An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4+ and CD8+ T cells by bone marrow-derived DC and Langerhans cells in vitro. Whereas nonconjugated Ag was preferentially cross-presented by splenic CD8α+ DC in vivo, M1-conjugated Ag was cross-presented by all dendritic subtypes assessed. Potent effector T cell responses with antitumor activity were elicited when M1 conjugates were injected together with an adjuvant. This method of Ag delivery has significant potential in therapeutic applications.

Capture of extracellular Ags by APCs is followed by Ag processing in phagolysosomal compartments and subsequent presentation of Ag-derived peptide fragments by MHC class II molecules to CD4+ T cells (1, 2, 3). In contrast, Ags that have been synthesized intracellularly (for example viral or tumor Ags) are processed in the cytosol and presented by MHC class I molecules to CD8+ T cells (2, 3, 4). A further mechanism operates in APC in which captured extracellular Ags are diverted into the MHC class I presentation pathway to stimulate CD8+ T cells (5, 6). This process of “cross-presentation” is of key significance for vaccine-driven antiviral and antitumoral immunity in which CD8+ T cell-mediated responses are required. In murine models, it has been shown that targeted delivery of soluble extracellular Ags to distinct dendritic cell (DC)3 subsets in the spleen can lead to different pathways of Ag uptake and intracellular routing to either MHC class I or MHC class II molecules (7). The CD8α+ DC subtype has been shown to be superior at cross-presentation (8, 9), whereas CD8α DC are superior at presentation on MHC class II molecules (10).

We investigated whether Ags that are specifically targeted to MHC class II molecules on the cell surface of APC can be processed and presented to T cells to enhance tumor rejection. We reasoned whether the ubiquitous MHC class II binding capacity of bacterial superantigens could be used to efficiently deliver Ag to APC once the toxicity due to polyclonal T cell activation had been eliminated. Mutations were therefore introduced into the TCR binding domain of the superantigen streptococcal mitogenic exotoxin Z-2 (SMEZ-2) (11, 12), and a site for chemical ligation introduced to allow efficient conjugation of Ag. Analysis of Ag presentation with this novel delivery system exposed a pathway of efficient cross-presentation operating in all DC populations assessed, which can be exploited in targeted vaccination procedures.

Breeding pairs of the inbred strains C57BL/6 (CD45.2+) and B6.SJL-PtprcaPepcb/BoyJ (CD45.1+) were obtained from The Jackson Laboratory, and from the Animal Resource Centre (Canning Vale, Western Australia). OT-I mice and OT-II mice expressing transgenic Vα2, Vβ5.1/5.2 TCR-specific for an H-2 Kb-binding peptide of OVA (OVA257–264) and an I-Ab-binding peptide of OVA (OVA323–339), respectively (13, 14), were provided by Professor F. Carbone (Melbourne University, Melbourne, Australia) and B6Aa0/Aa0 MHC class II-deficient (MHC class II−/−) mice (15) were provided by Dr. H. Bluethmann (Hoffmann-La Roche, Basel, Switzerland). All mice were bred at the animal facility of the Malaghan Institute of Medical Research (Wellington, New Zealand). All experimental protocols were approved by the Victoria University of Wellington Animal Ethics Committee and performed according to institutional guidelines.

Culture medium (complete medium) was IMDM supplemented with 5% FBS, 2 mM glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME (all Invitrogen). Endotoxin-free OVA protein (16) was provided by Professor T. M. Moran (Mount Sinai School of Medicine, New York, NY). The tumor cell line used was B16.OVA cell line, a derivative of B16.F0 that expresses OVA (17). Peptides OVA257–264, OVA265–280, and OVA323–339 were from Mimotopes. The invariant CD1d-dependent NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) was manufactured as described (18) and solubilized in 150 mM NaCl, 0.5% Tween 20.

M1, mutated SMEZ-2 with no TCR binding ability, is a mutant toxoid of the wild-type SMEZ-2 isolated from Streptococcus pyogenes strain 2035 (19) containing three alterations in the TCR binding site, W75L.K182Q.D42C. The D42C mutation introduces an exposed cysteine into the former TCR binding site for direct conjugation of Ags. DM, the mutated SMEZ-2 with no TCR binding or MHC binding ability, is a protein defective in both MHC class II binding and TCR binding and contains the mutations Y18A.D42C.H202A.D204A. The ability of both M1 and DM to stimulate human T cells in an in vitro PBL stimulation assay was reduced by a factor of 105-fold. Recombinant M1 and DM proteins were produced as thioredoxin fusion proteins as previously described from a 5L fermentation from Escherichia coli (AD494(DE3)pLysS) from the vector pET32a-3C (20). The fusion protein was purified from crude bacterial lysate by Ni2+ affinity chromatography, cleaved using recombinant 3C protease, and subjected to Ni2+ affinity chromatography to remove the thioredoxin. M1 and DM proteins were further purified by cation exchange chromatography on a 50-ml column of carboxymethyl Sepharose (Amersham Biosciences) as previously described (21). Typically, 300 mg of each protein was purified from a single 5L fermentation.

Purified M1 or DM toxoid (200 nM) was resuspended in 1 ml of 50 mM Na2HPO4 (pH 7.0), and reduced by the addition of freshly prepared 1 M DTT to a final concentration of 0.1 M just before conjugation. The DTT was removed on a G10 desalting column (Bio-Rad) equilibrated with degassed PBS (pH 7.4), 1 mM EDTA. OVA (200 nM) was resuspended in 1 ml of PBS (pH 7.4) and treated for 2 h at room temperature with 2000 nM sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; Pierce Chemicals) that had been freshly dissolved in 50% dimethylformamide:water. Protein was separated from sulfo-SMCC on a G10 desalting column. Reduced M1 or DM was immediately mixed with the derivatized OVA (molar ratio of 1:1) and incubated overnight at 4°C. Conjugated protein was separated from monomers by FPLC size exclusion chromatography on a Superdex 200 GL 10/30 column (GE pharmaceuticals). High molecular weight fractions were pooled and concentrated to ∼2 mg/ml (20 nM) by ultrafiltration then subjected to Triton X-114 extraction to remove trace endotoxin (22). The OVA-conjugated proteins M1-OVA or DM-OVA were sterilized through a 0.22-μM filter and stored in aliquots at 4°C. Samples tested negative in a Limulus amoebocyte lysate test.

The modified superantigen constructs M1 and DM, and whole OVA protein, were labeled with Alexa488 (Invitrogen Life Technologies) according to the manufacturer’s instruction. The fluorescent molecules were i.v. injected into C57BL/6 or MHC class II−/− mice (50 μg per animal), and then uptake of fluorescence was assessed in different tissues by flow cytometry 3 h later. Experiments presented were repeated a minimum of two times.

Bone marrow cells were flushed from femur and tibia of mice and cultured in complete medium supplemented with 20 ng/ml IL-4 and 10 ng/ml GM-CSF. Fresh complete medium supplemented with cytokines was added every 3 days. On day 6 varying concentrations of OVA, M1-OVA, or DM-OVA were added to the cultures together with 100 ng/ml LPS. After overnight incubation, nonadherent mature bone marrow-derived DC (BMDC) were used for in vitro proliferation assays, at a concentration of 104 cells per well in a 96-well plate. LC were prepared from murine ear tissue and trunk skin that had been incubated with 0.8% trypsin (Sigma-Aldrich) for 25–45 min. The epidermal cell suspensions were then incubated for 6 h with different concentrations of OVA or M1-OVA, washed thoroughly, and transferred into fresh complete medium. LC matured spontaneously in this culture within 3 days and were enriched on a Nycodenz gradient (Sigma-Aldrich) as described (23). This method provided suspensions of LC with a purity of at least 50%, which were used at a concentration of 5 × 103 cells per well in a 96-well plate for in vitro proliferation assays.

Spleens and lymph nodes from OT-I or OT-II mice were teased through a cell strainer, RBC were lysed with ammonium chloride buffer, and then CD8+ or CD4+ cells were positively selected (from OT-I and OT-II, respectively) using magnetic beads (Miltenyi Biotec). The T cell suspensions (2 × 106 cells/ml) were then cultured with BMDC or LC for 64 h, with proliferation measured by incorporation of [3H]thymidine (1 mCi/ml; Amersham Biosciences) during the last 16 h. All experiments presented were repeated between two and four times. Statistical differences between treatment groups were determined by two-way ANOVA.

T cells were isolated from TCR transgenic OT-I and OT-II mice as described and labeled with 2 μM CFSE (Molecular Probes). Groups of three CD45.1 congenic mice (B6.SJL-PtprcaPepcb/BoyJ) were i.v. injected with a mix of CFSE-labeled OT-I and OT-II cells (2 × 106 cells of each) and then 1 day later injected i.v. with 20 pM of one of the Ags OVA, M1-OVA, or DM-OVA in the presence or absence of 200 ng of α-GalCer. T cell proliferation was assessed in splenic cell suspensions 3 days later by monitoring the decrease in CFSE fluorescence intensity as described (24). Injected T cells were identified with CD4-APC (clone GK1.5), CD45.2-PE (clone 104e, both eBioscience) and CD8-PerCP (clone Ly2; BD Pharmingen).

Mice were injected i.v. with 2 μg of either M1-OVA or DM-OVA, or with 1 or 200 μg nonconjugated OVA. Spleens were removed 16 h later and digested with Liberase and DNase I (both Roche) to provide a single cell suspension, which was then passed over a Lymphoprep gradient (Axis-Shield). An enriched DC fraction (>80% purity) was obtained by positive selection with anti-CD11c-coated magnetic beads (Miltenyi Biotec). The CD11c fraction was enriched for macrophages by plastic adherence for 2 h at 37°C. Adherent cells were washed with PBS and harvested using cell scrapers in the presence of 5 mM EDTA. A purity of >40% macrophages was achieved, as determined by staining with anti-CD68 Ab. The nonadherent cells were enriched for B cells by positive selection using anti-CD45R-PE (B220, clone 6B2, made in house) followed by magnetic sorting with anti-PE microbeads (Miltenyi Biotec). A purity of >97% B cells was achieved. A varying number of each of the APC populations was then used in in vitro proliferation assays as described.

Groups of five mice each were i.v. injected with 62 μg of either M1-OVA or DM-OVA, or with 2 mg nonconjugated OVA (∼65-fold molar excess) with or without 200 ng of α-GalCer. In some experiments lower doses of M1-OVA were used, as indicated in the text. Also, in some experiments animals were injected i.v. with 5 mg of Cyt c (Sigma-Aldrich) at 8 and 32 h before Ag administration. Spleens were removed 14–16 h after Ags were administered, digested with Liberase and DNase I to provide a single cell suspension, and enriched for DC using a Nycodenz gradient as previously described (23). Cells were further enriched with magnetic sorting (CD11c-MACS MicroBeads, clone N418; Miltenyi Biotec), followed by electronic sorting using a FACSVantage DiVA cell sorter (BD Pharmingen) and the Abs CD8α-FITC (FITC, clone Ly-2), CD4-PE (PE, clone L3T4), CD11c-allophycocyanin (clone HL3). All were purchased from BD Pharmingen. The three different splenic DC subsets (double negative CD4CD8 DC, single positive CD4+ DC, and single positive CD8+ DC) were sorted to a purity >97% for use in in vitro proliferation assays.

Magnetically sorted CD11c+ BMDC were incubated with Alexa488-labeled M1-OVA or Alexa488-labeled OVA for the indicated times, and were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with anti-early endosomal Ag (EEA)1 marker (rabbit polyclonal serum from Affinity BioReagents). The primary Ab was detected with a goat anti-rabbit secondary Ab conjugated to the fluorochrome Alexa555 (Invitrogen). The specificity of the secondary Ab was confirmed using an isotype mismatched primary Ab. Cells were visualized with Leica TCS SP2 confocal microscope (Leica Microsystems). Images were acquired at room temperature using a photomultiplier tube and Leica Confocal software. Images were processed using NIH ImageJ image manipulation software. Figures were generated using Adobe Photoshop.

The cytotoxic capacity of induced CD8+ T cell responses was measured by VITAL assay as described (25). Syngeneic splenocyte populations were loaded with 50 nM OVA257–264 peptide and labeled with 1.65 nM CFSE. A control population without Ag was labeled with 10 mM chloromethyl-benzoyl-aminotetramethyl-rhodamine (Molecular Probes). Equal proportions of each population were mixed together and injected i.v. into groups of immune and naive mice (n = 3–5). Specific lysis of the peptide-loaded targets was assessed 24 h later in lymph node tissue by flow cytometry. The mean percentage for survival of peptide-pulsed targets was calculated relative to that of the control population, and cytotoxic activity was expressed as a percentage of specific lysis using the calculation 100 − mean percentage of survival of peptide-pulsed targets.

Splenocytes were isolated from groups of immune or naive mice (n = 5) and restimulated with OVA257–264 peptide, OVA265–280 peptide, or an irrelevant peptide for 40 h in vitro. In all experiments stimulation with 10 μg/ml Con A (Sigma-Aldrich) served as a positive control. ELISPOT assays were performed using an IFN-γ ELISPOT PLUS kit according to the manufacturer’s instructions (Mabtech).

Mice were challenged with 1.5 × 105 B16.OVA tumor cells injected s.c. into the flank and, 7 days later when tumors were palpable, treated i.v. with 2 μg of either M1-OVA or DM-OVA, or with 200 μg of nonconjugated OVA (∼200-fold molar excess) in the presence or absence of 200 ng of α-GalCer. Tumor size was assessed three times per week by measuring the short and long tumor diameters using calipers, and is expressed as mean product ± SEM of tumor diameters. Five mice were used in each group. Experiments were repeated between two and four times.

We investigated whether conjugation of chicken OVA to a modified form of SMEZ-2 that has no TCR binding function (M1) would promote targeting to MHC class II+ APC and improve presentation to CD4+ and CD8+ T cells. OVA was also conjugated to a mutated form of SMEZ-2 that does not bind TCR or MHC class II (DM), to establish the degree to which Ag presentation was attributable to MHC class II binding. Varying concentrations of each of the conjugates (M1-OVA and DM-OVA), or nonconjugated OVA, were incubated overnight with BMDC and cocultured with OVA-specific CD8+ and CD4+ T cells (from OT-I and OT-II transgenic mice, respectively) to assess induction of T cell proliferation (Fig. 1,A). Approximately 10,000-fold less M1-OVA was required to achieve levels of CD4+ T cell proliferation observed with DM-OVA or nonconjugated OVA, indicating that presentation of Ag to CD4+ T cells was significantly improved by targeting to MHC class II molecules. Approximately 1000-fold less M1-OVA was required to achieve levels of CD8+ T cell proliferation observed with nonconjugated OVA. Although some of this increased activity could be attributed to targeting to MHC class II molecules, Ag presentation was also increased when OVA was conjugated to DM. This MHC class II-independent targeting activity was also observed when BMDC from MHC class II-deficient animals were used, implying the existence of an additional cellular receptor for superantigen SMEZ-2, which is currently under investigation. Proliferation of CD4+ and CD8+ T cells was not enhanced when BMDC were incubated with a combination of modified superantigen and nonconjugated OVA (data not shown), highlighting the importance of superantigen-mediated targeting of APC to improve immune responses. Presentation of M1-conjugated Ag was also assessed in LC that had been freshly isolated from the skin (Fig. 1 B). Proliferation of OVA-specific CD4+ and CD8+ T cells was increased in excess of 1000-fold when M1-OVA was used relative to nonconjugated OVA, confirming the capacity of M1 to improve Ag presentation.

FIGURE 1.

Ag conjugated to M1 is efficiently presented by DC in vitro. A, BMDC cultures were prepared from C57BL/6 or MHC class II−/− mice and supplemented on day 6 with 100 ng/ml LPS together with varying concentrations of the modified superantigen conjugates M1-OVA or DM-OVA, or OVA protein. After overnight culture the BMDC were combined with OVA-specific CD4+ or CD8+ T cells that had been positively selected from OT-II or OT-I mice, respectively, and proliferation assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. B, LC were prepared from murine epidermis, incubated for 6 h with titrated doses of OVA or M1-OVA, and then cultured with OVA-specific CD4+ or CD8+ cells as in A. Results are shown as average ± SEM of triplicate wells.

FIGURE 1.

Ag conjugated to M1 is efficiently presented by DC in vitro. A, BMDC cultures were prepared from C57BL/6 or MHC class II−/− mice and supplemented on day 6 with 100 ng/ml LPS together with varying concentrations of the modified superantigen conjugates M1-OVA or DM-OVA, or OVA protein. After overnight culture the BMDC were combined with OVA-specific CD4+ or CD8+ T cells that had been positively selected from OT-II or OT-I mice, respectively, and proliferation assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. B, LC were prepared from murine epidermis, incubated for 6 h with titrated doses of OVA or M1-OVA, and then cultured with OVA-specific CD4+ or CD8+ cells as in A. Results are shown as average ± SEM of triplicate wells.

Close modal

To establish whether the MHC class II binding capacity of M1 leads to its efficient acquisition by MHC class II+ APC in vivo, M1, DM, or OVA proteins were labeled with the fluorescent dye Alexa488 and injected into C57BL/6 or MHC class II-deficient mice (Fig. 2,A). Extensive fluorescence was associated with MHC class II+ cells in the spleens of C57BL/6 mice when M1-Alexa488 was administered, which was significantly reduced in MHC class II-deficient recipients. Only low level cellular fluorescence was seen when DM-Alexa488 or OVA-Alexa488 were used, reflecting a low efficiency uptake mechanism independent of MHC class II targeting. The cells targeted with M1-Alexa488 included B cells, macrophages, and DC (Fig. 2 B). Significant fluorescence was observed in all major splenic DC subsets (CD4+CD8 DC, CD4CD8+ DC, and CD4CD8 DC). Interestingly, injection of DM-Alexa488 resulted in weak fluorescence in DC that was greater than that observed with OVA-Alexa488, again providing evidence for an MHC-independent uptake mechanism for DM.

FIGURE 2.

M1 binds to MHC class II+ cells in vivo. The modified superantigen constructs M1 and DM, and OVA protein, were labeled with the fluorescent dye Alexa488 and i.v. injected individually into mice. A, Cellular fluorescence was assessed 3 h later in spleen tissue by flow cytometry with Abs for MHC class II and Thy1.2. B, Cellular fluorescence was assessed (open histogram) in splenic B cells (B220+), T cells (Thy1.2+), macrophages (F4/80+), and the major splenic DC subtypes (CD11c+ CD4+, CD11c+ CD8+, and CD11c+ CD4CD8). Similarly stained and gated cells in untreated animals are indicated (gray-filled histogram). C, Expression of maturation markers CD40, CD80, and CD86 and MHC class II was assessed on gated splenic DC (CD11c+ cells) from animals treated with the fluorescent molecules. Similarly stained and gated cells in untreated animals are indicated (gray-filled histogram).

FIGURE 2.

M1 binds to MHC class II+ cells in vivo. The modified superantigen constructs M1 and DM, and OVA protein, were labeled with the fluorescent dye Alexa488 and i.v. injected individually into mice. A, Cellular fluorescence was assessed 3 h later in spleen tissue by flow cytometry with Abs for MHC class II and Thy1.2. B, Cellular fluorescence was assessed (open histogram) in splenic B cells (B220+), T cells (Thy1.2+), macrophages (F4/80+), and the major splenic DC subtypes (CD11c+ CD4+, CD11c+ CD8+, and CD11c+ CD4CD8). Similarly stained and gated cells in untreated animals are indicated (gray-filled histogram). C, Expression of maturation markers CD40, CD80, and CD86 and MHC class II was assessed on gated splenic DC (CD11c+ cells) from animals treated with the fluorescent molecules. Similarly stained and gated cells in untreated animals are indicated (gray-filled histogram).

Close modal

Expression of CD40, CD80, and CD86 was unchanged on DC targeted by M1-Alexa488 (Fig. 2 C), suggesting that interaction with MHC class II molecules does not induce any obvious maturation of these cells.

We next investigated whether Ag that has been conjugated to M1 is efficiently processed and presented to T cells in vivo. As a readout of Ag presentation, we examined the proliferation of a cohort of CFSE-labeled, OVA-specific CD8+ T cells and CD4+ T cells (from OT-I and OT-II transgenic mice) that were transferred into recipients 1 day before Ag administration. Equivalent molar doses of M1-OVA, DM-OVA, or OVA were injected in the presence or absence of α-GalCer, which serves as an immune adjuvant by invoking iNKT cell-mediated activation of DC (26, 27). Proliferation of CFSE+ T cells was assessed in the spleen 72 h later by flow cytometry, as represented by progressive halving of cellular fluorescence with every cell division completed (24). In the absence of α-GalCer, injection of M1-OVA induced proliferation of both CD4+ and CD8+ T cells (Fig. 3 A). In contrast, DM-OVA or OVA alone did not elicit any T cell proliferation in absence of adjuvant. Administration of α-GalCer increased proliferation of CD8+ T cells in all treatment groups, but did not enhance CD4+ T cell responses. Some of the enhancement of CD8+ T cell proliferation was observed in the absence of Ag, which may be driven by cytokines released following iNKT cell activation (28). Overall, we conclude that Ags conjugated to M1 are efficiently presented by APC in vivo, including effective cross-presentation to CD8+ T cells.

FIGURE 3.

Ag conjugated to M1 is efficiently presented to T cells in vivo. A, OVA-specific CD4+ or CD8+ cells were positively selected from OT-II and OT-I mice, respectively, labeled with CFSE, and transferred together into congenic CD45.1+ recipients. One day later the recipients were injected i.v. with one of the following: 2 μg of M1-OVA, 2 μg of DM-OVA, or 1 μg of OVA protein (equivalent molar doses of OVA protein). Half of the animals also received α-GalCer in the injected bolus. Animals that received CFSE-labeled T cells, but were not administered with protein Ag, served as negative controls (Nil). Proliferation of the CFSE-labeled T cells was assessed by flow cytometry of spleen tissue 72 h after Ag administration, using Abs for CD45.2, CD4, and CD8. A representative profile from each of the treatment groups (n = 3 mice per group) is presented. B, Ag conjugated to M1 is efficiently presented by DC. Animals were injected with M1-OVA, DM-OVA, or OVA as in A, and an additional group was injected with 200 μg of OVA. Spleens were collected 16 h later, and populations enriched for DC, macrophages, or B cells were cultured with OVA-specific CD4+ or CD8+ cells. Proliferation was assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. Results are shown as the average ± SEM of triplicate wells.

FIGURE 3.

Ag conjugated to M1 is efficiently presented to T cells in vivo. A, OVA-specific CD4+ or CD8+ cells were positively selected from OT-II and OT-I mice, respectively, labeled with CFSE, and transferred together into congenic CD45.1+ recipients. One day later the recipients were injected i.v. with one of the following: 2 μg of M1-OVA, 2 μg of DM-OVA, or 1 μg of OVA protein (equivalent molar doses of OVA protein). Half of the animals also received α-GalCer in the injected bolus. Animals that received CFSE-labeled T cells, but were not administered with protein Ag, served as negative controls (Nil). Proliferation of the CFSE-labeled T cells was assessed by flow cytometry of spleen tissue 72 h after Ag administration, using Abs for CD45.2, CD4, and CD8. A representative profile from each of the treatment groups (n = 3 mice per group) is presented. B, Ag conjugated to M1 is efficiently presented by DC. Animals were injected with M1-OVA, DM-OVA, or OVA as in A, and an additional group was injected with 200 μg of OVA. Spleens were collected 16 h later, and populations enriched for DC, macrophages, or B cells were cultured with OVA-specific CD4+ or CD8+ cells. Proliferation was assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. Results are shown as the average ± SEM of triplicate wells.

Close modal

We investigated the role of different splenic APC in the processing and presentation of M1-conjugated Ag. Mice were injected with equivalent molar doses of M1-OVA, DM-OVA, or OVA or a 200-fold molar excess of OVA, and then B cells, macrophages, and DC were isolated from the spleen to assess their ability to stimulate proliferation of OVA-specific T cells in vitro (Fig. 3 B). Of these ex vivo APC populations, CD11c+ DC from animals injected with M1-OVA were the most efficient stimulators, with 10- to 100-fold less cells required to initiate proliferation of both CD4+ and CD8+ T cells compared with macrophages or B cells. When DM-OVA was injected, or an equivalent molar dose of OVA, only limited T cell proliferation was observed from any ex vivo APC population, highlighting the importance of MHC class II targeting. Increasing the dose of injected OVA resulted in some proliferation of both CD4+ and CD8+ T cells, with DC being the most effective APC. However, 10- to 100-fold more cells were required to induce this response relative to M1-OVA-targeted cells.

Murine splenic DC can be divided into the three distinct subtypes based upon expression of CD4 and CD8: CD4+CD8 DC, CD4CD8+ DC, and CD4CD8 DC. Although all three populations are capable of stimulating CD4+ T cells, it has been reported that the CD4CD8+ DC subtype is the major subtype contributing to cross-presentation to CD8+ T cells (8, 9, 29). We investigated the role of the different splenic DC subtypes in the processing and presentation of Ag conjugated to M1. Mice were injected with OVA, M1-OVA, or DM-OVA, and then the different DC subtypes were electronically sorted from the spleen to assess their ability to present processed Ag- to OVA-specific T cells in vitro. On the basis of the previous in vitro studies showing significantly enhanced presentation of OVA by M1, we chose to compare the injection of a 65-fold lower molar dose of M1-OVA to nonconjugated OVA. We also examined the effect of administering Ag together with α-GalCer (Fig. 4). Following injection of nonconjugated OVA protein, all three DC subtypes stimulated low levels of CD4+ T cell proliferation. Proliferation of CD8+ T cells was also observed, with this cross-presentation limited only to the CD4CD8+ DC subtype, which is in agreement with published studies (Fig. 4,A). Injection of M1-OVA resulted in particularly efficient processing and presentation of Ag, with T cell responses similar to, or exceeding, those induced with a 65-fold higher dose of OVA. Moreover, all of the different DC subsets were capable of presenting M1-conjugated Ag to both CD4+ and CD8+ T cells, although in the absence of α-GalCer, CD4CD8+ DC were weak stimulators of CD4+ T cells (Fig. 4, A and B). The unique cross-presentation profile of M1-OVA, involving all DC subsets, was highly reproducible (Fig. 4,C), and directly attributed to the targeting of MHC class II molecules because injection of DM-OVA produced results that were similar to OVA alone (Fig. 4 D).

FIGURE 4.

Ag conjugated to M1 is efficiently presented, and cross-presented, by all of the major splenic DC subtypes in vivo. A, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein (a 65-fold molar excess of OVA relative to M1-OVA), and DC subtypes electronically sorted from spleen tissue 14–16 h later. A varying number of each of the indicated DC subtypes were then incubated with OVA-specific CD4+ or CD8+ cells, and proliferation assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. DC from untreated animals did not induce Ag-specific T cell proliferation (data not shown). Results are the average ± SEM of triplicate wells. B, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein as in A except 200 ng of α-GalCer was included in the injected bolus. C, Data combined from three to seven experiments are expressed as a percentage of maximum value for CD8+ DC in each experiment. Data points that significantly deviate from one another are indicated. ∗∗∗, p < 0.001 and ∗∗, p < 0.01. D, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein as in A, except an equimolar dose of DM-OVA (62 μg) was used relative to M1-OVA.

FIGURE 4.

Ag conjugated to M1 is efficiently presented, and cross-presented, by all of the major splenic DC subtypes in vivo. A, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein (a 65-fold molar excess of OVA relative to M1-OVA), and DC subtypes electronically sorted from spleen tissue 14–16 h later. A varying number of each of the indicated DC subtypes were then incubated with OVA-specific CD4+ or CD8+ cells, and proliferation assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. DC from untreated animals did not induce Ag-specific T cell proliferation (data not shown). Results are the average ± SEM of triplicate wells. B, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein as in A except 200 ng of α-GalCer was included in the injected bolus. C, Data combined from three to seven experiments are expressed as a percentage of maximum value for CD8+ DC in each experiment. Data points that significantly deviate from one another are indicated. ∗∗∗, p < 0.001 and ∗∗, p < 0.01. D, Mice were injected i.v. with 62 μg of M1-OVA or 2 mg of OVA protein as in A, except an equimolar dose of DM-OVA (62 μg) was used relative to M1-OVA.

Close modal

When lower doses of M1-OVA were used (650- and 6500-fold lower molar doses than OVA) there was a trend for the CD4CD8+ DC subset to show superior cross-presentation activity over the other DC subsets (Fig. 5,A). Thus, the efficiency of M1-conjugated Ags to be cross-presented may reflect at least two activities; one that is enhanced in the CD4CD8+ DC subset, and another that is common to all subsets that predominate at higher Ag doses. To further investigate the APC involved in cross-presentation at the higher doses, recipients were depleted of APC capable of recycling acquired Ags through the cytosol, a function known to be required for cross-presentation of nonconjugated OVA. Thus, animals were injected with Cyt c before Ag administration, as cells that can efficiently recycle Cyt c to the cytosol are killed by Apaf-1-dependent apoptosis (30). As has been published, this treatment did indeed abolish subsequent cross-presentation of OVA. In contrast, cross-presentation of M1-OVA was less severely affected, with some activity retained by all DC subtypes (Fig. 5 B). A proportion of the cross-presentation of M1-OVA is therefore attributable to DC of each subtype without the capacity to efficiently recycle acquired proteins such as Cyt c through the cytosol.

FIGURE 5.

Ag conjugated to M1 is cross-presented by more than one mechanism in vivo dependent on dose. A, Mice were injected i.v. with 2 mg of OVA protein, or 62-μg (hi), 6.2-μg (med), or 0.6-μg (low) doses of M1-OVA (representing 65-, 650-, and 6500-fold lower molar doses relative to OVA). DC subtypes were electronically sorted from spleen tissue 14–16 h later and analyzed for capacity to stimulate OVA-specific CD8+ cells in vitro. Results from three experiments are combined and expressed as a percentage of the maximum value for CD8+ DC in the OVA group for each experiment. B, Mice were treated with Cyt c, or left untreated, and then injected with 2 mg of OVA or a 62-μg (hi) dose of MI-OVA. DC subtypes were electronically sorted from spleen tissue 14–16 h later and analyzed as in A. Results from three experiments are combined and expressed as a percentage of the maximum value for CD8+ DC in the OVA group in each experiment.

FIGURE 5.

Ag conjugated to M1 is cross-presented by more than one mechanism in vivo dependent on dose. A, Mice were injected i.v. with 2 mg of OVA protein, or 62-μg (hi), 6.2-μg (med), or 0.6-μg (low) doses of M1-OVA (representing 65-, 650-, and 6500-fold lower molar doses relative to OVA). DC subtypes were electronically sorted from spleen tissue 14–16 h later and analyzed for capacity to stimulate OVA-specific CD8+ cells in vitro. Results from three experiments are combined and expressed as a percentage of the maximum value for CD8+ DC in the OVA group for each experiment. B, Mice were treated with Cyt c, or left untreated, and then injected with 2 mg of OVA or a 62-μg (hi) dose of MI-OVA. DC subtypes were electronically sorted from spleen tissue 14–16 h later and analyzed as in A. Results from three experiments are combined and expressed as a percentage of the maximum value for CD8+ DC in the OVA group in each experiment.

Close modal

The improved cross-presentation of OVA when conjugated to M1, particularly at higher doses, may reflect a different intracellular trafficking and processing route. Confocal microscopy was therefore used to examine the redistribution of fluorescent M1-OVA and OVA following uptake by BMDC in vitro (Fig. 6). Given that entry into the early endosomes has previously been associated with cross-presentation of OVA (31) we specifically assessed whether the fluorescent Ags colocalized with the EEA1 marker. Analysis 10 and 30 min after internalization showed fluorescent OVA colocalized with EEA1, whereas the majority of fluorescent M1-OVA remained in EEA1-negative compartments at both time points. Thus, OVA and M1-OVA segregate to different intracellular compartments after uptake.

FIGURE 6.

Fluorescent OVA colocalizes with EEA1 early after internalization, whereas fluorescent M1-OVA remains in EEA1-negative compartments. Magnetically sorted CD11c+ BMDC were incubated with Alexa488-labeled OVA (left panels) or Alexa488-labeled M1-OVA (right panels) for the indicated times, and were then fixed and stained with anti-EEA1 for analysis by confocal microscopy. Two representative cells for each treatment group are presented.

FIGURE 6.

Fluorescent OVA colocalizes with EEA1 early after internalization, whereas fluorescent M1-OVA remains in EEA1-negative compartments. Magnetically sorted CD11c+ BMDC were incubated with Alexa488-labeled OVA (left panels) or Alexa488-labeled M1-OVA (right panels) for the indicated times, and were then fixed and stained with anti-EEA1 for analysis by confocal microscopy. Two representative cells for each treatment group are presented.

Close modal

It remained to be established whether injection of M1-Ag conjugates elicits full effector function in responding T cells. This result was assessed by measuring the cytotoxic capacity of induced responses against Ag-loaded fluorescent syngeneic targets in vivo (Fig. 7,A). Cytotoxic activity was only observed when Ags were coadministered with the adjuvant α-GalCer, indicating that targeted delivery of Ag via MHC class II molecules is insufficient in itself to produce effector cells. Nevertheless, the combined injection of α-GalCer and M1-OVA produced a cytotoxic response at a molar dose 200 times lower than OVA. This enhanced cytotoxic T cell response was dependent on targeting of MHC class II molecules, as no cytotoxicity was observed when DM-OVA was used (Fig. 7,B). Injection of α-GalCer alone did not induce Ag-specific cytotoxic activity (data not shown). When varying doses of M1-OVA or OVA were injected into groups of animals together with a fixed dose of α-GalCer, M1-OVA was at least 50 times more efficient at inducing a cytotoxic response than OVA (Fig. 7 C).

FIGURE 7.

Ag conjugated to M1 induces effector function in T cells in the presence of an adjuvant. A and B, Groups of mice (n = 3–5 each group) were injected i.v. with 2 μg of M1-OVA, 2 μg of DM-OVA, or 200 μg of OVA protein (a 200-fold molar excess of OVA relative to M1-OVA), in the presence or absence of α-GalCer as indicated. Seven days later cytotoxicity was assessed against i.v. injected CFSE-labeled syngeneic splenocytes loaded with OVA257–264 peptide as described in Materials and Methods. Percentage of specific lysis ± SEM for each group of mice is presented. C, Cytotoxicity was assessed in groups of mice (n = 3) injected with varying doses of M1-OVA or OVA protein in the presence of α-GalCer. D, Groups of mice (n = 5) were injected i.v. with 2 μg of M1-OVA or 200 μg of OVA protein in the presence or absence of α-GalCer as indicated. After 10 days, IFN-γ-producing cells were enumerated in spleen tissue following 40 h restimulation in vitro with one of the following: OVA265–280 peptide (a CD4+ T cell epitope), OVA257–264 peptide (a CD8+ T cell epitope), or an irrelevant peptide (Control). Results are shown as the mean ± SEM of triplicate samples. E, Varying doses of M1-OVA or OVA protein were injected into individual mice, and IFN-γ-producing cells were enumerated in spleen tissue 10 days later as in D.

FIGURE 7.

Ag conjugated to M1 induces effector function in T cells in the presence of an adjuvant. A and B, Groups of mice (n = 3–5 each group) were injected i.v. with 2 μg of M1-OVA, 2 μg of DM-OVA, or 200 μg of OVA protein (a 200-fold molar excess of OVA relative to M1-OVA), in the presence or absence of α-GalCer as indicated. Seven days later cytotoxicity was assessed against i.v. injected CFSE-labeled syngeneic splenocytes loaded with OVA257–264 peptide as described in Materials and Methods. Percentage of specific lysis ± SEM for each group of mice is presented. C, Cytotoxicity was assessed in groups of mice (n = 3) injected with varying doses of M1-OVA or OVA protein in the presence of α-GalCer. D, Groups of mice (n = 5) were injected i.v. with 2 μg of M1-OVA or 200 μg of OVA protein in the presence or absence of α-GalCer as indicated. After 10 days, IFN-γ-producing cells were enumerated in spleen tissue following 40 h restimulation in vitro with one of the following: OVA265–280 peptide (a CD4+ T cell epitope), OVA257–264 peptide (a CD8+ T cell epitope), or an irrelevant peptide (Control). Results are shown as the mean ± SEM of triplicate samples. E, Varying doses of M1-OVA or OVA protein were injected into individual mice, and IFN-γ-producing cells were enumerated in spleen tissue 10 days later as in D.

Close modal

The development of effector function in T cells was also assessed by examining cytokine production. In the presence of α-GalCer, M1-OVA elicited IFN-γ-producing T cells with a 200-fold lower molar dose than OVA (Fig. 7,D). Analysis of dose response showed that M1-OVA coadministered with α-GalCer was ∼50 times more efficient at inducing IFN-γ-producing cells than OVA with α-GalCer (Fig. 7 E).

Finally, we investigated the therapeutic potential of initiating immune responses with Ag conjugated to M1. The cell line B16.OVA, which is a derivative of the melanoma cell line B16.F0 that expresses OVA cDNA (17), was injected s.c. into the flanks of mice and allowed to reach palpable size. A group of recipients was left to show the rate of subsequent tumor progression. The remaining groups were immunized with a single dose of 2 μg of M1-OVA, or 200 μg of OVA (representing a 200-fold molar excess of tumor-specific Ag) in the presence or absence of α-GalCer. Significant retardation of tumor growth was observed when M1-OVA was administered with adjuvant (Fig. 8,A), which was similar to that observed with a 200-fold higher dose of OVA plus adjuvant. No significant antitumor activity was seen in animals treated with α-GalCer as a single agent (Fig. 8,B). The enhanced antitumor activity of M1-OVA was clearly dependent on MHC class II binding because a comparable dose of DM-OVA plus α-GalCer did not induce any significant antitumor activity (Fig. 8 B). These experiments highlight the enhanced potency of Ag coupled to M1 and the potential for immunotherapeutic strategies based upon this simple modification to Ags.

FIGURE 8.

Ag conjugated to M1 induces antitumor activity in the presence of an adjuvant. Groups of mice (n = 5) were challenged with a s.c. injection of B16.OVA tumor cells in the flank, and then 7 days later, a single injection of 2 μg of M1-OVA, 2 μg of DM-OVA, or 200 μg of OVA protein was administered i.v. in the presence or absence of α-GalCer as indicated. In each experiment a group of animals was left untreated to serve as a control for tumor growth. Mean tumor size ± SEM per group is shown.

FIGURE 8.

Ag conjugated to M1 induces antitumor activity in the presence of an adjuvant. Groups of mice (n = 5) were challenged with a s.c. injection of B16.OVA tumor cells in the flank, and then 7 days later, a single injection of 2 μg of M1-OVA, 2 μg of DM-OVA, or 200 μg of OVA protein was administered i.v. in the presence or absence of α-GalCer as indicated. In each experiment a group of animals was left untreated to serve as a control for tumor growth. Mean tumor size ± SEM per group is shown.

Close modal

Conjugating Ag to the modified SMEZ-2 superantigen construct M1 enables specific targeting to MHC class II+ APC in vivo without the toxic polyclonal T cell response associated with direct TCR engagement. Our results show that conjugated Ag is efficiently processed and presented to CD4+, and cross-presented to CD8+ T cells. Of particular significance, this form of Ag delivery provides access to pathway of cross-presentation operating in all DC populations assessed, including the three major DC subtypes in murine spleen. This finding contrasts with many other studies in which splenic CD4CD8+ DC have been shown to be superior at cross-presentation than both of the CD8 DC subtypes (8, 9, 29). The knowledge that Ag can be efficiently targeted to promote cross-presentation in a variety of DC subtypes should be particularly useful in the design of vaccines against intracellular pathogens and tumors in which CD8+ T cell responses are desirable.

It has previously been reported that Ag incorporated into immune complexes can be cross-presented by CD8 DC, but in this situation, activating FcγRI and FcγRIII were required, implying some degree of DC maturation (32). By contrast, delivery of M1-conjugated Ag was not associated with any obvious maturation of the targeted cells, although they were capable of processing the Ag and inducing proliferation of CFSE-labeled Ag-specific T cells (Fig. 3,A). However, this short-term assay of T cell reactivity does not distinguish between eventual induction of tolerance or activation, as both of these processes are preceded by proliferation (33). In fact, full effector function in responder T cells was shown to be dependent upon co-delivery of M1-conjugate with α-GalCer, which serves to invoke iNKT cell activity resulting in activated APC (26, 27). Accordingly, T cell responses induced in the presence of α-GalCer were particularly potent, providing IFN-γ-producing CD4+ and CD8+ T cells, and Ag-specific cytotoxic responses capable of significantly delaying the growth of established Ag-positive tumors (Figs. 7 and 8).

Analysis of T cell responses to M1-conjugated Ag in vitro showed a 3 to 4 log increase in efficiency of presentation to CD4+ and CD8+ T cells relative to Ag alone. When assessed in vivo, the relative enhancement in T cell effector function by M1 was reduced, with a 50- to 100-fold advantage of M1-OVA over nonconjugated Ag (Fig. 7). It is possible that the uptake of M1-OVA conjugates in vivo by weak APC, such as B cells and macrophages, serves as an Ag “sink,” thereby reducing the Ag available for potent APC such as splenic DC. In support of this conclusion, fluorescent M1 was absorbed by virtually all MHC class II+ cells in vivo, although DC were shown to be the most efficient at presentation to T cells (Fig. 3 B).

Other studies have delivered Ag to APC in vivo by targeting DNGR-1 (34), CD205 (DEC205) (10, 35, 36), Dectin-1 and Dectin-2 (37, 38), DCIR2 (10), CD11c (39), and Langerin (40). Current interpretation of these Ag-targeting experiments points to distinct, intrinsic, Ag-presenting functions in DC subsets in vivo (7), with the suggestion that specialized cross-presentation machinery may be enriched in the splenic CD8+ DC subset (10). In contrast, the splenic CD8 subset favors presentation to CD4+ T cells (10). For example, Ags targeted to CD205 (DEC205), which is highly expressed on CD8+ DC, are efficiently cross-presented, whereas Ags conjugated to 33D1 mAb that preferentially targets DCIR2 on CD8 DC promote presentation on MHC class II molecules. In instances in which equivalent levels of absorption of Ag to the different DC subsets were firmly established, for example in experiments in which Ag was attached to latex beads, the CD8+ DC was still the predominant cross-presenting APC (29). By way of explanation for this pattern of Ag presentation, it has been noted that the CD8+ DC subset expresses higher levels of molecules involved in MHC class I presentation than CD8 DC (10). In accordance with these previous studies, we did observe superior cross-presentation by CD8+ DC when nonconjugated Ag was used.

Our results point to an ability of all DC subsets to cross-present Ag under circumstances forced by targeting of MHC class II molecules with modified superantigen M1. There have been previous reports of targeting Ag to MHC class II molecules with anti-MHC class II Abs (35, 39, 41, 42), or as recombinant “Troybodies” (43). Although these targeting studies focused primarily on Ab or CD4+ T cell responses, evidence of limited cross-presentation to CD8+ T cells was observed in one study (39), whereas cross-presentation was absent in another (35). It is possible therefore that the pathway of efficient cross-presentation accessed by M1 conjugates reflects a property unique to superantigens such as SMEZ-2, rather than simply binding MHC class II molecules per se. Our confocal microscopy studies showed fluorescent OVA colocalized with EEA1 both 10 and 30 min after internalization, whereas fluorescent M1-OVA remained in EEA1-negative compartments at both time points (Fig. 6). These data indicate that OVA and M1-OVA segregate to different intracellular compartments after uptake, and although the nature of the superantigen-targeted compartment awaits further investigation, it is not associated with the early endosomal marker EEA1. It is possible that uptake of M1-OVA is related to the normal process of internalization of MHC class II molecules. A recent study of internalization of peptide/MHC class II complexes from the plasma membrane highlighted a clathrin-independent pathway involving Arf6+, Rab65+, EHD1+ endosomal tubules (44). Interestingly, internalized MHC class I molecules can also recirculate through Arf6+ endosomes (45), suggesting that reloading of both classes of MHC molecule may be possible in these compartments. Certainly our studies suggest that a proportion of the cross-presentation of M1-OVA in vivo occurs by a mechanism that is different from nonconjugated OVA. Although the latter was shown to be entirely dependent on APC with capacity to transfer acquired Ags to the cytosol (Fig. 5 B), some cross-presentation of M1-OVA was still observed in animals selectively depleted of APC with this capability. Furthermore, this residual activity was not restricted to the CD4CD8+ DC subtype, but was evident in all DC subtypes assessed in the spleen. Further studies to establish the mechanism behind this efficient cross-presentation process are ongoing.

Targeting of Ag to APC by conjugation to M1 permits the use of lower Ag doses to stimulate T cell responses, and as such should be a useful strategy for immunotherapy. In this context, M1 conjugates could be used to load cultured DC before injection into patients, or to target DC in situ. All superantigens examined so far display a higher affinity toward human MHC class II molecules than mouse MHC class II (46), so it is likely that the efficiency of Ag uptake will be superior in human cells. This method of Ag delivery therefore has significant potential in clinical vaccination procedures, particularly where cross-presentation is required.

We thank Drs. E. Lord and J. G. Frelinger, and Prof. F. Carbone for generously providing cell lines and mouse strains used in this study, Dr. G. F. Painter and A. Lee for providing the α-GalCer, the staff of the Malaghan Experimental Research Facility for animal husbandry and care, and the staff of the Malaghan Institute for useful suggestions and discussion.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the New Zealand Health Research Council, the Cancer Society of New Zealand, the Wellington Medical Research Foundation, and the Genesis Oncology Trust. P.S. was supported by the Erwin Schroedinger Auslandsstipendium Grant FWF-J2479 from the Austrian Science Fund. I.F.H. was supported by a New Zealand Health Research Council Sir Charles Hercus Fellowship.

3

Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; LC, Langerhans cell; α-GalCer, α-galactosylceramide; iNKT, invariant NKT; SMEZ, streptococcal mitogenic exotoxin Z; Cyt c, cytochrome c; EEA, early endosomal Ag.

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