Tyrp1 DNA constructs.
Immunization against multiple epitopes is associated with improved immunity in infectious disease models (26
). We decided to investigate active immunization against multiple MHC class I epitopes using the melanocyte-specific self antigen mouse Tyrp1. This autoantigen has only been connected to antibody-dependent, CD8-independent immunity, and to our knowledge no MHC class I–restricted epitopes have been previously identified in Tyrp1 (12
). The strategy was to induce a potentially broad CD8+
T cell response against Tyrp1. To do this, we introduced a series of mutations in mouse Tyrp1, creating multiple altered peptide ligands for increased MHC presentation. Antigen processing and presentation were further enhanced by creating deficient glycosylation sites. The panel of Tyrp1 DNA plasmid constructs included nonmutated syngeneic (wild-type) mouse Tyrp1 (Tyrp1); epitope-enriched Tyrp1 (Tyrp1ee), incorporating 10 point mutations to create potential altered peptide ligands (Table ); Tyrp1 mutated at 6 positions to generate deficient Asn-linked glycosylation sites (Tyrp1ng), but not optimized for MHC class I binding; and combined epitope-enriched, glycosylation-deficient Tyrp1 (Tyrp1ee/ng). Construction and characterization of these vectors is described in the following paragraphs.
Tyrp1 peptide optimization for MHC class I binding
The Tyrp1ee DNA constructs incorporated multiple amino acid mutations that were predicted to increase epitope binding to MHC class I molecules H2-Kb
. To design this construct, we identified 10 self peptides in Tyrp1 with predicted low-level binding to MHC class I molecules using existing matrices (28
). T cells recognizing low-affinity, tissue-specific self peptides are expected to escape thymic deletion. Mutated peptides with single amino acid changes at candidate MHC anchor sites that were predicted to create altered peptide ligands with enhanced MHC class I binding were compared with nonmutated wild-type peptides. Eight of 10 mutated peptides had enhanced MHC class I binding compared with their wild-type counterparts by MHC class I stabilization assays (Figure ), using the transporter associated with antigen processing 2–deficient (TAP2–deficient) RMA-S cell line (29
). The Tyrp1ee cDNA variant was constructed by introducing single nucleotide mutations by site-directed mutagenesis into full-length mouse Tyrp1 cDNA (22
) (Table , H2-Db
– and H2-Kb
Rational optimization of peptide anchor residues in mouse Tyrp1 enhances MHC class I binding.
Tyrp1 with deficient Asn-linked glycosylation.
Tyrp1 is a transmembrane protein that, in its mature state, contains complex Asn-linked sialylated sugars. Tyrp1 carbohydrates are processed from high mannose to intermediate and complex glycans during movement from the ER through the Golgi complex (32
). Tyrp1 contains 6 potential Asn-linked glycosylation sites in the lumenal domain (33
). Our prior studies have shown that these glycans differentially determine egress from the ER, movement through the Golgi to the endosomal pathway, stability in the endosomal pathway, and overall stability of Tyrp1 (34
Six mutations (Asn→Gln) were introduced into the potential glycosylation sites of Tyrp1 and Tyrp1ee to produce deficient Asn-linked sites Tyrp1ng and Tyrp1ee/ng (see Methods), the latter protein incorporating both sets of mutations. We predicted that these mutations would promote ER retention, enhanced susceptibility to proteolytic digestion, and decreased stability, thereby increasing MHC class I antigen processing, perhaps guided by an ER misfolded protein response (34
Biosynthesis of epitope-enriched and glycosylation-deficient Tyrp1 mutants.
To assess the stability of Tyrp1ee and Tyrp1ee/ng proteins compared with wild-type Tyrp1, Cos-7 cells were transiently transfected with DNA encoding each of the Tyrp1 constructs containing a Flag tag at the carboxyl terminus. New protein synthesis was blocked with muconomycin A at 24 hours. At this time point (designated 0), wild-type Tyrp1 and Tyrp1ee were detected as single bands of approximately 75 kDa (Figure A). Interestingly, Tyrp1ee/ng was consistently observed as a ladder of bands ranging from approximately 75 kDa to approximately 59–60 kDa, always with a weak top 75-kDa band that increased in intensity over the 6-hour chase (Figure A). These results were reproduced in 3 different experiments for wild-type Tyrp1 and Tyrp1ee and 4 experiments for Tyrp1ee/ng.
Biosynthesis and stability of wild-type Tyrp1, Tyrp1ee, and Tyrp1ee/ng.
Deficient maturation of carbohydrate moieties on Tyrp1ee and Tyrp1ee/ng.
Although the Tyrp1ee/ng ladder might represent intermediate degradation products, particularly because glycosylation sites had been mutated, we suspected that the ladder instead represented different unstable glycosylated forms of Tyrp1 based on a close resemblance to our prior results with partial digestion of Tyrp1 by N-glycanase (33
). Digestion with the enzymes endoglycosidase H (Endo H) and N-glycanase collapsed the ladder to a single band of approximately 59–60 kDa (Figure B). These results show that sugars on Tyrp1ee/ng were not processed beyond the ER (based on Endo H sensitivity), with the ladder representing different glycosylated polypeptides of Tyrp1ee/ng. Thus Asn→Gln conversion did not completely abrogate glycosylation, but rather created a series of ER-retained, differentially glycosylated polypeptides (with glycans at 0, 1, 2, 3, or 4 Asn sites). The highest-mass 75-kDa band, which increased over 6 hours, was consistent with further posttranslational processing. In addition, Tyrp1ee glycans were also sensitive to Endo H, showing that the mutations in Tyrp1ee led to ER retention — probably through misfolding — with no further processing in the Golgi, followed by proteasome degradation (shown below; Figure B).
Tyrp1ee/ng mutants are degraded in a proteasome-dependent pathway.
Protein stability and degradation of wild-type Tyrp1, as assessed over a 24-hour chase, were nearly identical to what we have previously reported in mouse melanocytic cells, consistent with little or no effect of the Flag tag or expression in Cos-7 cells on stability or degradation (33
) (Figure A). We investigated the effects of chloroquine (to inhibit degradation in acidic compartments) and the proteasome inhibitor MG132. The fate of mature wild-type Tyrp1 was minimally affected by either inhibitor (Figure A). A short-lived 17-kDa carboxyterminal degradation fragment of wild-type Tyrp1 was detected in the presence of inhibitors but was not detected for Tyrp1ee and Tyrp1ng.
The stability of Tyrp1ee was essentially the same as for wild-type Tyrp1, despite ER retention. The majority of Tyrp1ee underwent proteasome degradation, with a minor proportion reaching acidic compartments for delayed degradation (Figure A). Thus the mutations in Tyrp1ee altered its trafficking, leading to ER retention and proteasome degradation.
Total degradation of Tyrp1ee/ng was modestly delayed by proteasome inhibition over 12 hours (Figure A). When higher and lower Tyrp1ee/ng bands were examined, the higher-mass glycoforms (~75 and 69 kDa) were less stable than wild-type Tyrp1 and were degraded largely via acidic compartments, presumably the endosomal pathway (Figure A). The 3 lower-mass forms (~65–66, 61–63, and 59–60 kDa, representing glycoforms with 1 or 2 carbohydrate moieties and the naked polypeptide) were very unstable, retained in the ER, and not rescued by either proteasome inhibition or chloroquine (Figure A). In summary, Tyrp1ee/ng generates multiple unstable products that are degraded both in the endosomal pathway (high-mass forms) and through proteasomes (low-mass forms).
Increased immunogenicity of Tyrp1ee mutants.
Although candidate altered peptide ligands of Tyrp1ee had enhanced binding to MHC class I (Figure ), we needed to ascertain whether these epitopes could be naturally processed and presented. To increase immunogenicity further, Tyrp1 mutants were ligated to the translocation domain of Pseudomonas aeruginosa
’s exotoxin A, which enhances CD8+
T cell responses in other immunization models (40
). The exotoxin A domain was fused upstream of Tyrp1 (exo-Tyrp1), Tyrp1ee (exo-Tyrp1ee), and Tyrp1ee/ng (exo-Tyrp1ee/ng); alternatively, exotoxin A alone was noncovalently mixed with Tyrp1ee. Immunogenicity was initially addressed by assessing the ability of immunization with DNA constructs encoding full-length wild-type or mutated Tyrp1 constructs to induce CD8+
T cell responses against wild-type Tyrp1 peptides in C57BL/6 mice. Mice were immunized with 4 μg DNA 4 times by ballistic bombardment (14
). Purified CD8+
T cells were tested for IFN-γ secretion in ELISPOT assays after 18-hour stimulations to detect responses to wild-type Tyrp1 epitopes and to intact syngeneic melanoma cells.
Immunization with wild-type Tyrp1, exo-Tyrp1, and Tyrp1ng did not induce any detectable T cell response, but CD8+ T cell responses were seen in mice immunized with Tyrp1ee, Tyrp1ee/ng, exo-Tyrp1ee, and exo-Tyrp1ee/ng (Figure , A–C). Immunization with Tyrp1ee induced responses against 3 wild-type Tyrp1 peptides, 455, 481, and 522 (Figure , A and C). We believe these to be novel MHC class I–restricted Tyrp1 epitopes revealed by this DNA immunization strategy. Immunization with Tyrp1ee/ng induced a substantially higher CD8+ T cell response against peptides 455, 481, and 522, increasing responses by 70%, 90%, and 95%, respectively, compared with Tyrp1ee (Figure A).
Immunization with optimized Tyrp1 induces epitope-specific CD8+
T cell responses.
Exo-Tyrp1ee elicited higher CD8+ responses than did Tyrp1ee against peptide 455 (Figure , B and C). However, fusion of the exotoxin A domain into Tyrp1ee/ng substantially diminished its potency down to Tyrp1ee levels (Figure C). Notably, CD8+ T cells primed by immunization with Tyrp1ee, Tyrp1ee/ng, exo-Tyrp1ee, and exo-Tyrp1ee/ng were able to respond to intact B16 melanoma cells, with Tyrp1ee/ng and Tyrp1ee eliciting the strongest response and exo-Tyrp1ee/ng the lowest (Figure C). Immunization with DNA encoding the exotoxin A fragment mixed with Tyrp1ee DNA showed no enhancement of CD8+ T cell responses compared with Tyrp1ee immunization alone (data not shown), indicating that the effect of exotoxin A is not mediated simply through the expression of exotoxin A (e.g., exotoxin helper epitopes). These results demonstrate that Tyrp1ee, Tyrp1ee/ng, exo-Tyrp1ee, and exo-Tyrp1ee/ng generated strong functional CD8+ T cell responses that recognized naturally processed and presented Tyrp1 epitopes expressed by B16 melanoma. Through this approach, we identified what we believe to be novel CD8+ T cell epitopes in a tissue self antigen, demonstrating a proof of concept for discovery of subdominant and cryptic epitopes in self antigens that can be processed and presented.
Antitumor effects following immunization with optimized DNA.
To investigate the in vivo effects of these rationally designed Tyrp1 DNA constructs, we assessed their ability to protect mice against lethal challenge with B16 melanoma cells. Palpable intradermal tumors were detected between days 4 and 8 following B16 challenge in naive mice with a tumor challenge that was 50- to 100-fold greater than the minimum lethal dose. No delay was observed in mice immunized with Tyrp1, Tyrp1ng, or exo-Tyrp1 (Figure A; Tyrp1ng data not shown). Notably, tumor protection was observed in approximately 40–45% of mice immunized with Tyrp1ee (P < 0.05, log-rank analysis) and 75–90% of mice immunized with Tyrp1ee/ng (P < 0.001; Figure A), with the same results observed in 4 independent experiments. Immunization with exo-Tyrp1ee and Tyrp1ee/ng led to greater tumor protection compared with Tyrp1ee (~40–45% to ~75–90%; P < 0.001; Figure , A and B). Ligating exotoxin A to Tyrp1ee/ng substantially diminished antitumor effects to below 20% tumor protection (Figure A), and mixing Tyrp1ee with exotoxin A showed no enhancement of tumor immunity (Figure B). These results demonstrated the effectiveness of immunization with epitope-enriched self antigens and revealed increased potency when the self antigen was further destabilized by modifying glycosylation and conjugating to exotoxin A.
Immunization with optimized Tyrp1 rejects tumors and induces autoimmunity.
Autoimmunity manifested as patchy coat hypopigmentation was observed in 42 of the 45 mice immunized with Tyrp1ee, Tyrp1ee/ng, exo-Tyrp1ee, and exo-Tyrp1ee/ng, but in none of the 30 mice immunized with wild-type Tyrp1, in 3 experiments (data not shown). The most marked hypopigmentation was observed in mice immunized with Tyrp1ee/ng, with a representative mouse shown in Figure C. Mice with autoimmune hypopigmentation after surviving tumor challenge remained otherwise healthy for up to 12 months or more.
Immunity induced by optimized Tyrp1 vaccines is CD8 dependent.
Tumor immunity was completely abrogated in mice immunized with Tyrp1ee, exo-Tyrp1ee, and Tyrp1ee/ng following depletion of CD8+
T cells, but not with depletion of CD4+
cells or NK cells (Figure , A–C); to our knowledge, no difference in tumor-free survival in mice immunized with wild-type Tyrp1 with or without CD8+
depletion has been previously reported (14
). These results showed that the immune responses induced by the optimized vaccines were CD8 dependent, without a requirement for CD4+
or NK cells for tumor rejection.
Immunization with Tyrp1ee, exo-Tyrp1ee, and Tyrp1ee/ng protects mice from tumor challenge through a mechanism dependent on CD8+
Immunization with optimized Tyrp1 vaccines in a treatment model.
Immunization with optimized Tyrp1 constructs was examined in a treatment model by initiating immunization 4 days after challenge with B16 cells. Significant delays in tumor growth and prolonged survival were observed in mice treated with Tyrp1ee/ng (P < 0.05; Figure A) and exo-Tyrp1ee (P < 0.01; Figure B). No treatment effects were detected in mice immunized with Tyrp1ee, exo-Tyrp1, or Tyrp1.
Immunization with Tyrp1ee/ng and exo-Tyrp1ee significantly prolongs tumor-free survival in a treatment model.
Epitope optimization for HLA-A*0201.
To design a vaccine that might be clinically applied to people with melanoma, an epitope-enriched mouse Tyrp1 DNA construct (Tyrp1ee/A*0201) was created. This construct contained 6 mutations optimized for HLA-A*0201 binding within polypeptide regions that are highly conserved between mouse and human Tyrp1. HLA-A*0201/Kb
transgenic mice (43
) were immunized with plasmid DNA encoding wild-type Tyrp1 or Tyrp1ee/A*0201. Purified CD8+
T cells from immunized mice were assessed for responses to Tyrp1 epitopes by secretion of IFN-γ following stimulation with either TAP2-deficient T2 cells or HLA-negative K562 cells transfected with HLA-A*0201 (44
) and pulsed with wild-type Tyrp1 peptides. Strong CD8+
T cell responses against 2 wild-type Tyrp1 peptides, 188–196 and 213–221, were detected only in mice immunized with Tyrp1ee/A*0201 (Figure A). CD8+
T cells against wild-type Tyrp1 peptide 213–221 responded to HLA-A*0201–positive, but not –negative, human melanoma cells, supporting processing and presentation of the 213–221 epitope by human melanoma cells (Figure , B and C). CD8+
T cells against the Tyrp1 peptide 188–196 reacted with HLA-A*0201–positive melanoma cells but also responded weakly to HLA-A*0201–negative melanoma cells, making inferences about HLA-restricted processing and presentation for this epitope more difficult (data not shown). Of note, HLA-A*0201 transgenic mice express the mouse heavy chain α3 domain, while HLA-A*0201 molecules expressed by T2 and transfected K562 cells contain only the human heavy chain α3 domain, suggesting that newly generated Tyrp1-specific CD8+
T cells have high avidity because they respond independently of CD8 coengagement (45
Immunization with optimized Tyrp1 induces HLA-A*0201–restricted CD8+
T cells against Tyrp1.