Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.

Antigen dose as well as the type and amount of the provided costimulatory signals influence strongly the outcome of Ag-specific CD8+ and CD4+ T cell responses (1, 2). Priming of CD8+ T cells with APCs providing insufficient densities of surface epitope/MHC class I complexes results in weak responders exhibiting impaired cytokine secretion and a dramatically decreased CTL memory size (3, 4). Analogously, low Ag doses favor the development of less efficient Th2-type CD4+ T cell responses (5, 6, 7).

Accordingly, the knowledge of how to leverage MHC class I and class II Ag presentation efficiency is of particular interest for the development of effective vaccines.

Various factors inherent to the Ag itself as well as to the series of steps in Ag processing and presentation affect the density of epitope/MHC complexes that eventually are presented on the cell surface. Recent studies have shown that presentation of MHC class I epitopes is a relatively inefficient process (8, 9, 10). Even for a high-affinity MHC class I ligand, only 1 peptide of 10,000 degraded molecules is presented, providing considerable space for improvement by engineered vaccines. Whereas all endogenous proteins are subject to MHC class I presentation, only a subset of intracellular Ags is loaded on MHC class II molecules. The major proportion of MHC class II epitopes is generated by cleavage and processing of peptides by endosomal and lysosomal proteases, and is therefore mainly limited to engulfed proteins and Ags, which reside in or travel through the endocytic pathway. Proteins without direct access to the endocytic pathway (e.g., Ags naturally located in the cytoplasm, in nonendocytic organelles, or in the nucleus) are in general poorly presented to CD4+ T cells.

Several strategies have been explored over the past few years to optimize MHC class II presentation. Ags have been linked to the trafficking sequences of endosomal or lysosomal proteins known to reside in MHC class II Ag processing compartments, such as invariant chain (11, 12, 13, 14), lysosome-associated membrane proteins (LAMP1,4 LAMP2), and dendritic cell (DC)-LAMP (15, 16, 17, 18, 19). Such trafficking signals attached to viral and tumor Ags improved in vitro stimulation of Ag-specific CD4+ T cells. In line with the relevance of cognate T cell help, this was associated with robust CTL responses in vivo (20). The common molecular principle behind these approaches is that the cytoplasmic tails of these proteins contain tyrosine- or leucine-based signals, which interact with adaptor protein complexes and mediate translocation into defined subcellular compartments. Whereas invariant chain fusion proteins are targeted to early endosomes, LAMP proteins are predominantly located in acidified late endosomes and lysosomes (21). Each of the compartments within the MHC class II processing route has its peculiar composition of cleavage activities. This may explain that the kind of MHC class II epitopes, which preferentially profit from such maneuvers, depends on the trafficking signal to which the Ag is fused (22).

Interestingly, MHC class I molecules themselves reside in or travel through all cellular compartments involved in MHC class II Ag processing and presentation, including endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, early and late endosomes, as well as lysosomes. A recent study established that MHC class I molecules contain a sequence located in the cytoplasmic domain, which controls their recycling between different endolysosomal compartments (23). We speculated that the MHC class I trafficking domain may assist in achieving our objective and be a suitable targeting signal for redirecting Ags into these compartments to allow generation of a comprehensive set of MHC class II epitopes.

The human erythromyeloblastoid leukemia cell line K562, the murine thymoma cell line EL4, and the TAP-deficient cell line RMAS (24) were cultured under standard conditions.

Immature DCs were differentiated from human monocytes of healthy blood bank donors by culture in RPMI 1640 with 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, nonessential amino acids, and 10% heat-inactivated human AB serum (all Invitrogen Life Technologies) supplemented with 1000 U/ml GM-CSF (Essex) and 1000 U/ml IL-4 (Strathmann Biotech). Maturation of DCs was induced by culturing for 2 days with 500 U/ml IL-4, 800 U/ml GM-CSF, 10 ng/ml IL-1β (BD Pharmingen), 10 ng/ml TNF-α (Sigma-Aldrich), 1000 U/ml IL-6 (Strathmann Biotech), and 1 μg/ml PG-E2 (Sigma-Aldrich). Murine bone marrow-derived DCs (BMDCs) for in vitro or in vivo stimulation were generated, as described by Lutz et al. (25), by culturing bulk cells obtained from bone cavities of BALB/c mice in medium supplemented with 200 U/ml murine rGM-CSF (Peprotech/Tebu). For maturation, DCs were treated for 4 h with poly(I:C) (50 μg/ml; Amersham Biosciences).

BALB/cThy1.1+ and C57BL/6 mice were 6–9 wk old at the initiation of experiments. RAG-2−/− TCR-influenza hemagglutinin (HA) mice (H-2d; Thy1,2+) transgenic for an influenza virus HA107–119 peptide-specific, I-Ed-restricted TCR were provided by U. Hartwig (University Mainz, Mainz, Germany) (26). TCR transgenic OT-I mice recognizing the H2-Kb-restricted epitope SIINFEKL from chicken OVA (OVA257–264) were provided by H. Schild (University Mainz, Mainz, Germany). Mice were maintained at the university animal facility. Experimental procedures were performed according to German federal and state regulations.

In vitro transcription (IVT) constructs were based on the pST1-A120 vector, a variant of pCMV-Script-Vektor (Stratagene) created by introduction of a T7 promotor, a 120-bp poly(A) tail, a 3′β-globin UTR, and the neomycin-resistance gene, as already described elsewhere (27). Thereby, we could omit enzymatic poly(A) tailing, which reduces variations in translation efficacy of different RNA constructs. A MHC class I signal peptide fragment (78 bp, secretion signal (sec)) and the transmembrane and cytosolic domains including the stop-codon (MHC class I trafficking signal (MITD), 168 bp) both amplified from activated PBMC were inserted (sec sense, 5′-aag ctt agc ggc cgc acc atg cgg gtc acg gcg ccc cga acc-3′; sec antisense, 5′-ctg cag gga gcc ggc cca ggt ctc ggt cag-3′; MITD sense, 5′-gga tcc atc gtg ggc att gtt gct ggc ctg gct-3′; and MITD antisense, 5′-gaa ttc agt ctc gag tca agc tgt gag aga cac atc aga gcc-3′).

Enhanced GFP (eGFP), sec-eGFP, and eGFP-MITD vectors (Fig. 1 a) were obtained by cloning a BamHI-site-flanked eGFP fragment amplified from peGFP-C1 vector (BD Biosciences; eGFP sense, 5′-gga tcc acc atg gtg agc aag ggc gag gag-3′; eGFP-No-Stop antisense, 5′-gga tcc ctt gta cag ctc gtc cat gcc g-3′; or eGFP-Stop antisense, 5′-gga tcc tta ctt gta cag ctc gtc cat gcc g-3′) into the respective backbones.

FIGURE 1.

Trafficking of sec/MITD-tagged fusion proteins into endolysosomal compartments. a, Vector templates used for generation of IVT RNA-encoding eGFP, sec-eGFP, and eGFP-MITD contain the signal peptide (sec), the eGFP open reading frame (eGFP), and the MHC class I trafficking domain (MITD), which consists of the TM and the cytoplasmic tail of this molecule. All this is downstream to a T7 promotor. b, K562 cells were analyzed by fluorescence microscopy 18 h after transfection with 20 μg of IVT RNA. c, Cellular distribution of eGFP-MITD protein in immature DCs (iDCs) and mature DCs (mDCs) analyzed 18 h after transfer of 30 μg of IVT RNA. Cells were stained with Abs against HLA class I, HLA class II, Golgi, and Lamp 1 molecules (red). eGFP signal was enhanced with a polyclonal anti-eGFP Ab (green) and nuclei stained with Hoechst 33342 (blue).

FIGURE 1.

Trafficking of sec/MITD-tagged fusion proteins into endolysosomal compartments. a, Vector templates used for generation of IVT RNA-encoding eGFP, sec-eGFP, and eGFP-MITD contain the signal peptide (sec), the eGFP open reading frame (eGFP), and the MHC class I trafficking domain (MITD), which consists of the TM and the cytoplasmic tail of this molecule. All this is downstream to a T7 promotor. b, K562 cells were analyzed by fluorescence microscopy 18 h after transfection with 20 μg of IVT RNA. c, Cellular distribution of eGFP-MITD protein in immature DCs (iDCs) and mature DCs (mDCs) analyzed 18 h after transfer of 30 μg of IVT RNA. Cells were stained with Abs against HLA class I, HLA class II, Golgi, and Lamp 1 molecules (red). eGFP signal was enhanced with a polyclonal anti-eGFP Ab (green) and nuclei stained with Hoechst 33342 (blue).

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For engineering pp65 and pp65-MITD constructs (Fig. 4 a), the sequence of human CMV (HCMV) UL83 (pp65) was amplified from a lysate of HCMV-infected fibroblasts (BioWhittaker) with and without stop codon (pp65 sense, 5′-gga tcc acc atg gag tcg cgc ggt cgc cgt tgt ccc gaa atg-3′; pp65-No-Stop antisense, 5′-gga tcc acc tcg gtg ctt ttt ggg cgt cga ggc gat gc-3′; pp65-Stop antisense, 5′-gga tcc tca acc tcg gtg ctt ttt ggg cgt cga ggc-3′) and cloned into the respective vector backbones.

FIGURE 4.

Induction of antitumoral immunity in a therapeutic mouse model. a, BALB/c mice (n = 10) were inoculated with lethal dose of A20-HA tumor cells. After 17 days, when tumors had a diameter of 2–3 mm, mice received five s.c. immunizations in three daily intervals either with 1 × 106 BMDCs transfected with 20 μg of HA-MITD IVT RNA or with 50 μg of HA518–526 peptide in IFA. Mice were sacrificed when tumors reached a mean diameter of 15 mm. b, Data are shown as percent survival at different time points after termination of treatment.

FIGURE 4.

Induction of antitumoral immunity in a therapeutic mouse model. a, BALB/c mice (n = 10) were inoculated with lethal dose of A20-HA tumor cells. After 17 days, when tumors had a diameter of 2–3 mm, mice received five s.c. immunizations in three daily intervals either with 1 × 106 BMDCs transfected with 20 μg of HA-MITD IVT RNA or with 50 μg of HA518–526 peptide in IFA. Mice were sacrificed when tumors reached a mean diameter of 15 mm. b, Data are shown as percent survival at different time points after termination of treatment.

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An analogous cloning strategy was used to obtain vectors NY-ESO-I and NY-ESO-I-MITD (Fig. 4 a) after amplifying the open reading frame from human testis tissue (NY-ESO-Stop sense, 5′-gga tcc gcc acc atg cag gcc gaa ggc cgg ggc aca-3′; NY-ESO-Stop antisense, 5′-gga tcc tta gcg cct ctg ccc tga ggg agg ctg agc-3′; NY-ESO-No-Stop antisense, 5′-gga tcc gcg cct ctg ccc tga ggg agg-3′).

To generate HA (Fig. 2 a) constructs, the entire 1698-bp open reading frame of HA derived from influenza strain A/PR/8/34 (H1N1) was amplified from a transfected cell line (A20-HA cells provided by U. Hartwig, University Mainz, Mainz, Germany) (InflHA-HindIII sense, 5′-aag ctt atg aag gca aac cta ctg gtc ctg tta-3′; InflHA-BamHI antisense, 5′-gga tcc tca gat gca tat tct gca ctg caa aga-3′) and cloned into pST1-A120 to generate the HA-WT vector. The secreted version HA Δ transmembrane domain (TM) was generated by deleting the TM of HA to obtain a 1587-bp 3′-truncated fragment (InflHA-HindIII sense, 5′-aag ctt atg aag gca aac cta ctg gtc ctg tta-3′; InflHA-BamHI antisense, 5′-gga tcc aat ctg ata gat ccc cat tga ttc caa-3′). Ligation of the MITD fragment into the HAΔTM vector resulted in the HA-MITD construct, whereas the respective insert for the HA-CD27 construct, namely the transmembrane and cytosolic domains of CD27 (GenBank accession number M63928; bp 565–651), were synthesized with BamHI and XhoI sites by a commercial provider (GENEART). Analogously, bp 766–801 (aa 255–266) of the OVA sequence flanked by BamHI sites was synthesized and cloned into the respective backbones to obtain vector variants featuring the SIINFEKL epitope.

FIGURE 2.

Improved MHC class II presentation of the HA S1 epitope mediated by sec/MITD trafficking signals. a, Vector templates for generation of IVT RNA-encoding HA variants. Signal peptide and TM designate the autochthonous leader peptide and TM of HA. The hatched box represents the S1 epitope HA110–119. b, BMDCs either transfected with IVT RNA or loaded with S1 peptide (2 μg/ml) were used as stimulators of naive CD4+ TCR-HA cells in vitro. NY-Eso-I-MITD was used as control RNA. For analysis of proliferation, TCR-HA cells (0.4 × 105/well) were stimulated for 3 days with DCs (0.3 × 105/well) and then pulsed with [3H]thymidine for 16 h. Representative data from three independent experiments are shown as mean values from triplicates ± SEM. For quantification of cytokine secretion, supernatants of TCR-HA cells were harvested 40 h after stimulation. Data shown are representative for two independent experiments. c–e, Thy1.1+ BALB/c mice were immunized i.p. with 1 × 106 HA-WT- or HA-MITD-transfected BMDCs or BMDCs loaded with HA110–119 peptide (n = 3 per group) on day 1 after i.v. transfer of 0.75 × 106 CFSE-labeled Thy1.2+ CD4+ TCR-HA cells. c, CFSE profiles of Thy1.2+ spleen cells from representative mice obtained by flow cytometry on day 6 are shown. d, Fraction of Thy1.2+ CD4+ TCR-HA cells in the spleen after subtraction of the baseline frequency of CD4+ TCR-HA lymphocytes induced by immunization with control RNA-transfected (NY-Eso-I-MITD) BMDCs. Data are shown as mean values + SEM. ∗, p < 0.025. e, Cytokine profiles determined in the supernatants of spleen cells from immunized mice after 40 h of incubation with S1 HA107–119 peptide (2 μg/ml). Data are shown as mean values + SEM. ∗, p < 0.025; ∗∗, p < 0.001.

FIGURE 2.

Improved MHC class II presentation of the HA S1 epitope mediated by sec/MITD trafficking signals. a, Vector templates for generation of IVT RNA-encoding HA variants. Signal peptide and TM designate the autochthonous leader peptide and TM of HA. The hatched box represents the S1 epitope HA110–119. b, BMDCs either transfected with IVT RNA or loaded with S1 peptide (2 μg/ml) were used as stimulators of naive CD4+ TCR-HA cells in vitro. NY-Eso-I-MITD was used as control RNA. For analysis of proliferation, TCR-HA cells (0.4 × 105/well) were stimulated for 3 days with DCs (0.3 × 105/well) and then pulsed with [3H]thymidine for 16 h. Representative data from three independent experiments are shown as mean values from triplicates ± SEM. For quantification of cytokine secretion, supernatants of TCR-HA cells were harvested 40 h after stimulation. Data shown are representative for two independent experiments. c–e, Thy1.1+ BALB/c mice were immunized i.p. with 1 × 106 HA-WT- or HA-MITD-transfected BMDCs or BMDCs loaded with HA110–119 peptide (n = 3 per group) on day 1 after i.v. transfer of 0.75 × 106 CFSE-labeled Thy1.2+ CD4+ TCR-HA cells. c, CFSE profiles of Thy1.2+ spleen cells from representative mice obtained by flow cytometry on day 6 are shown. d, Fraction of Thy1.2+ CD4+ TCR-HA cells in the spleen after subtraction of the baseline frequency of CD4+ TCR-HA lymphocytes induced by immunization with control RNA-transfected (NY-Eso-I-MITD) BMDCs. Data are shown as mean values + SEM. ∗, p < 0.025. e, Cytokine profiles determined in the supernatants of spleen cells from immunized mice after 40 h of incubation with S1 HA107–119 peptide (2 μg/ml). Data are shown as mean values + SEM. ∗, p < 0.025; ∗∗, p < 0.001.

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The pST1-A120-based plasmids were linearized with EciI, SapI, or BpiI restriction enzyme, purified by phenol chloroform extraction and sodium acetate precipitation, and used as templates for IVT. IVT was performed after purification with T7 polymerase using the mMESSAGE mMachine Ultra T7 Kit (Ambion). RNA concentration and quality were assessed by spectrophotometry and agarose/formaldehyde gel electrophoresis.

A total of 1–10 × 106 cells was suspended in 250 μl of X-VIVO-15 medium (Cambrex) and transferred into a sterile electroporation cuvette (Bio-Rad). IVT RNA was added and cells were electroporated (DCs, BMDCs, EL-4, RMA-S: 300 V/150 μF; K562: 250 V/300 μF) with a Gene-Pulser II apparatus (Bio-Rad). To test for interprobe variance in transfection efficacy, we performed parallel to the transfection with the gene of interest a cotransfection with eGFP in BMDCs (n = 10; data not shown). Thereby, we could calculate that the coefficient of variance is 6.3%, showing a high degree of reproducibility for RNA transfection.

The HA peptides derived from HA (I-Ed; S1 epitope comprising aa 110–119; H2-Kd epitope comprising aa 518–526), the SIINFEKL peptide from OVA (aa 257–264), a peptide pool representing overlapping 15-mer peptides with 11-aa overlap covering the entire HCMV pp65 sequence, the entire NY-ESO-I sequence, and a HLA-A*0201-restricted peptide (aa 41–49, KASEKIFYV) derived from the human tumor Ag SSX2 were all purchased from a commercial provider (Jerini). The peptides were dissolved in PBS/10% DMSO and stored at –20°C.

For peptide pulsing, DCs were diluted to a final density of 2–6 × 106 cells/ml in culture medium containing 2 μg/ml single peptides or 1.75 μg/ml peptide pool and were incubated for 3 h at 37°C, washed, and used for T cell stimulation.

CD4+ as well as CD8+ T cells were isolated from PBMC of HCMV-seropositive healthy donors by magnetic cell sorting (Miltenyi Biotec). A total of 2 × 105 autologous DCs either electroporated with IVT RNA or pulsed with peptide pools was washed and cocultured in 24-well plates with 1 × 106 CD4+ or CD8+ effector T cells in complete medium supplemented with 10% AB serum (BD Biosciences), 10 U/ml IL-2, and 5 ng/ml IL-7 (R&D Systems).

As described elsewhere (28), microtiter plates (MAHA S4510; Millipore) were coated with 0.1 μg/ml anti-IFN-γ Ab 1-D1k (Mabtech). After blocking with 2% human albumin, 1–3 × 104 effector cells were added per well, followed by 2 × 104 target cells. After 18 h at 37°C, the plates were washed with PBS/0.05% Tween 20 and then incubated with biotinylated anti-IFN-γ detection Ab (7-B6-1; Mabtech). Plates were washed with PBS/0.05% Tween 20 and stained with streptavidin-peroxidase (Vactastain Elite Kit; Vector Laboratories) and the substrate 3-amino-9-ethyl carbazole (Sigma-Aldrich). Spots were counted with a special assisted video imaging analysis system (Carl Zeiss Vision), using the KS-ELISPOT software version 4.4.35 (Carl Zeiss Vision). For murine ELISPOT, the anti-IFN-γ Abs AN18 and R4-6A2 (Mabtech) were used. Expansion of CD4+ RAG-2−/−TCR-HA cells in mice after immunization was tested using whole spleen cells (2 × 105/well) seeded after pulsing with HA peptide (4 μg/ml).

The cytokine profile of CD4+ RAG-2−/−TCR-HA cells was determined with the murine Th1/Th2 cytokine cytometric bead array kit, according to the manufacturer’s instructions (BD Biosciences). To this aim, T cells were expanded in vitro using whole spleen cells (2 × 105/well) seeded with HA peptide (4 μg/ml) for 40 h.

Cytotoxic effector function was measured in a standard 51Cr release assay. Autologous DCs were loaded with various concentrations of pp65 peptide pool or with SSX2 peptide (1.75 μg/ml) and labeled with 100 μCi of 51Cr (NEN Life Science) for 1 h at 37°C/5% CO2. A total of 5 × 10351Cr-labeled DCs was washed and coincubated with effector T cells (E:T ratio 20:1) in 200 μl of X-Vivo-15 medium for 4.5 h. A total of 100 μl of the supernatant was harvested, and released 51Cr was measured with a scintillation counter. Spontaneous release was <7% of maximal release. SD of the means of triplicate wells was <5%. Percent specific lysis was calculated using the following equation: ((experimental release – spontaneous release)/(maximum release – spontaneous release)) × 100.

After 12 days of stimulation, human responder cells were incubated in triplicates in round-bottom 96-well plates (Corning Glass) with autologous DCs loaded with pp65 peptide pool (1.75 μg/ml). [3H]Thymidine (1 μCi/well) was added at day 4. Proliferation was determined after 16 h by measuring [3H]thymidine incorporation using a Microbeta scintillation counter (Wallac). The same protocol was applied to measure proliferation of mouse T cells.

To assess in vivo cytoloytic activity, splenocytes from OT-I mice were adoptively transferred i.v. into C57BL/6 mice. Recipient mice were immunized i.p. or s.c. with 1 × 106 RNA-transfected BMDCs activated by poly(I:C) (50 μg/ml for 4 h). As targets for readout, C57BL/6 splenocytes were labeled with either low (0.5 μM) or high (5 μM) amounts of CSFE (Invitrogen Life Technologies), and 2 × 107 cells were adoptively transferred into mice in a 1:1 ratio (CSFElow:CSFEhigh) after peptide pulsing (10 μM) the CSFEhigh cells. Splenocytes from host mice were analyzed 16 h later by flow cytometry. Specific lysis was calculated as follows: specific lysis = (1 – (percentage of cells pulsed with peptide)/percentage of cells nonpeptide pulsed) × 100. Naive C57BL/6 mice were immunized with transfected BMDCs at days 0 and 3. In vivo cytotoxicity was analyzed at day 8. For quantification of SIINFEKL-specific T cells, peripheral blood was stained with H-2Kb/SIINFEKL tetramer (Beckman Coulter) and CD8 Ab (Caltag Laboratories). To assess the in vivo proliferation of CD4+ T cells, RAG-2−/− Thy1.2+ TCR-HA spleen cells were labeled with CSFE (5 μM) and adoptively transferred to Thy1.1+ BALB/c mice. The mice were immunized s.c. with 1 × 106 RNA-transfected or peptide-loaded BMDCs. Effector cells were quantified on day 5 by staining for CD4 and Thy1.2 (BD Biosciences) double-positive cells. For depletion of CD4+ T cells, mice received purified YTS191 Ab by i.p. injection (29). Evaluation of depletion efficacy showed that no CD4+ T cells were detectable up to 10 days after the last Ab injection (data not shown).

For therapeutic treatment models in non-TCR transgenic mice, we used an A20 cell line (A20-HA) transfected with the HA gene from the Mt. Sinai strain of the PR8 influenza virus (HA-phbApr-neo) provided by U. Hartwig (University of Mainz, Mainz, Germany). A total of 1 × 105 tumor cells was inoculated s.c. into the flanks of BALB/c mice. Advanced tumors were established, and vaccination of mice was initiated 17 days after inoculation. Mice received five immunizations in three daily intervals. Immunization was performed s.c. with 1 × 106 BMDCs electroporated with 20 μg of HA-MITD IVT RNA. For peptide immunization, 50 μg of the H2-Kd-restricted HA518–526 peptide was administered s.c. in IFA.

For subcellular localization studies, monocyte-derived DCs were matured for 24 h, transfected with IVT RNA, and grown on coverslips coated with Alcian blue for 18 h. Thereafter, cells were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Applichem). Labeling was performed with a polyclonal rabbit anti-eGFP serum (BD Biosciences), and costaining was performed with either murine anti-HLA class I (clone w6/32 donated by W. Herr, Mainz, Germany), anti-HLA class II (L243; BD Biosciences), anti-Lamp-I (H4A3; BD Biosciences), or anti-Golgi (58K-9; Abcam) Abs. Goat anti-rabbit FITC and goat anti-mouse Cy3 (both Jackson ImmunoResearch Laboratories) were used as secondary Abs, and cell nuclei were counterstained with Hoechst 33342. Microscopy was performed with a Leica immunofluorescence microscope with a ×40/0.4 NA objective lens using the QFISH software (Leica Microsystems).

For quantification of the SIINFEKL peptide presented on the Kb molecule, mouse thymoma EL4 cells or RMA-S cells transfected with SIINFEKL IVT RNA variants were harvested after 16 h, washed with cold buffer (PBS-1% FCS), blocked with PBS-10% FCS, stained for 30 min at 4°C with 25.D1.16 Ab, which specifically detects OVA257–264 peptide SIINFEKL in conjunction with H-2 Kb molecules (16), and thereafter stained with goat anti-mouse APC secondary Ab (Jackson ImmunoResearch Laboratories). For quantification of SIINFEKL-specific T cells, peripheral blood was stained with anti-CD8 (Caltag Laboratories) and SIINFEKL tetramer (H-2Kb/SIINFEKL 257–264; Beckman Coulter). Flow cytometric analysis was performed on a FACSCalibur analytical flow cytometer using CellquestPro (BD Biosciences) or FlowJo software (Tree Star).

The significance of the results was determined using Student’s t test or Tukey’s multiple comparison test. Values of p < 0.05 were considered statistically significant.

To test the assumption that the MHC class I trafficking domain may assist in redirecting Ags into compartments, in which MHC class II epitopes are generated, we constructed a series of vector templates for IVT RNA-encoding fusion proteins. In these, the marker of interest was N-terminally preceded by a sec mediating translocation into the endoplasmatic reticulum and C-terminally linked to the MITD consisting of the transmembrane and cytoplasmic domain of a MHC class I molecule. First, we determined the influence of trafficking domains by fluorescence microscopy of cells transfected with IVT RNA-encoding wild-type (WT) eGFP or eGFP fusion proteins (Fig. 1,a). In transfected K562 cells, WT eGFP displayed the expected diffuse cellular distribution in cyto- and nucleoplasm, whereas sec-eGFP showed a fluorescence pattern consistent with secretory organelles. For eGFP-MITD, however, we observed a staining of vacuolar compartments as well as accentuation of the cell surface membrane (Fig. 1,b). To determine the nature of these compartments, eGFP-MITD-transfected APCs were costained with a polyclonal rabbit anti-eGFP Ab and mAbs against defined compartment markers. Analysis of immature human monocyte-derived DCs revealed a nearly complete overlap of the eGFP-MITD fluorescence signal with the staining pattern of HLA class I molecules (Fig. 1 c). Most interestingly, eGFP-MITD mimics the characteristic maturation-dependent trafficking dynamics of MHC class I molecules (30). In immature DCs, eGFP-MITD colocalizes with the Golgi apparatus, whereas upon maturation it redistributes to the cell surface. Additionally, we found a strong colocalization of eGFP-MITD with intracellular and cell surface anti-HLA-DR staining and a partial colocalization with anti-LAMP-1 fluorescence pattern. In summary, these data indicate that the sec/MITD enables eGFP to enter into different endocytic compartments.

To analyze the effect of the sec/MITD signals on MHC class II peptide presentation, we resorted to HA as a model Ag. The HA molecule contains a well-characterized H2-ED-restricted MHC class II epitope (S1, aa 107–119) in its globular domain, which is generated by proteolytic cleavage in late endosomal compartments (31). As a class I transmembrane protein, HA is equipped with an autochthonous N-terminal signal peptide, a C-terminal TM, and a short cytoplasmic tail. We compared the Ag-specific CD4+ T cell stimulatory capacity of IVT RNA encoding the natural protein (HA-WT) with variants, in which the original TM was either entirely deleted (HAΔTM) or substituted by the transmembrane and cytosolic domains of either MHC class I (HA-MITD) or of the human CD27 (HA-CD27; Fig. 2,a). BMDCs transfected with these IVT RNA species or loaded with a synthetic HA110–119 peptide were used for in vitro stimulation of TCR transgenic HA-specific (TCR-HA) CD4+ T cells. A substantial improvement of stimulation of TCR-HA CD4+ T cells by HA-MITD was documented by increased proliferation and cytokine responses (Fig. 2,b). Titration of the amounts of transfected RNA showed that in comparison with the control Ags, 10- to 20-fold less HA-MITD RNA is sufficient to induce the same response, indicating that the stimulatory superiority of HA-MITD is most likely an effect of higher epitope density. These observations were further confirmed by in vivo studies, for which Thy1.1+ BALB/c mice were immunized with engineered Ag-presenting DCs after transfer of CFSE-labeled Thy1.2+ TCR-HA cells. Immunization with HA-MITD-transfected DCs induced a maximal proliferative response in mice comparable to DCs loaded with saturating doses of synthetic S1 HA107–119 peptide (Fig. 2,c), resulting in a strong expansion of TCR-HA cells (∗, p < 0.025; Fig. 2,d) and secretion of higher amounts of cytokines upon stimulation (Fig. 2 e). In contrast, immunization with DCs transfected with HA-WT resulted in moderate in vivo proliferation and expansion, whereas immunization with DCs transfected with unrelated control RNA did not induce proliferation at all (data not shown).

To determine the impact of sec/MITD trafficking signals on the amount of presented MHC class I-restricted epitopes, we used the OVA-derived SIINFEKL epitope. SIINFEKL/H2-Kb complexes on the cell surface can be directly quantified with the 25D1.16 Ab (32). A cDNA fragment encoding the peptide OVA255–266, which encompasses the original SIINFEKL epitope together with neighboring amino acids to enable processing in the original context, was cloned in frame into the pre-established expression cassettes for expression of unmodified (OVA255–266), secreted (sec-OVA255–266), and sec/MITD-tagged (OVA255–266-MITD) miniepitopes (Fig. 3,a). Flow cytometric quantification of SIINFEKL/H2-Kb complexes in BMDCs or EL4 cells (data not shown) 12 h after transfection with equimolar IVT RNA demonstrated a strongly increased epitope density for OVA255–266-MITD as compared with the unmodified OVA255–266 fragment (Fig. 3,b, left). Interestingly, a significant increase of epitope densities could be already obtained by the secretion signal alone, although to a lesser extent than with the OVA255–266-MITD variant. Analysis of SIINFEKL/H2-Kb at later time points showed a persistence of increased epitope densities (Fig. 3 b, right), excluding that the observed effect is a transient burst of epitopes due to an accelerated degradation of the fusion Ags.

FIGURE 3.

Improved MHC class I epitope presentation mediated by sec/MITD trafficking signals. a, Vector templates encoding OVA aa 255–266 variants comprising the H2-Kb-restricted SIINFEKL epitope and neighboring amino acids. b, Median fluorescence intensities of BMDCs stained with 25D1.16 Ab to measure SIINFEKL/H2-Kb complexes after transfection with IVT RNA 12 h (left) or 24 h (right) after transfection. Data represent mean values of two independent experiments + SEM. ∗, p < 0.05; ∗∗, p < 0.01. c, TAP-deficient EL4 and RMA-S cells were electroporated with 100 or 10 (data not shown) pmol RNA, cultured for 16 h, and stained with 25D1.16. Epoxomycin (0.5 μM) was used for proteasome inhibition. Both cell lines do present synthetic peptides delivered by external loading (4.5 nM). d, In vivo killing by TCR transgenic CD8+ cytolytic effector cells after immunization with IVT RNA-transfected BMDCs. The cytolytic activity of TCR-transgenic SIINFEKL-specific CD8+ OT-I cells (1 × 105/mouse) adoptively transferred into C57BL/6 mice (n = 3) was measured by flow cytometric quantification of CSFE+ target cells on day 6 after i.p. immunization with 0.75 × 106 BMDCs transfected with 15 pmol RNA. Data are representative for two independent experiments showing mean values + SEM. ∗∗, p < 0.01. e and f, De novo priming of naive T cells of C57BL/6 mice (n = 3) after two s.c. immunizations (d0, d3) with 1 × 106 BMDCs transfected with 100 pmol RNA. e, The frequency of Ag-specific T cells in peripheral blood was determined by flow cytometric quantification on day 8. Data are representative for two independent experiments showing mean values + SEM. Representative dot plots are shown. f, The lytic activity of de novo primed cytolytic effectors was determined in vivo on day 8 after priming. Representative histograms of the CSFE+ target cells are shown, and individual percentage of specific lysis is depicted. g, Relevance of CD4+ T cells for de novo priming was tested in C57BL/6 mice after depletion of CD4+ T cells (1 mg of YTS191 i.p.; days −3, 1, and 9). The mice (n = 4) were immunized by three s.c. immunizations (d0, d3, d10) with 1 × 106 BMDCs transfected with 100 pmol sec-OVA255–266 RNA. The frequency of SIINFEKL-specific CD8+ T cells in peripheral blood was quantified by tetramer staining (d8, d15). Data are shown as mean + SEM. ∗∗, p < 0.01.

FIGURE 3.

Improved MHC class I epitope presentation mediated by sec/MITD trafficking signals. a, Vector templates encoding OVA aa 255–266 variants comprising the H2-Kb-restricted SIINFEKL epitope and neighboring amino acids. b, Median fluorescence intensities of BMDCs stained with 25D1.16 Ab to measure SIINFEKL/H2-Kb complexes after transfection with IVT RNA 12 h (left) or 24 h (right) after transfection. Data represent mean values of two independent experiments + SEM. ∗, p < 0.05; ∗∗, p < 0.01. c, TAP-deficient EL4 and RMA-S cells were electroporated with 100 or 10 (data not shown) pmol RNA, cultured for 16 h, and stained with 25D1.16. Epoxomycin (0.5 μM) was used for proteasome inhibition. Both cell lines do present synthetic peptides delivered by external loading (4.5 nM). d, In vivo killing by TCR transgenic CD8+ cytolytic effector cells after immunization with IVT RNA-transfected BMDCs. The cytolytic activity of TCR-transgenic SIINFEKL-specific CD8+ OT-I cells (1 × 105/mouse) adoptively transferred into C57BL/6 mice (n = 3) was measured by flow cytometric quantification of CSFE+ target cells on day 6 after i.p. immunization with 0.75 × 106 BMDCs transfected with 15 pmol RNA. Data are representative for two independent experiments showing mean values + SEM. ∗∗, p < 0.01. e and f, De novo priming of naive T cells of C57BL/6 mice (n = 3) after two s.c. immunizations (d0, d3) with 1 × 106 BMDCs transfected with 100 pmol RNA. e, The frequency of Ag-specific T cells in peripheral blood was determined by flow cytometric quantification on day 8. Data are representative for two independent experiments showing mean values + SEM. Representative dot plots are shown. f, The lytic activity of de novo primed cytolytic effectors was determined in vivo on day 8 after priming. Representative histograms of the CSFE+ target cells are shown, and individual percentage of specific lysis is depicted. g, Relevance of CD4+ T cells for de novo priming was tested in C57BL/6 mice after depletion of CD4+ T cells (1 mg of YTS191 i.p.; days −3, 1, and 9). The mice (n = 4) were immunized by three s.c. immunizations (d0, d3, d10) with 1 × 106 BMDCs transfected with 100 pmol sec-OVA255–266 RNA. The frequency of SIINFEKL-specific CD8+ T cells in peripheral blood was quantified by tetramer staining (d8, d15). Data are shown as mean + SEM. ∗∗, p < 0.01.

Close modal

SIINFEKL/H2-Kb complexes derived from RNA OVA255–266-MITD variant encoding the SIINFEKL epitope together with neighboring amino acids were not detectable in epoxomycin (33)-treated EL4 or in TAP-deficient RMA-S cells, proving that the generation of epitopes from the sec-modified precursors depends on proteasomal degradation and TAP-driven transport of cleavage products from the cytosol (Fig. 3 c).

Together, these results demonstrate that by sec/MITD-modified chimeric Ags, not only presentation of MHC class II, but also of MHC class I epitopes is improved. Whereas for superior MHC class II presentation the combination of sec/MITD is required, a secretion signal alone significantly enhances the presentation efficiency of Ag-derived MHC class I epitopes, which may be further increased by MITD.

Next, we investigated whether the increase in MHC class I/epitope complexes by the modified variants has an effect on induction of effector functions of T cells in vivo. In a first setting, BMDCs transfected with equimolar amounts of either OVA255–266 or sec-OVA255–266-MITD were used for immunization of mice after transfer of TCR-transgenic SIINFEKL-specific CD8+ OT-I T cells. Testing effector functions by an in vivo cytotoxicity assay administering CFSE-labeled peptide-pulsed spleen cells as targets demonstrated clearly improved lytic activity in mice immunized with sec-OVA255–266-MITD (∗∗, p < 0.01; Fig. 3 d).

Even more pronounced effects were observed when measuring induction of autologous T cell responses in naive C57BL/6 mice rather than stimulation of adoptively transferred TCR transgenic T cells after immunization with RNA-transfected BMDCs. Frequencies of primed SIINFEKL-specific CD8+ T cells as measured by tetramer technology were higher in mice immunized with sec-OVA255–266-MITD (Fig. 3,e), as was their lytic activity in in vivo cytotoxicity assays (Fig. 3 f).

Previous studies using, for example, LAMP or DC-LAMP fusion Ags, which share the N-terminal sec modification, reported improved in vivo CD8+ T cell responses as well, but attributed this to augmented CD4+ T cell help (34, 35). The SIINFEKL minigene used in our studies does not code for an apparent CD4+ epitope, making it unlikely that Ag-specific CD4+ T cell help is responsible for the improved CTL response. Moreover, depletion of CD4+ T cells before immunization with sec-SIINFEKL did not abrogate this effect (Fig. 3 g). In contrast, we observed an even higher expansion of OVA-specific CD8+ cells in CD4+ T cell-depleted mice, most likely due to the concomitant depletion of regulatory T cells.

Preclinical and clinical protocols exploit IVT RNA for treatment of cancer by using this format for Ag delivery into autologous DCs, which are subsequently adminstered s.c. into the respective host (36, 37, 38). To assess the therapeutic efficacy of a MITD-tagged Ag-specific vaccine, we used HA as Ag in an analogous model for treatment of advanced tumors. BALB/c mice were challenged with a lethal dose of A20-HA cells and subjected to five rounds of immunization starting 17 days after inoculation, when tumors were grown to a diameter of 2–3 mm (Fig. 4,a). Whereas untreated mice all died within 30 days after termination of treatment, 70% of mice vaccinated with HA-MITD-transfected BMDCs were still alive at that time (Fig. 4 b). Forty percent of the mice had complete remissions and survived the tumor challenge. Survival rates achieved by immunization with the H2-Kd-restricted immunodominant HA518–526 peptide in combination with IFA as gold standard were even lower.

To assess a broader panel of epitopes, we analyzed responses to HCMV pp65, the immunodominant Ag of human CMV (39, 40), and NY-ESO-1 (41, 42), an immunodominant tumor Ag expressed in a wide range of human cancers. Multiple MHC class I- and class II-restricted epitopes have been described for these Ags. DCs generated from blood of HCMV-seropositive donors were transfected with varying amounts of IVT RNA (0.1–10 μg) encoding either unmodified pp65, a sec/MITD pp65 variant (pp65-MITD), or an unrelated control Ag (Fig. 5,a) and used for in vitro stimulation of autologous T cells. The expansion of preformed Ag-specific CD4+ T cells was determined by IFN-γ ELISPOT and proliferation assay using autologous DCs loaded with a pool of overlapping 15-mer peptides covering the entire pp65 protein. HCMV pp65 is a nuclear protein (43), which does not directly access compartments for MHC class II loading. Accordingly, we observed no significant expansion of pp65-specific IFN-γ-secreting CD4+ T cells with pp65 IVT RNA. In contrast, even with nanogram amounts of pp65-MITD, strong expansion of pp65-specific T cells was achieved (Fig. 5,b). The magnitude of expansion induced by pp65-MITD corresponded to that obtained by stimulation with DCs loaded with saturating doses of a pool of overlapping pp65 peptides, indicating that the entirety of immune relevant CD4+ epitopes is generated by processing of the modified pp65 variant (Fig. 5,c). Similarly, NY-ESO-I-specific CD4+ T cells were efficiently expanded from bulk T cell populations of NY-ESO-I-seropositive cancer patients using sec/MITD-modified NY-ESO-I RNA, but not by stimulation with RNA encoding the unmodified tumor Ag (Fig. 5 d).

FIGURE 5.

Improved expansion of Ag-specific CD4+ and CD8+ T cells from bulk lymphocyte populations by stimulation with sec/MITD fusion Ags. a, Vectors for generation of IVT RNA-encoding variants of the viral HCMV pp65 protein and NY-ESO-I tumor Ag. b and c, Purified CD4+ T lymphocytes obtained from healthy HCMV-seropositive donors were cocultured in vitro with autologous DCs transfected with indicated amounts of RNA-encoding HCMV pp65 variants or an unrelated control. b, IFN-γ ELISPOT was performed on day 7 with autologous DCs loaded with a pool of overlapping CMV pp65 peptides (pp65 pool) as Ag presenters. Data are representative for three independent experiments and are shown as mean ± SEM of triplicates. c, Proliferation was measured on day 12. Data are representative for three independent experiments and are shown as mean + SEM of triplicates. ∗, p < 0,025. d, Expansion and IFN-γ secretion of sorted CD4+ T cells from two NY-ESO-I-seropositive cancer patients upon stimulation with autologous DCs transfected with 20 μg of IVT RNA. Results are shown as mean + SEM of triplicates. e, Quantification of IFN-γ-secreting pp65-reactive CD8+ T lymphocytes from nine HCMV-seropositive donors determined on day 7 after stimulation with autologous DCs transfected with 20 μg of RNA. No significant spot counts were detected when lymphocytes from CMV-seronegative donors were used. Data are shown as mean of triplicates after background substraction. f, Dose-dependent expansion of IFN-γ-secreting CD8+ pp65-reactive T lymphocytes cultured for 1 wk with autologous DCs transfected with indicated amounts of RNA. Data are representative for three independent experiments and are shown as mean ± SEM of triplicates. g, Cytolytic effector function of CD8+ bulk T cell populations determined on day 7 after stimulation with autologous DCs either loaded with pp65 peptide pool (1.75 μg/μl) or transfected with 20 μg of Ag-encoding RNA. Cytotoxicity was determined in a 4.5-h 51Cr release assay using 51Cr-labeled autologous DCs loaded with indicated total peptide concentrations of the pp65 peptide pool (E:T ratio = 20:1). The results are representative for three independent experiments and are shown as mean ± SEM of triplicates.

FIGURE 5.

Improved expansion of Ag-specific CD4+ and CD8+ T cells from bulk lymphocyte populations by stimulation with sec/MITD fusion Ags. a, Vectors for generation of IVT RNA-encoding variants of the viral HCMV pp65 protein and NY-ESO-I tumor Ag. b and c, Purified CD4+ T lymphocytes obtained from healthy HCMV-seropositive donors were cocultured in vitro with autologous DCs transfected with indicated amounts of RNA-encoding HCMV pp65 variants or an unrelated control. b, IFN-γ ELISPOT was performed on day 7 with autologous DCs loaded with a pool of overlapping CMV pp65 peptides (pp65 pool) as Ag presenters. Data are representative for three independent experiments and are shown as mean ± SEM of triplicates. c, Proliferation was measured on day 12. Data are representative for three independent experiments and are shown as mean + SEM of triplicates. ∗, p < 0,025. d, Expansion and IFN-γ secretion of sorted CD4+ T cells from two NY-ESO-I-seropositive cancer patients upon stimulation with autologous DCs transfected with 20 μg of IVT RNA. Results are shown as mean + SEM of triplicates. e, Quantification of IFN-γ-secreting pp65-reactive CD8+ T lymphocytes from nine HCMV-seropositive donors determined on day 7 after stimulation with autologous DCs transfected with 20 μg of RNA. No significant spot counts were detected when lymphocytes from CMV-seronegative donors were used. Data are shown as mean of triplicates after background substraction. f, Dose-dependent expansion of IFN-γ-secreting CD8+ pp65-reactive T lymphocytes cultured for 1 wk with autologous DCs transfected with indicated amounts of RNA. Data are representative for three independent experiments and are shown as mean ± SEM of triplicates. g, Cytolytic effector function of CD8+ bulk T cell populations determined on day 7 after stimulation with autologous DCs either loaded with pp65 peptide pool (1.75 μg/μl) or transfected with 20 μg of Ag-encoding RNA. Cytotoxicity was determined in a 4.5-h 51Cr release assay using 51Cr-labeled autologous DCs loaded with indicated total peptide concentrations of the pp65 peptide pool (E:T ratio = 20:1). The results are representative for three independent experiments and are shown as mean ± SEM of triplicates.

Close modal

The same set of pp65 IVT RNA species and analogous assays was used to explore the impact on stimulation of pp65-specific CD8+ T cells. We demonstrated a profound increase in expansion of pp65-specific CD8+ T cells from nine different CMV+ donors by stimulation with autologous DCs transfected with the pp65-MITD variant (Fig. 5,e). The extent of improvement of CD8+ T cell expansion varied between donors ranging from 1.5- to 50-fold, most likely indicating an impact of epitope specificities of the pre-established T cell repertoire. Titration studies demonstrated that loading of APCs with pp65-MITD IVT RNA amounts several logs lower than with WT pp65 was sufficient for even better T cell expansion (Fig. 5,f). Moreover, we observed that T cells stimulated with pp65-MITD IVT RNA-loaded DCs, in contrast to responders generated with WT pp65 RNA and pp65 peptide pool, lyse target cells pulsed with low nanomolar amounts of antigenic peptide, suggesting improved cytotoxic effector functions and higher TCR avidity (Fig. 5 g).

For a comprehensive mapping of epitope specificities of expanded T lymphocytes, we loaded the single peptide species from the overlapping peptide pool on autologous DCs and tested each of them as Ag presenters for induction of IFN-γ secretion in ELISPOT. Investigating several HCMV-seropositive donors, we found that stimulation with pp65-MITD resulted in a polyepitopic expansion of Ag-specific T cells. Interestingly, reactivity patterns of CD8+ and CD4+ T cells differed substantially. The pp65-reactive CD8+ T cells were directed against a few distinct peptides governed by known immunodominant epitopes such as the previously described HLA-B7-restricted pp65417–426 peptide (Fig. 6,a) (44). In contrast, the repertoire of preformed Ag-specific CD4+ T cells was surprisingly broad, featuring a diversity of epitopes per donor and varying sets of epitopes in independent individuals (Fig. 6 b). Apparently, nearly all regions of the pp65 molecule are subject to recognition by CD4+ T cells. Moreover, this observation proves an efficient presentation of all regions using pp65-MITD IVT RNA as Ag format. With HCMV-seronegative donors, in contrast, we observed no significant reactivity (data not shown), proving that the observed reactivity pattern indeed reflects T cell specificities preformed by HCMV infection.

FIGURE 6.

Potent polyepitopic expansion of preformed pp65-reactive CD8+ and CD4+ T cells by stimulation with sec/MITD fusion Ags. a, Mapping of epitopes recognized by CD8+ T lymphocytes of three HCMV-seropositive, HLA-B7-positive donors. Purified CD8+ T cells were expanded for 7 days with pp65-MITD RNA-transfected DCs. IFN-γ secretion was measured by ELISPOT using DCs loaded with single 15-mer peptides. Background reactivity obtained by stimulating with DCs loaded with NY-ESO-I peptide pool was subtracted. Noteworthily, all donors recognize peptides 104 (aa 413–427) and 105 (aa 417–431), which contain the immunodominant HLA-B7-restricted PRPLPLAGA epitope. b, Mapping of epitopes recognized by MACS-sorted CD4+ T lymphocytes of HCMV-seropositive donors after 7 days of expansion with pp65-MITD RNA-transfected DCs.

FIGURE 6.

Potent polyepitopic expansion of preformed pp65-reactive CD8+ and CD4+ T cells by stimulation with sec/MITD fusion Ags. a, Mapping of epitopes recognized by CD8+ T lymphocytes of three HCMV-seropositive, HLA-B7-positive donors. Purified CD8+ T cells were expanded for 7 days with pp65-MITD RNA-transfected DCs. IFN-γ secretion was measured by ELISPOT using DCs loaded with single 15-mer peptides. Background reactivity obtained by stimulating with DCs loaded with NY-ESO-I peptide pool was subtracted. Noteworthily, all donors recognize peptides 104 (aa 413–427) and 105 (aa 417–431), which contain the immunodominant HLA-B7-restricted PRPLPLAGA epitope. b, Mapping of epitopes recognized by MACS-sorted CD4+ T lymphocytes of HCMV-seropositive donors after 7 days of expansion with pp65-MITD RNA-transfected DCs.

Close modal

The dissection of Ag-processing pathways has paved the way for rational approaches to increase the number of MHC class II/peptide complexes and thus improve CD4+ T cell responses by targeting Ags into distinct MHC class II compartments. An important observation in this context is that MHC class I molecules occur in virtually all compartments and organelles involved in MHC class II processing. Therefore, we assumed that coupling an Ag to domains contributing to trafficking of MHC class I molecules would reroute it into the compartments in which protein breakdown for loading of MHC class II molecules takes place. Our studies with eGFP-MITD confirm this assumption. The sec/MITD-modified fluorescence reporter not only translocates into diverse Ag-processing compartments harboring MHC class I and class II molecules, but also mimics the dynamic natural trafficking properties of MHC molecules in immature and mature DCs. Studying the effects of this modification with four different well-defined Ags, we made the following major findings.

First, sec/MITD fusion proteins are highly efficient for stimulation and expansion of Ag-specific CD4+ T cells. As shown in the HA system, this effect is mediated by the MHC class I trafficking domain itself. Based on this observation, one would expect that peptides derived from MHC class I molecules are frequently presented on MHC class II. Intriguingly, there is evidence from sequencing peptide ligands eluted from MHC class II molecules that >15% of these epitopes are actually derived from processing of MHC class I molecules (45). Given the fact that several thousand different proteins undergo processing for MHC class II presentation, this is an enormous proportion, further supporting the validity of our rationale.

Second, we found a substantial increase in MHC class I epitope presentation efficiency as well. Our studies with a set of OVA variants demonstrate that this effect is mediated at least in part by the secretion signal alone. An obvious explanation for this finding is that translocation into the ER leads to misfolding and accelerated degradation of proteins. This, however, is not compatible with the fact that increased OVA epitope densities are observed not only at early, but also at late time points after transfection. It is well known that Ag processing is an inefficient process. The vast majority (>99%) of peptide fragments generated by the proteasome are intercepted on their way to MHC class I processing and loading compartments and degraded by cytosolic exo- and endopeptidases (46). It is conceivable that this rate-limiting step depends on the diffusion time needed by proteasomal digestion products to reach the TAP transporter. In contrast to cytosolic or nuclear polypeptides, misfolded proteins in the secretory pathway are eliminated by ER-associated degradation after retranslocation into the cytosol (47). A recent report (48) demonstrated that such proteins are preferentially digested by proteasomes in the immediate vicinity of the ER. A difficult to prove hypothesis is that cleavage products of such ER adjacent proteasomes may be salvaged from degradation by cytosolic peptidases due to shorter distance and thus faster access to TAP transporter molecules. Several other genetic modifications known to enhance MHC class II presentation, such as LAMP or DC-LAMP fusion proteins, also incorporate a secretion signal at the N terminus. Improved CTL induction after vaccination with such Ags is often interpreted as a mechanism mediated by concomitant stimulation of CD4+ Th cells. Our studies demonstrate that better expansion of Ag-specific CD8+ T cells and improved CTL responses in vitro and in vivo do not depend on CD4+ T cells. Rather, increased MHC class I Ag presentation efficiency induced by attachment of a secretion signal may be the underlying mechanism.

Third, we show that the improved MHC class I and II Ag presentation efficiency mediated by the combined sec/MITD trafficking signal is not limited to single epitopes, but can be applied for simultaneous stimulation and expansion of multiple CD8+ and CD4+ T cell subsets. In the case of HCMV pp65, our approach apparently allowed us to expand even Ag-specific populations of low frequency. This led to the surprising observation that a plentitude of epitopes covering nearly all regions of the molecule is subject to recognition by CD4+ T cells of individual pp65 Ag-experienced donors. HCMV-specific CD8+ T cell repertoires, in contrast, seem to be skewed to single immunodominant epitopes, as exemplified by the three donors sharing the HLA-B7 allele.

Altogether, our findings establish the MITD as a novel powerful tool to amplify Ag presentation efficiency of recombinant Ags introduced into DCs. Such chimeric fusion Ags may be applicable as improved genetic vaccines enabling the induction of combined cellular immune responses, or may be used for diagnostic purposes to map Ag-specific immune repertoires and quantify Ag-specific T cells, as previously demonstrated by us for CMVpp65 (49).

We thank M. Holzmann for expert technical assistance.

U.S., Ö.T., and S.K. hold a patent related to the work described in the present study. The other authors have no financial or other interests with regard to the submitted manuscript.

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 Combined Project Grant SFB432 and Heisenberg Scholarship TU 115/2-1 of the Deutsche Forschungsgemeinschaft, as well as the Immunology Cluster of Excellence at the University Mainz.

4

Abbreviations used in this paper: LAMP, lysosome-associated membrane protein; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; eGFP, enhanced GFP; ER, endoplasmic reticulum; HA, influenza hemagglutinin; HCMV, human CMV; IVT, in vitro transription; MITD, MHC class I trafficking signal; sec, secretion signal; TM, transmembrane domain; WT, wild type.

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