, and its capacity to render DC maturation in vivo.

In addition to showing the capacity of a vaccine adjuvant to render DCs immunogenic in vivo, our results provide ways to help identify those innate stimuli and their combinations that can provide the link between innate and the desired adaptive immunity.

Materials and methods

Mice

C57BL/6, B6.TLR4−/−, and CD11c-DTR mice were purchased from Jackson Laboratory. Mice in specific pathogen-free conditions were studied at 6–10 weeks according to institutional guidelines and approval of the Rockefeller University institutional animal care and use committee (IACUC).

DC innate responses

Mice were injected s.c. with 20 μg of GLA-SE or as control, oil-in-water SE (Immune Design, Seattle, WA). Spleens and lymph nodes were collected 6 or 18 h later and treated with collagenase D (400 U/mL) for 20 min at 37°C. DC maturation was analyzed by increased expression of CD80, CD86, and CD40 after gating on CD11c+ MHCII+ DCs. For cytokine production, spleens were harvested 4 h after in vivo stimulation. CD11c+ MHCII+ DCs were purified by cell sorting (FACSAria; BD Biosciences) and plated at 5×104 cells/well in a 96-well plate for 18 h prior to assay of cytokines in the supernatants by multiplex ELISA (Meso Scale Discovery, Gaithersburg, MD).

DC antigen presentation

To test allostimulatory capacity, spleen and node CD11c+ MHCII+ DCs were cell-sorted 12 h after GLA-SE or SE injection. C57BL/6 DCs were fixed with 1% PFA (paraformaldehyde) for 10 min at 4°C and added in graded numbers to 2×105 carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled (Molecular Probes, Eugene, OR) Balb/C T cells. After 5 days, cell proliferation was analyzed by CFSE dilution in CD3+CD4+ cells. For DC antigen presentation in vivo, WT and MHCII−/− mice were injected with 5 μg of gag-p24 together with 20 μg of GLA-SE or control adjuvant SE. After 4 h, splenic CD11c−/− DCs were purified and adoptively transferred into naïve mice (i.v). Antigen-specific responses were evaluated by intracellular IFN-γ after prime-boost.

Vaccination and immune cell responses

Mice were immunized as previously described with some modifications 26. Briefly, mice were primed and boosted with 5 μg of HIV gag-p24 and 10 μg of HIV gag-p24 plus 20 μg of GLA-SE or adjuvant negative control SE. For CD11c-DTR, mice were injected 2 days pre-immunization, with 100 ng of DT s.c. After 1 week, splenocytes and lymph node cells were restimulated with p24 or p17 mix as negative control and 2 μg/mL of αCD28 for 5 h in the presence of Brefeldin A (10 μg/mL; Sigma-Aldrich). Cells were stained with Live/Dead Fixable Violet viability dye, Alexa Fluor 700-α-CD3, and PerCPCy5.5-α-CD4 for 20 min at 4°C. Cells were fixed and permeabilized (Cytofix/Cytoperm Plus; BD Biosciences) and stained with allophycocyanin-anti-IFN-γ mAbs for 15 min RT (BD Biosciences). IFN-γ+ T cells were analyzed by flow cytometer (BD LSR II). Antibody titers were measured as previously described 4.

Lamina propria and lung tissue processing

To prepare single intestinal cell suspensions, part of the small bowel including jejunum and ileum, or large bowel (cecum and colon) were excised. Peyer's patches were removed from the small intestinal tissue. Intestinal lumen was exposed by a longitudinal incision and the tissue was cut to a pasty consistency. Next, intestinal tissues were incubated in Roswell Park Memorial Institute medium (RPMI) with 1.3 mM EDTA (Cellgro) in a 37°C shaker for 1 h. The supernatants containing intestinal epithelial cell (IEC) with some superficial villous cells were discarded. Tissue was washed thrice with RPMI to remove EDTA. Tissue was digested with 0.2 mg/mL of type IV collagenase (Sigma-Aldrich) at 37°C for 1 h. Tissue was then homogenized, filtered, and washed. The resulting cell suspension was layered on a 44%/66% percoll (GE Biochemicals) gradient and the interface was collected to obtain an enriched mononuclear cell population. Cells were washed and resuspended in complete medium at a density of 2–5×106 cells/mL. One week after boost, lungs were perfused with PBS and the lobes extracted and stored in PBS on ice. Lungs were minced into small pieces and digested in collagenase D (Roche) for 20 min at 37°C. Following digestion, lungs were passed through a cell strainer and centrifuged at 1500 RPM for 5 min. Recall responses were examined as described in Vaccination and immune cell responses.

Statistical analysis

Data reported in the figures represent the average of at least three independent experiments. Statistical significance was determined by unpaired t-test with 95% confidence interval. Error bars represent the means±SD. Data were analyzed and figures were generated using Prism 5 (GraphPad Software).

Acknowledgements

We are grateful to Dr. Steven G. Reed, Infectious Disease Research Institute, and Immune Design Corp., Seattle, USA, for providing GLA-SE, and we thank J. Adams for graphics. Grant support was provided by NIAID AI13013 to R.M.S., The Robert Mapplethorpe Foundation, the Human Science Frontiers Program to M.P.L., New York Community Trust's Francis Florio funds to C.C., and NCRR UL1RR024143 to A.P.

Conflict of interest: R.M.S. is an advisor to Celldex Therapeutics, which is developing DEC-targeted protein vaccines.

Ancillary