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The Journal of Immunology, 2006, 177: 1534-1542.
Copyright © 2006 by The American Association of Immunologists

Priming Protective CD8 T Cell Immunity by DNA Vaccines Encoding Chimeric, Stress Protein-Capturing Tumor-Associated Antigen1

Reinhold Schirmbeck2,*, Petra Riedl*, Mark Kupferschmitt*, Ursula Wegenka*, Hansjörg Hauser{dagger}, Jason Rice{ddagger}, Andrea Kröger{dagger} and Jörg Reimann*

* Department of Internal Medicine I, University of Ulm, Ulm, Germany; {dagger} German Research Centre for Biotechnology, Braunschweig Germany; and {ddagger} Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals Trust, Southampton, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
DNA vaccines encoding heat shock protein (hsp)-capturing, chimeric peptides containing antigenic determinants of the tumor-associated Ag (TAA) gp70 (an envelope protein of endogenous retrovirus) primed stable, specific, and tumor-protective CD8 T cell immunity. Expression of gp70 transcripts was detectable in most normal tissues but was particularly striking in some (but not all) tumor cell lines tested (including the adenocarcinoma cell line CT26). An ~200 residue gp70 fragment or its Ld-binding antigenic AH1 peptide cloned in-frame behind an hsp-capturing (cT272) or noncapturing (T60) N-terminal large SV40 tumor Ag sequence was expressed as either hsp-binding or -nonbinding chimeric Ags. Only hsp-capturing, chimeric fusion proteins were expressed efficiently in transfected cell lines and primed TAA-specific CD8 T cell immunity. This immunity mediated protection in the CT26 and mKSA models. A vaccination strategy based on delivering antigenic, hsp-associated TAA fragments can thus prime protective CD8 T cell immunity even if these TAA are of low intrinsic immunogenicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although DNA vaccine-encoded Ags often are quite potent immunogens in mice, new strategies to enhance their immunogenicity especially for T cells by codelivering cytokines or costimulator molecules are an active area of research. Heat shock proteins (hsp)3 are abundant intracellular proteins involved in activation of APCs, indirect (cross)-presentation of class I-restricted epitopes, and chaperoning of peptides during Ag presentation. These properties of hsp have been used in novel immunotherapeutic approaches for cancers and infections (1, 2). In addition to using complexes of hsp with peptide/protein Ags as vaccines, DNA vaccines encoding hsp/Ag fusion proteins have been shown to display enhanced immunogenicity of the Ag for T cells (3, 4). We developed an hsp-facilitated expression system to produce chimeric Ags with enhanced immunogenicity for the humoral and cellular immune system (reviewed in Ref. 5). The system is based on N-terminal sequences of the large SV40 tumor Ag (T-Ag) that show strong, noncovalent binding to hsp73/hsc70 through the T1–77 DnaJ (hsp40) homology domain. This domain binds to, and regulates substrate binding of, the constitutively expressed cytosolic hsp73/hsc70. The ability to bind hsp73 is lost when this 77 residue domain is truncated. Chimeric proteins with an N-terminal, hsp73-binding domain and various C-terminal antigenic sequences can be expressed efficiently with a long half-life in immunogenic form (5, 6). In contrast, expression of fusion products between a non-hsp-binding domain (e.g., the T1–60) and C-terminal antigenic protein fragments is not (or only barely) detectable and induces no (or only weak) immune responses. We demonstrated the usefulness of the system in priming tumor-protective T cell responses in mice (6, 7). This was achieved using viral reporter Ags but not the more informative but less immunogenic tumor-associated Ags (TAA). T cell responses to TAA are difficult to establish because most TAA are self-proteins with low intrinsic immunogenicity, to which the specific immune system has been tolerized. Therefore, we tested if specific and protective CD8 T cell immunity can be primed by vaccination with hsp-capturing TAA in a well-described, experimental tumor system.

Cancer vaccines are designed to prime T cells that mediate antitumor responses in vivo. The protective and/or therapeutic value of TAA-specific T cell immunity is usually assessed in preclinical models by testing if 1) immune T cells can specifically protect against growth of transplanted tumor cells; 2) adoptive immune T cell transfer can reject growing tumors; or 3) vaccine-induced T cells can limit growth of (or reject) an established tumor. The many-sided interactions between cells of the immune system and transformed cells during emergence and progression of cancers in immunocompetent hosts are complex and described in terms of "immune surveillance," "immune editing," or "immune tolerance" (8). The simplest test to ascertain the relative efficacy of CD8 T cell responses to TAA in vivo is the resistance to a challenge of transplantable tumor cells.

Members of the human and mouse endogenous retroviruses are expressed preferentially in fresh tumors and tumor cell lines (9, 10). The antigenic, Ld-restricted peptide AH1 has been identified in the colonic epithelial CT26 tumor model as a dominant epitope of the envelope protein gp70 of the endogenous, ecotropic murine leukemia virus emv-1 integrated as a provirus in the mouse germline DNA (11). Also in the EL4 thymoma model, the Kb-restricted AFV8 peptide of gp70 is a dominant TAA (12). Although envelope proteins of endogenous retroviruses rarely give rise to stable proteins, they may effectively generate antigenic peptides from defective, nascent translation products (13, 14, 15). Attempts have been made to enhance the immunogenicity of this TAA to make it an attractive cancer vaccine candidate (1, 16, 17). We used an expression system that supports high-level and long-lasting expression of chimeric, hsp-capturing Ags from DNA vaccines to efficiently prime tumor-protective, gp70 (AH1)-specific CD8 T cell immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/cJ mice (H-2d) obtained from Janvier were kept under standard pathogen-free conditions in the animal colony of Ulm University. Female mice were used at 12–16 wk of age. All animal experiments were approved by the respective Institutional Animal Care and Use Committees in accordance with the applicable federal, provincial, and local regulations.

Cells

The N-nitroso-N-methylurethane-induced, undifferentiated colon carcinoma cell line CT26 (CRL-2638), the mastocytoma cell line P815 (TIB-64), and the T cell lymphoma RBL5/EL4 (TIB-39) were obtained from the American Tissue Culture Collection. The BALB/c-derived mKSA was provided by Dr. W. Deppert (Heinrich Pette Institute, Hamburg, Germany). The spontaneous mammary adenocarcinoma cell line TS/A (18) and the epithelial cell line TC-1 from a B6 mouse cotransformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes (19) were provided by the German National Resource Centre for Biological Material.

DNA vaccines

Four DNA vaccines were constructed, as indicated in Fig. 1B. For vaccine 1, pCI/cT272-gp70{Delta}, the gp70-encoding, 594-bp fragment was amplified from a CT26-tumor cell-derived cDNA by PCR with the forward primer (AAA GGT ACC AAC CTT TGT CCG AAG TGA CCG) containing a KpnI site and a reverse primer (AAA GCG GCC GCT TAT TGT ACC AAT CCT GTG TGG TCG) containing a NotI site. The product was cloned C-terminally in-frame behind the cT272 (229 T-Ag residues containing the 272 N-terminal residues minus the nuclear localization sequence (T110–152)) fragment generating the pCI/cT272-gp70{Delta} plasmid. For vaccine 2, pCI/T60-gp70{Delta}, the identical 594-bp fragment was cloned C-terminally in-frame behind the 60 N-terminal T-Ag residue (T60) fragment generating the pCI/T60-gp70{Delta} plasmid. For vaccine 3, pCI/cT272-AH1, the cT272-encoding fragment was amplified from the pCI/cT272 by PCR using a forward primer (AAA GCT AGC ACC ATG GAT AAA GTT TTA) containing a NheI site and a reverse primer (TTT GGT ACC TCA AAA TTG GTG GTA AAC ATA ACT AGG GGA TTC AAG CTT CCA GGA CAC TT) containing an AH1 epitope-encoding, 27-bp fragment and a KpnI site. The italics in the sequence indicate the 27-bp-encoding AH1 epitope. The fragment was cloned into pCI vector, generating the pCI/T272--AH1 plasmid. For vaccine 4, pCI/T60-AH1, the T60-encoding fragment was amplified by PCR using a forward primer (AAA GCT AGC ATG GAT AAA GTT TTA AAC AG) containing NheI site and a reverse primer (TTT GGT ACC TCA AAA TTG GTG GTA AAC ATA ACT AGG GGA AGT CGA CTG CAG AAT TCG AA) containing the AH1 epitope-encoding, 27-bp fragment and a KpnI site. The italics in the sequence indicate the 27-bp-encoding AH1 epitope. The fragment was cloned into pCI vector, generating the pCI/T60-AH1 plasmid.


Figure 1
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  		FIGURE 1. A, Map of the gp70 sequences used for generation of DNA vaccines. B, Characterization of DNA vaccines. DNA encoding a 198-residue fragment of gp70 (residue 376–573) was cloned in frame C-terminally to a mutant, hsp-binding cT272 fragment or to a T60 fragment (not binding hsp), generating the plasmids pCI/cT272-gp70{Delta} (vaccine 1) or pCI/T60-gp70{Delta} (vaccine 2), respectively. DNA encoding the minimal 9-residue AH1 epitope of gp70 was cloned in frame C-terminally to the cT272 fragment or to the T60 fragment, generating the plasmids pCI/cT272-AH1 (vaccine 3) or pCI/T60-AH1 (vaccine 4), respectively. C, Expression of fusion Ags was tested in vitro. LMH cells were transiently transfected with constructs 1–4 or the control pCI DNA. Cells were labeled with [35S]methionine and immunoprecipitated for T-Ag (using a monoclonal mouse anti-T-Ag mAb) and analyzed by SDS-PAGE, followed by fluorography of the gels. Alternatively, nonlabeled cell extracts were immunoprecipitated with an anti T-Ag mAb and processed for hsp73- or T-Ag-specific Western blotting. The positions of cT272-gp70{Delta}, cT272-AH1, the cellular hsp73, and the IgG/HC or IgG/LC of the anti-T mAb are indicated.

 
The control DNA vaccine pCI/cT272-S1 has been described previously (6, 20).

Generation of short- term AH1-specific CD8 T cell lines and coculture with tumor cells

BALB/c mice were immunized and boosted with pCI/cT272-gp70{Delta} plasmid. Ten days after the last injection, spleen cells (107 cells/ml) were prepared and cultured in RPMI 1640 medium supplemented with 100 µM 2-ME, antibiotics, 10% FCS, and a selected batch of Con A-stimulated rat spleen cell supernatant (2% v/v). To stimulate gp70-specific CD8 T cells, 10–30 µg/ml of the Ld-binding SPSYVYHQF gp70 AH1 peptide (JPT Peptide Technologies) was added. Responder T cells were harvested from a 4-day culture, washed, and 5 x 105 responder cells were cocultured with 5 x 104 tumor cells for 24 h in 200 µl. Cell supernatants were collected and tested for IFN-{gamma} content by a commercial ELISA system (catalog no. 551216/554410; BD Biosciences).

Expression of chimeric Ag from DNA vaccines

Cells were transfected with plasmids using the Ca2PO4 method. Two days after transfection, cells were metabolically labeled for 12 h with [35S]methionine and extracted with lysis buffer (120 mM NaCl, 1% aprotinin (Trasylol; Bayer), leupeptin, 0.5% Nonidet P-40, and 50 mM Tris-hydrochloride (pH 8.0)) for 30 min at 4°C. Extracts were cleared by centrifugation and immunoprecipitated for T-Ag using the mAb PAb108 and protein A-Sepharose. This mAb is directed against the extreme N terminus of the T-Ag and was used to immunoprecipitate T1–60- or T1–77-based fusion proteins (6, 21). Immune complexes bound to protein A-Sepharose were purified with wash buffer (300 mM LiCl, 1% Nonidet P-40, and 100 mM Tris-hydrochloride (pH 8.5)), followed by two washes with PBS and 0.1x PBS. Immune complexes were recovered from protein A-Sepharose with elution buffer (1.5% SDS, 5% mercaptoethanol, and 7 mM Tris-hydrochloride (pH 6.8)) and processed for SDS-PAGE. Levels of immunoprecipitated proteins were analyzed by fluorography of the gels. Alternatively, nonlabeled cell extracts were immunoprecipitated as described above and processed for hsp73- or T-Ag-specific Western blotting as described previously (22, 23). Briefly, immunoprecipitates were processed by SDS-PAGE and blotted onto nitrocellulose paper. Nitrocellulose sheets were incubated for 6 h with 1/500 diluted rabbit anti T serum (to detect the cT272 fragment) or 1/750 diluted rat anti-hsp73 mAb (catalog no. SPA815; StressGene), followed by a second (1 h) incubation with a rabbit anti-rat IgG serum (catalog no. 612-4102; Rockland) to detect hsp73. Sheets were washed and incubated with 0.5 µCi of 35S-labeled protein A (Amersham Biosciences), washed, dried, soaked in 20% 2,5-diphenyloxazolol in toluol, and again dried. Radiolabeled immune complexes were detected by fluorography.

DNA vaccination

We injected 50 µl of PBS containing 1 µg/µl plasmid DNA into each tibialis muscle for nucleic acid immunization. All mice received bilateral i.m. injections as described previously (24, 25).

In vivo suppression of CD4 or CD8 T cells

CD4 or CD8 T cells were suppressed in mice by three injections of the anti-CD4 mAb YTS 191 or the anti-CD8 mAb YTS 169. Three and 1 days before, and 5 days after the tumor graft, mice were i.p. injected with 200 µl of PBS containing 100 µg of Ab. Flow cytometry (FCM) analyses of peripheral blood mononuclear cell populations demonstrated that >98% of the CD4 or CD8 T cells were deleted after two injections and remained deleted up to 5 days after the last Ab injection.

Tumor cell transplantation

Tumor cells were washed three times in PBS, and 50 µl of the cell suspension (containing the indicated number of cells) was injected s.c. into the shaved right flank of mice (five mice per experimental group). Tumor development was followed by serial measurements of tumor size at two perpendicular diameters.

Determination of specific CD8 T cell frequencies by FCM

For determination of CD8 T cell frequencies, spleen cells (107 cells/ml) were restimulated in the presence of 2.5 µg/ml brefeldin A (catalog no. 15870; Sigma-Aldrich) for 4 h in RPMI 1640 medium with 1 µg/ml of the Ld-binding gp70 peptide SPSYVYHQF or the Ld-binding control peptide HBsAg28–39 IPQSLDSWWTSL (26). Cells were harvested, washed, and stained with PE-conjugated anti-CD8 mAb (catalog no. 553032; BD Biosciences). Surface-stained cells were fixed (2% paraformaldehyde in PBS), resuspended in permeabilization buffer (HBSS, 0.5% BSA, 0.5% saponin, and 0.05% sodium azide), incubated with FITC-conjugated anti-IFN-{gamma} (catalog no. 554411; BD Biosciences) for 30 min at room temperature, and washed twice in permeabilization buffer. Stained cells were resuspended in PBS supplemented with 0.3% w/v BSA and 0.1% w/v sodium azide. The number of cytokine-expressing CD8 T cells per 105 splenic CD8 T cells was determined by FCM.

Gp70-specific, biotinylated Ld/SPSYVYHQF monomers were provided by the National Institute of Allergy and Infectious Diseases Tetramer Facility (Emory University Vaccine Center at Yerkes, Atlanta, GA). Tetramers were formed with streptavidin R-PE conjugate (catalog no. S-866; Molecular Probes). Freshly isolated cells were washed twice in PBS/0.3% w/v BSA/0.1% w/v sodium azide. Nonspecific binding of Abs to FcR was blocked by preincubating cells with mAb 2.4G2 (catalog no. 01241D; BD Biosciences) directed against the Fc{gamma}RIII/II CD16/CD32 (0.5 µg mAb/106 cells/100 µl). Cells were incubated for 30 min at 4°C with APC-conjugated anti-CD8 mAb (catalog no. 553035; BD Biosciences) and PE-conjugated tetramers. Cells were washed twice in FACS buffer and analyzed by FCM.

Semiquantitative RT-PCR

RNA was prepared using TRIzol (catalog no. 15596-026; Invitrogen Life Technologies) and the RNeasy Mini kit (catalog no. 74104; Qiagen). Samples were treated with RNase-free DNase I (catalog no. 1010395; Qiagen) to eliminate contaminating genomic DNA. RNA (2 µg) was reverse transcribed with Superscript II reverse transcriptase (catalog no. 18064-014; Invitrogen Life Technologies) using random primers according to the supplier’s instructions. beta-actin PCR signals were used to equalize cDNA amounts between preparations. Primers were as follows: beta-actin (forward), 5'-GGG AAT GGG TCA GAA GGA CT-3', and beta-actin (reverse), 5'-TTT GAT GTC ACG CAC GAT TT-3'; and gp70 (forward), 5'-CAC CAA TTT GAA AGA CGA GCC-3', and gp70 (reverse), 5'-CAA TTC CGC CCA TAG TGA GTC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Construction of DNA vaccines

We used a previously described fragment of the gp70 gene (27, 28) that encodes 198 residues (residue 376–573) of this TAA from an endogenous retrovirus. This fragment contains the well-defined AH1 (SPSYVYHQF) determinant recognized by CD8 T cells from H-2d mice in the context of Ld (Fig. 1A). The 198-residue encoding fragment was cloned in frame behind the 229 residue N terminus cT272 of the cytoplasmic SV40 large T-Ag to generate the DNA vaccine pCI/cT272-gp70{Delta} (vaccine 1; Fig. 1B). The cT272 fragment contains the 77-residue DnaJ-homologous domain that captures cytosolic, constitutively expressed hsp73 (5). To check Ag expression from this DNA vaccine, cells were transiently transfected with this vector, labeled with [35S]methionine, and lysates were used in immunoprecipitation studies with the mAb PAb108 binding the extreme N terminus of the T-Ag. These studies showed readily detectable expression of a product of the expected 46-kDa size (Fig. 1C). cT272-gp70/hsp73 complexes were confirmed by hsp73- and T-Ag- specific Western blotting (Fig. 1C). Cloning the same fragment behind a 60-residue (T1–60) N terminus of the T-Ag (that does not capture hsp because it has no intact DnaJ like domain) generated the DNA vaccine pCI/T60-gp70{Delta} (vaccine 2; Fig. 1B). We were unable to detect the T60-gp70{Delta} Ag in short-term (1–2 h) or long-term (12–18 h) [35S]methionine-labeled cells or in Western Blot analyses (Fig. 1C). This confirmed that only the N-terminal DnaJ-like domain of T-Ag facilitates stable expression of hsp-capturing, chimeric Ag fragments, as we have reported previously in other Ag systems (5).

Furthermore, we fused the sequence encoding the antigenic (Ld-binding) 9-mer peptide AH1 of gp70 in-frame behind the cT272-encoding fragment. This generated the DNA vaccine pCI/cT272-AH1 (vaccine 3; Fig. 1B). Cells transiently transfected with this DNA efficiently expressed an hsp73-binding protein of ~34-kDa molecular mass, i.e., the expected size of the fusion protein (Fig. 1C). In addition, the AH1 determinant was fused to the T1–60 N terminus of the T-Ag to generate the DNA vaccine pCI/T60-AH1 (vaccine 4; Fig. 1B). As expected, no product could be immunoprecipitated from lysates of cells transiently transfected with this plasmid (Fig. 1C). Hence, an intact N-terminal DnaJ domain (of the T-Ag) greatly facilitates expression of hsp-binding, chimeric Ag fragments or its epitopes.

DNA vaccines encoding hsp-capturing tumor Ag fragments are immunogenic for CD8 T cells

Each of the four vaccines was tested for immunogenicity in vivo by i.m. injection into BALB/c mice. Mice were boosted 4 wk postpriming by an i.m. injection of the same dose of the same DNA constructs. Ten days after the second injection, specific (tetramer+) CD8 T cells were detected in the spleens of mice immunized with the hsp-capturing constructs (Fig. 2, groups 3 and 5). Hsp-bound fusion Ags encoding either the nonamer AH1 epitope or the 198-residue gp70 fragment (containing the AH1 determinant) induced comparable Ld-restricted T cell responses: ~2–5% of all splenic CD8 T cells were specific for the AH1 epitope. The non-hsp-capturing vaccines primed only low levels of specific CD8+ T cells (Fig. 2, groups 2 and 4). No specific CD8 T cells were present in mice injected with an equal dose of "empty" pCI plasmid DNA (Fig. 2, group 1). A similar pattern of vaccine-primed AH1-specific CD8 T cell responses was obtained when IFN-{gamma}+CD8+ T cell frequencies were determined ex vivo by intracellular cytokine staining (Fig. 2A). Specifically inducible IFN-{gamma}+CD8+ T cell frequencies were 3- to 5-fold lower than the frequencies of tetramer+CD8+ T cells (Fig. 2A). Significant responses were only detected in mice injected with vaccines encoding hsp-capturing sequences. Thus, high expression of the hsp-bound constructs (Fig. 1C) correlates with their efficacy to prime CD8 T cell responses, suggesting that hsp73 acts as heterologous "help" for CD8 T cell priming.


Figure 2
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  		FIGURE 2. DNA vaccination primes Ld-restricted, gp70-specific CD8+ T cell responses. BALB/c mice were vaccinated i.m. with pCI (group 1), T60-gp70{Delta} (group 2), cT272-gp70{Delta} (group 3), cT60-AH1 (group 4), or cT272-AH1 (group 5). Mice were boosted after 4 wk by an i.m. injection of the same dose of the same DNA constructs. The specific CD8 T cell response was analyzed 10 days after the second immunization (A and B) using either AH1 (SPSYVYHQF)/Ld tetramer staining (A and B) or 4-h ex vivo stimulation with the specific AH1 (SPSYVYHQF) peptide (or the HBsAg-derived negative control IPQSLDSWWTSL peptide), followed by determination of IFN-{gamma}+CD8+ T cell frequencies (A). The mean numbers of specific (tet+ or IFN-{gamma}+) CD8 T cells/105 splenic CD8 T cells of five mice per group (±SD) (A and B) or the percentage of tet+CD8+ T cells from two representative mice of each group (B) are shown.

 
Expression of gp70 transcripts is detectable in most normal tissues but is particularly striking in some tumor cell lines

The envelope protein gp70 of endogenous retroviruses is present in the mouse genome. Its expression pattern is variable. Conventional RT-PCR detected gp70 transcripts in all normal tissues tested and in some (but not all) tumor cell lines tested (Fig. 3A). Quantitative RT-PCR revealed striking differences in the transcript levels. All normal tissues, including mitogen-stimulated, proliferating T blasts expressed low levels of gp70 (Fig. 3A). Low transcript levels were also detected in the thymus, although a previous report (11) indicated no expression of this retroviral product in this organ. In contrast, some tumor cell lines, especially the adenocarcinoma cell lines CT26 and TS/A, expressed 5- to 30-fold higher transcript levels than normal cells (Fig. 3A). Other transplantable tumor cell lines, e.g., the fibrosarcomas TC-1 or mKSA or the T lymphoma RBL5, expressed low levels of gp70 transcripts (Fig. 3A).


Figure 3
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  		FIGURE 3. A, Expression of gp70 in normal tissues and tumor cells. Total RNA was prepared from the indicated organs or splenic T blasts of BALB/c mice and from the indicated tumor cell lines. RNA was reverse transcribed, and equal amounts of the cDNA preparations were analyzed by RT-PCR (left) or quantitative SybrGreen real-time TaqMan analysis (qPCR, right). mRNA was analyzed with the same primers to exclude genomic DNA contamination. No signal was observed with RNA as template (data not shown). For the semiquantitative gp70 RT-PCR, equal cDNA input was controlled by beta-actin RT-PCR (data not shown). cDNA amounts in quantitative PCR were normalized by measuring the housekeeping gene cyclophilin in each sample. Quantitative PCR data are expressed as n-fold expression of gp70 relative to gp70 mRNA level in Hepa1–6 cells (relative expression). At least three independent samples of each tissue/cell line were analyzed (mean + SD). B, AH1-specific epitope presentation of tumor cells. Short-term, splenic CD8 T cell lines specific for the Ld/AH1 epitope of gp70 were established from immunized BALB/c mice. CD8 T cells were cocultured in vitro with H-2d tumor cell lines (CT26, mKSA, P815, and TS/A), and their specifically inducible IFN-{gamma} secretion was determined by ELISA. The levels of IFN-{gamma} are given in nanograms per milliliter. C, Ld expression by tumor cells. Ld expression by tumor cells was analyzed by FCM using biotin-conjugated anti-Ld Ab 28-14-8 (BD Biosciences) and PerCP-conjugated streptavidin (BD Biosciences). Mean fluorescence intensities (MFI) are shown.

 
Short-term, splenic CD8 T cell lines specific for the Ld/AH1 epitope were established from immunized BALB/c mice. When these CD8 T cells were restimulated in vitro with H-2d tumor cell lines (CT26, mKSA, P815, and TS/A), their specifically inducible IFN-{gamma} response correlated with the level of gp70 transcripts detected by quantitative RT-PCR (Fig. 3, A and B). However, as expected, surface expression of the restricting Ld molecule also played a major role in the presentation of the AH1 epitope. Although CT26 and TS/A cells expressed comparable levels of gp70 transcripts, the low surface display of Ld molecules by TS/A cells limited their presentation of the AH1 epitope to CD8 T cells (Fig. 3, B and C). Hence, expression of the Ag as well as its presenting molecule determines the ability of the epitope to specifically (re)activate CD8 T cells.

DNA vaccines encoding hsp-capturing gp70 fragments protect mice from tumors

The pCI/cT272-gp70{Delta} DNA vaccine efficiently primed AH1-specific CD8 T cells in BALB/c mice. We tested if this specific immunity protects mice from growth of a transplantable tumor. We primed and boosted BALB/c mice and s.c. transplanted groups of immune animals (2 wk after the boost immunization) with 104, 105, or 106 CT26 cells (that express high levels of gp70 transcripts). Control (nonimmune) groups of five mice were injected with an equal dose of "empty" pCI vector DNA.

Subcutaneous inocula of 104, 105, or 106 CT26 cells gave rise to tumors in all transplanted mice (Fig. 4A). Immunization with pCI/cT272-gp70{Delta} completely inhibited outgrowth of tumors from inocula of 104 or 105 CT26 cells. Transient growth of small s.c. nodules was seen in two of five immune mice transplanted with 105 CT26 cells, but these tumors were rejected rapidly. In the group of immune mice transplanted with 106 CT26 cells, all mice developed tumors. However, three of five immune/transplanted mice were able to reject this tumor, even after it had grown to readily palpable s.c. nodule.


Figure 4
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  		FIGURE 4. A, Growth of CT26 tumors in syngeneic, gp70-immune, or nonimmune hosts. BALB/c mice (five per group) were vaccinated and boosted i.m. with 100 µg of pCI or cT272-gp70{Delta} DNA. Mice were transplanted s.c. with 104, 105, or 106 CT26 into the right flank. Tumor growth was followed daily by serial measurements of tumor size at two perpendicular diameters. Mean values of five mice per group + SEM are shown. B, Growth of CT26 tumors in vaccinated, T cell-depleted mice. BALB/c mice (five per group) were vaccinated and boosted i.m. with cT272-gp70{Delta} DNA. CD4 or CD8 T cells were suppressed in mice by three injections of the anti-CD4 mAb YTS 191 or the anti-CD8 mAb YTS 169 starting 10 days after the last injection. Three and 1 days before, and 5 days after the tumor graft (3 x 104 CT26), mice were i.p. injected with 200 µl of PBS containing 100 µg of the respective Ab. A control group remained untreated. Tumor growth was followed as described. C, Gp70-specific rejection of tumors. BALB/c mice (five per group) were vaccinated and boosted i.m. with cT272-gp70{Delta} or cT272-S1 DNA. Mice were transplanted s.c. with 3 x 104 CT26 or 106 mKSA cells into the right flank. Tumor growth was followed as described.

 
Specific CD8 T cells primed by DNA vaccines encoding hsp-capturing gp70 fragments mediate protection from tumors

The protective effect of the vaccination was mediated by CD8 T cells. When CD4 or CD8 T cells were eliminated from immune mice before and following tumor cell challenge, vaccinated mice depleted of CD4 T cells (five of five) but not vaccinated mice depleted of CD8 T cells (one of five) suppressed CT26 tumor growth (Fig. 4B). The protective effect of CD8 T cells was specific because only the vaccination with the hsp-capturing gp70 Ag but not vaccination with an hsp-capturing hepatitis B surface Ag fragment (pCI/cT272-S1) mediated protection (Fig. 4C). Hence, vaccine-induced specific CD8 T cells control tumor growth in this system.

Vaccination with the pCI/cT272-gp70{Delta} conferred protection against outgrowth of CT26 tumor cells (that express high levels of gp70 transcripts) but also of mKSA tumor cells (that express low levels of gp70 transcripts) (Fig. 4C). Thus, the vaccine has the potential to control growth of different tumors.

Specific CD8 T cell immunity primed by DNA vaccines delivering either hsp-capturing TAA fragments or the hsp-capturing AH1 epitope was tumor protective. Mice immunized with the pCI/cT272-AH1 DNA vaccine (that encodes the minimal AH1 epitope with an hsp-capturing N-terminal DnaJ-like domain) were protected from a s.c. challenge of 104 CT26 cells (Fig. 5). Hence, specific CD8 T cell immunity primed by DNA vaccines encoding an hsp-capturing construct with either a gp70 fragment or the AH1 epitope is tumor protective. The non-hsp-capturing construct pCI/T60-gp70{Delta} has poor immunogenicity (Fig. 2) and does not prime tumor protection (Fig. 5). Specific CD8 T cell immunity to the AH1 epitope of the TAA gp70 that is primed by hsp-capturing DNA vaccines can thus mediate protection against different tumors.


Figure 5
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  		FIGURE 5. Epitope-specific CD8 T cell immunity primed by hsp-capturing chimeric proteins elicit specific tumor protection. BALB/c mice (five mice per group) were primed and boosted i.m. with 100 µg of DNA of the constructs: pCI plasmid DNA (not immunized); cT272-gp70{Delta}, cT272-AH1, or T60-gp70{Delta}. Mice were transplanted s.c. with 104 CT26 into the right flank. Tumor growth was followed daily by serial measurements of tumor size at two perpendicular diameters. Mean values of five mice per group ± SEM are shown.

 
Longevity of the CD8 T cell-mediated protective immunity

A group of five immune mice that had rejected a s.c. inoculum of 105 CT26 cells was followed for 5 mo. No tumor appeared in this group. These mice were then challenged by a s.c. injection of 107 CT26 cells (>103-fold the minimal tumorigenic dose). Of the five challenged mice, one did not develop a tumor, three developed tumors that were subsequently rejected, and one developed a progressively growing tumor (Fig. 6A). Mice that showed transient tumor growth followed by tumor rejection were found to have substantial numbers of gp70-specific CD8 T cells in their spleens (Fig. 6B). The rejection of the first tumor graft by immune mice may have 1) primed additional tumor-specific immunity (not related to gp70); and/or 2) boosted the gp70-specific immunity. Hence, DNA vaccination with the hsp-capturing, chimeric gp70 construct pCI/cT272-gp70{Delta} induces CD8 T cell immunity that when boosted by a tumor graft can generate long-lasting protection from tumors.


Figure 6
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  		FIGURE 6. Long-term protection from CT26 tumors. A group of five immune mice that had rejected a s.c. inoculum of 105 CT26 cells were followed for 5 mo. Mice were then challenged by s.c. injection of 107 CT26 cells. Tumor development was followed by serial measurements of tumor size at two perpendicular diameters (A). The three mice that showed transient tumor growth followed by tumor rejection had substantial numbers of AH1-specific tet+CD8+ T cells in the spleen (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Intramuscular DNA vaccination with constructs encoding hsp-capturing, chimeric gp70 efficiently primed (Ld/AH1) specific CD8 T cell immunity. This class I-restricted T cell response was readily detectable by tetramer staining in mice despite widespread expression of gp70 transcripts in most normal tissues. The levels of gp70 transcripts in most tumor cell lines tested was 10- to >30-fold higher than that in normal tissues, suggesting that the level of expression is critical for targeting the CD8 T cell response to tumors. The TAA-specific CD8 T cell immunity was tumor protective in the CT26 and mKSA models.

DNA vaccines expressing the complete gp70 (29) or its fragment (this article) without an "adjuvant" do not prime TAA-specific CD8 T cell immunity. Different strategies have therefore been used in the CT26 and TS/A adenocarcinoma, EL4 thymoma, or B16 melanoma models to increase the immunogenicity of gp70 for CD8 T cells and thereby enhance the tumor-protective efficacy of the response. Help requirements for CD8 T cell priming to this TAA have been studied extensively. Strategies to recruit help for priming protective CD8 T cell immunity included injection of either genetically modified tumor cells or optimizing cellular or genetic vaccines. Tumor cells expressing GM-CSF (11), IL-21 (30), or MHC class II (after transgene-driven CIITA transactivator expression) (31) primed gp70-specific CD8 T cells. Specific CD4 T cell help facilitates CD8 T cell priming to this TAA (29, 32, 33). Cellular vaccines (dendritic cells (DC)) that present the class I-restricted gp70 determinant are immunogenic for CD8 T cells in vivo if they produce GM-CSF (34), are preactivated in vitro by TLR ligands (35), or copresent immunogenic, class II-restricted determinants (e.g., from engineered CLIP peptides (36), OVA (33), tetanus toxoid (28), recombinant vaccinia (37), or adenovirus (38)). The priming efficiency of gp70-presenting DC can be enhanced by CTLA-4 blockade (39), CD40 ligation (10), or OX40 ligation (40). We used the hsp-facilitated expression system in a DNA vaccine to achieve the same goal by simpler, apparently more efficient, and more universally applicable means. Stress proteins seem to provide some type of help in this system, the nature of which is unresolved. Different factors may cooperate. Stress proteins can provide signals that bypass CD4 T cell help (4, 41), mature DC (42), stimulate the innate immune system through, for example, TLRs, CD14, or CD91 (43, 44, 45, 46), efficiently chaperone Ag processing and/or presentation (47), or stimulate cross-presentation (7, 48, 49). It is unknown which of these factors contribute in the system we describe to enhance the immunogenicity of the codelivered epitope.

Either an ~200 residue fragment of gp70 or only the Ld-binding AH1 epitope of gp70 were cloned as fusion proteins with either the hsp-binding (cT272 domain) or the nonbinding (T60) domain. We tested expression, hsp-association, and immunogenicity in vivo for CD8 T cell precursors. We used tetramer staining as well as 4-h ex vivo-specific IFN-{gamma} expression to enumerate specific and functional CD8 T cells. Confirming our previously published data, high expression levels and enhanced immunogenicity correlated well with hsp capture (6, 7, 23, 50, 51). It is likely that hsp association but not the expression level of the Ag are the critical factor for its enhanced immunogenicity because efficient CD8 T cell priming by DNA vaccines was observed with Ags that were expressed at low levels and/or showed a high turn over rate (52, 53, 54). However, no proteins produced from the non-hsp-binding pCI/cT60-gp70{Delta} and pCI/cT60-AH1 constructs were detectable in our expression assays, and the inefficiently induced AH1-specific CD8 T cell responses were not tumor protective. In this system, hsp association of the gp70 fragments is thus a critical prerequisite for its immunogenicity.

We detected rejection of CT26 tumors in immune BALB/c mice even after these tumors had grown to a size of ~5 mm in diameter. On the other hand, one of five immune mice challenged with 107 CT26 cells developed a progressively growing tumor. The number of tumor cells that are either injected into the mouse or develop in situ from an inoculum may have different susceptibility to specific T cell-mediated rejection. Additional (possibly local) factors apparently operate that make the rejection process (or the protective efficacy of the specific CD8 T cell immunity) unpredictable. This is apparent from the observation that mice subjected to the same immunization protocol and to the same tumor challenge differed in their ability to reject the tumor. This area remains a major challenge in the development of therapeutic vaccines.

Priming self-reactive CD8 T cell responses to TAA is expected to represent a break in specific tolerance. The data indicate that Ld-restricted T cell immunity to "self" gp70 is readily established if an "innate adjuvant" is codelivered, confirming previous reports (47). This suggests that self-tolerance to this retroviral product is not very tight and that an "innate" adjuvant (the role of which is to eliminate damaged, mutate, or truncated self-proteins) can support breaking of tolerance. The relationship between endogenous retroviruses and autoimmunity is unresolved in murine models and in human systemic autoimmune disease. Retroviral virions harboring nuclear Ags can trigger autoimmune responses to chromatin and gp70 (55). We detected no clinical evidence of autoimmune disease during a >8-mo observation period in which our mice were (repeatedly) vaccinated and challenged with large tumor inocula and developed high specific CD8 T cell immunity. Hence, gp70 is an attractive TAA, and its high expression in some tumors makes it an attractive choice as a target TAA.

The data demonstrate that simple, robust, and efficient vaccination protocols can be used to prime protective CD8 T cell immunity to TAA. They confirm that endogenous hsp molecules are efficient adjuvants but do not elucidate their mechanism of action. Instead of delivering hsp as an adjuvant with the Ag, the system confers efficient hsp capture to a chimeric Ag in the process of its in vivo translation. Therefore, it has extensive potential for the design of cancer vaccines based, for example, on genetic vaccination or infection with recombinant viruses.


   Acknowledgments
 
We greatly appreciate the expert technical assistance of Katrin Ölberger and Claudia Heilig. The generous support of the National Institute of Allergy and Infectious Diseases Tetramer Facility for providing the Ld/SPSYVYHQF monomers is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 grants from the Wilhelm-Sander-Stiftung (95.088.3) (to A.K., H.H., R.S., and J.R.); from the Deutsche Forschungsgemeinschaft (DFG Schi 505/2-2) (to R.S.); and from the Leukaemia Research Fund (0306) to (J.Ri). Back

2 Address correspondence and reprint requests to Dr. Reinhold Schirmbeck, Internal Medicine I, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany. E-mail address: reinhold.schirmbeck{at}uniklinik-ulm.de Back

3 Abbrevations used in this paper: hsp, heat shock protein; T-Ag, large SV40 tumor Ag; TAA, tumor-associated Ag; cT272, 229 T-Ag residues containing the 272 N-terminal residues minus the nuclear localization sequence (T110–152); T60, 60 N-terminal T-Ag residue; FCM, flow cytometry; DC, dendritic cell. Back

Received for publication November 30, 2005. Accepted for publication May 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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