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Isoaspartyl Post-translational Modification Triggers Anti-tumor T and B Lymphocyte Immunity

2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês

10.1074/jbc.m604847200

1083-351X

Hester A. Doyle, Jing Zhou, Martin Wolff, Bohdan P. Harvey, Robert M. Roman, Renelle J. Gee, Raymond A. Koski, Mark J. Mamula,

Cancer Immunotherapy and Biomarkers

A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens. A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens. Isoaspartyl (iso-Asp) 3The abbreviations used are: iso-Asp, isoaspartic acid; CFA, complete Freund's adjuvant; IFA, incomplete Freund's adjuvant; FCS, fetal calf serum; APC, antigen-presenting cells; CTL, cytotoxic T lymphocytes; CFSE, carboxy-fluorescein diacetate, succinimidyl ester; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; MHC, major histocompatibility complex; E:T, effector:target. 3The abbreviations used are: iso-Asp, isoaspartic acid; CFA, complete Freund's adjuvant; IFA, incomplete Freund's adjuvant; FCS, fetal calf serum; APC, antigen-presenting cells; CTL, cytotoxic T lymphocytes; CFSE, carboxy-fluorescein diacetate, succinimidyl ester; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; MHC, major histocompatibility complex; E:T, effector:target. post-translational protein modification occurs at physiological temperatures and pH values (1Clarke S. Stephenson R.C. Lowenson J.D. Aher T.J. Manning M. C Stability of Protein Pharmaceuticals. Plenum Publishing Corp, New York1992: 2-23Google Scholar), and a wide variety of intracellular and extracellular proteins has been identified with iso-Asp modifications. Iso-Asp formation is prevalent in stressed (heat shock) or aged cells (2Lowenson J. Clarke S. Blood Cells. 1988; 14: 103-118PubMed Google Scholar), and the presence of iso-Asp residues in proteins has been shown to decrease biological functions in some proteins (3Teshima G. Porter J. Yim K. Ling V. Guzzetta A. Biochemistry. 1991; 30: 3916-3922Crossref PubMed Scopus (47) Google Scholar). Iso-Asp residues can be repaired by the ubiquitous enzyme protein carboxyl methyltransferase (4Clarke S. Annu. Rev. Biochem. 1985; 54: 479-506Crossref PubMed Google Scholar). The importance of this enzyme to normal cellular biology is demonstrated in that mice with an insertional mutation in the protein carboxyl methyltransferase gene die at about 6 weeks of age (5Kim E. Lowenson J.D. MacLauren D.C. Clarke S. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6132-6137Crossref PubMed Scopus (248) Google Scholar). Another important, but under explored, consequence of isoaspartate formation is the altered immunogenicity of peptides/proteins. Our laboratory has shown that the spontaneous conversion of an aspartic acid to an isoaspartic acid (Fig. 1) induces both T and B cell immunity to model self-antigens (6Mamula M.J. Gee R.J. Elliot J.I. Sette A. Southwood S. Jones P. Blier P.R. J. Biol. Chem. 1999; 274: 22321-22327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The B lymphocyte and antibody-mediated immune response is directed at not only the iso-Asp-modified form of self-antigen but also the native form of the self-antigen (6Mamula M.J. Gee R.J. Elliot J.I. Sette A. Southwood S. Jones P. Blier P.R. J. Biol. Chem. 1999; 274: 22321-22327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Moreover, studies have shown that the post-translational modification of amino acids, such as phosphorylation, glycosylation, or citrullination, in self-antigens can induce an immune response to a self-antigen that was previously ignored by the immune system (7Utz P.J. Hottelet M. Schur P.H. Anderson P. J. Exp. Med. 1997; 185: 843-854Crossref PubMed Scopus (202) Google Scholar, 8Corthay A. Backlund J. Broddefalk J. Michaelsson E. Goldschmidt T.J. Kihlberg J. Holmadahl R. Eur. J. Immunol. 1998; 28: 2580-2590Crossref PubMed Scopus (147) Google Scholar, 9Masson-Bessiere C. Sebbag M. Girbal-Neuhauser E. Nogueira L. Vincent C. Senshu T. Serre G. J. Immunol. 2001; 166: 4177-4184Crossref PubMed Scopus (562) Google Scholar). In this study, we describe our efforts to apply this concept to developing immunity to tumor antigens. Many tumor-associated antigens are self-antigens that are often poorly recognized by the immune system. In an effort to make tumor-associated antigens more immunogenic, we introduced an isoaspartate into the peptide sequence of self-tumor proteins. We chose to look at the melanocyte differentiation antigen TRP-2 (tyrosinase-related protein-2) because melanoma continues to be an aggressive and difficult to treat cancer, with over 59,000 new cases diagnosed in 2005 in the United States alone. Based on our prior studies of mechanisms that induce pathologic autoimmunity, we examined anti-tumor immune responses elicited after immunization with modified melanoma peptides. In particular, we introduced an isoaspartic acid at position 183 of the TRP-2 peptide 181-188. We chose TRP-2 because it is a highly conserved protein found on both human and murine melanoma, and the native protein and peptide 181-188 are only weakly immunogenic in previous studies (10Overwijk W.W. Tsung A. Irvine K.R. Parkhurst M.R. Goletz T.J. Tsung K. Carroll M.W. Liu C. Moss B. Rosenberg S.A. Restifo N.P. J. Exp. Med. 1998; 188: 277-286Crossref PubMed Scopus (402) Google Scholar, 11van Elsas A. Sutmuller R.P. Hurwitz A.A. Ziskin J. Villasenor J. Medema J.P. Overwijk W.W. Restifo N.P. Melief C.J. Offringa R. Allison J.P. J. Exp. Med. 2001; 194: 481-489Crossref PubMed Scopus (290) Google Scholar). In this study, we demonstrate that immunization with iso-Asp post-translationally modified TRP-2-(181-188) induces a cytotoxic CD8+ T cell response capable of recognizing both the iso-Asp TRP-2 peptide as well as the native TRP-2 peptide. In addition, a CD4+ T cell response and anti-tumor antibodies are elicited by immunization with iso-Asp TRP-2-(181-188) peptide. As in models of autoimmunity, immunization with the modified self-tumor peptide provides a basis for immunotherapy of cancer. Peptides—Aspartyl and isoaspartyl forms of TRP-2-(181-188) peptide (VYDFFVWL) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) biochemistry in the Yale University/W. M. Keck Biotechnology Resource Laboratory. An isoaspartyl residue was introduced at position 183 (designated in boldface above). All peptides were >98% pure as indicated by analytical reverse phase high pressure liquid chromatography and were analyzed by mass spectroscopy, amino acid analysis, and amino acid sequencing. Chicken OVA-(257-264) peptide (SIINFEKL) was a gift from Dr. Paula Kavathas (Yale University, New Haven, CT). Lyophilized peptides were resuspended at 50 mg/ml in Me2SO and stored at -80 °C until used. Animals and Immunization—Female C57BL/6 (H-2b) mice, 6-8 weeks old, were obtained from the NCI (National Institutes of Health, Frederick, MD). All mice were housed at the Yale University School of Medicine Animal Facility. All protocols were consistent with accepted guidelines of the National Institutes of Health for the care and use of laboratory animals as well as approved by the Yale University Institutional Animal Care and Use Committee. Mice were immunized subcutaneously at the base of the tail and footpads with 50 μg of peptide emulsified 1:1 in CFA. Fourteen days later, mice were boosted with 50 μg of the immunizing peptide emulsified 1:1 in IFA. Seven days later, draining lymph nodes were excised and single cell suspensions prepared for experimental use. Cell Lines—The C57BL/6 murine melanoma line B16F10 was kindly provided by Dr. John Pawlek (Yale University, New Haven, CT) or purchased from ATCC (Manassas, VA). B16F10 cells express the melanoma protein TRP-2. EL-4, a lymphoma cell line of C57BL/6 origin, was a gift of Dr. Charles Janeway (Yale University, New Haven, CT). B16F10 cells were grown in Dulbecco's modified Eagle's medium adjusted with 1.5 g/liter sodium bicarbonate, 10% FCS, and antibiotics. EL-4 cells were maintained in Clicks medium supplemented with 5% FCS, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 5 × 105 m 2-mercaptoethanol. All cells lines were maintained at 37 °C in a humidified 5% CO2 environment. T Lymphocyte Proliferation Assay—Mice were sacrificed 7 days after peptide immunization. Draining lymph node T cells were isolated by negative selection using magnetically labeled antibodies against B220, CD11b, and MHC class II (Miltenyi Biotec, Auburn, CA). Antigen-presenting cells (APC) were syngeneic naive splenocytes that were depleted of T cells by magnetic bead separation. T cells (1 × 105 cells/well) were resuspended in complete Clicks medium with 5% FCS and co-cultured with irradiated (3000 rads) APC (5 × 105 cells/well) with various titrations of peptides in 96-well plates. After 3 days of incubation at 37 °C, 5% CO2, cells were pulsed with 1 μCi of [3Hrsqb]thymidine/well (ICN Chemicals, Irvine, CA) at the last 18 h of incubation and harvested onto filters with a semiautomatic cell harvester (PerkinElmer Life Sciences). Radioactivity was counted with a Betaplate liquid scintillation counter (PerkinElmer Life Sciences)). In Vitro CTL Assays—Effector cells were prepared from draining lymph nodes from peptide-immunized mice. Single cell suspensions of the draining lymph nodes were resuspended at 4 × 106 cells/ml and incubated with the immunizing peptide (10 μg/ml) in Clicks medium with 5% FCS. Following expansion for 5 days, dead cells were removed by Ficoll gradient separation, and the remaining live cells were used as effector cells (12Overwijk W.W. Restifo N.P. Coligan J.E. Kruisbeck A.M. Margulies D.H. Shevach E.M. Strober W Current Protocols in Immunology. John Wiley & Sons, Inc, New York2003: 20.1.1-20.1.29Google Scholar). EL-4 cells (targets) were pulsed with 10-5 m peptides in serum-free medium for 2 h at 37 °C, 5% CO2. EL-4 cells were washed twice before use. Effector cells were mixed with EL-4 targets at varying effector:target (E:T) ratios in triplicate in 96-well U-bottomed plates and incubated at 37 °C, 5% CO2 for 4 h. Cytotoxicity was measured as a function of lactose dehydrogenase release from dead cells using the CytoTox 96® non-radioactive cytotoxicity assay per the manufacturer's instructions (Promega, Madison, WI). In Vivo CTL Assays—Splenocytes from syngeneic, nonimmunized mice were incubated in serum-free media alone or with 1 μg/ml of either Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188) for 45 min at 37 °C, 5% CO2. Splenocytes were washed, counted, and resuspended in PBS. The unpulsed splenocytes were labeled with 0.5 μm CSFE, whereas the peptide-pulsed splenocytes were labeled with 5 μm CSFE in PBS for 10 min at 37 °C. Splenocytes were again washed and counted, and an equal amount of unpulsed and either Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188) peptide pulsed splenocytes were mixed together before injecting 2 × 107 cells intravenously into mice previously immunized with either Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188). Sixteen hours later, the draining lymph nodes were collected and cells isolated and fixed in 2% paraformaldehyde in PBS prior to analysis by flow cytometry. The ratio of specific killing (%) = ratio:%CFSE lo(unpulsed)/%CFSE hi(peptide-pulsed) (13Rice J. Buchan S. Dewchand H. Simpson E. Stevenson F.K. J. Immunol. 2004; 173: 4492-4499Crossref PubMed Scopus (24) Google Scholar). MHC Tetramers and Flow Cytometry—Production of Kb/β2-microglobulin/peptide multimers followed protocols described previously (14Devine L. Rogozinski L. Naidenko O.V. Cheroutre H. Kavathas P.B. J. Immunol. 2002; 168: 3881-3886Crossref PubMed Google Scholar). Briefly, Kb molecules with the BirA recognition sequence at the carboxyl terminus and human β2-microglobulin molecules were produced in Escherichia coli. The synthesized proteins were purified from inclusion bodies, denatured, mixed with the appropriate peptide, and the mixture allowed to renature in suitable oxidoreductive conditions over 48 h. Following dialysis and concentration, the folded monomer complexes were biotinylated using BirA (Avidity, Denver, CO) and purified via fast protein liquid chromatography on a Superdex 200 column (GE Healthcare). Following concentration of the appropriate size fractions, the biotinylated Kb-peptide complexes were mixed with fast protein liquid chromatography-purified streptavidin-phycoerythrin (Molecular Probes, Portland, OR) at a 4:1 molar ratio. Multimers were tested for specific binding over a range of doses by flow cytometry and were typically used at a concentration of 10-20 μg/ml. Ex vivo lymph node cells or CTLs expanded in vitro (as described above) were resuspended in 50 μl of FACS buffer (PBS, 5% (v/v) FCS, and 0.1% (w/v) sodium azide) with Kb-peptide tetramers (typically, 10-20 μl/ml), anti-CD8+ mAb (CT-CD8+a, Caltag, Burlingame, CA), and anti-CD3 mAb (Pharmingen) on ice for 2 h. CD16/CD32 mAb (Pharmingen) was used to block nonspecific antibody binding. After incubation, cells were washed with FACS buffer and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). Antibody Detection—C57BL/6 mice were immunized with 50 μg of peptide in CFA and boosted 14 days later with the 50 μg of immunizing peptide in IFA. Sera were collected 28 days after peptide immunization. B16F10 cells were detached by 0.25% EDTA in PBS, washed, and incubated with a 1:200 dilution of sera at 4 °C for 30 min. Cells were washed and incubated with fluorescein isothiocyanate-labeled anti-mouse IgG (1:40 dilution) (Sigma) at 4 °C for 30 min followed by three washes in PBS. Samples were run on a FACSCalibur flow cytometer and analyzed using CellQuest software. Antibody binding was analyzed by flow cytometry. Sera from naive C57BL/6 mice served as the negative control. In addition, serum antibodies were examined by standard enzyme-linked immunosorbent assay technology. In brief, peptides were dissolved in carbonate coating buffer and plated at 10 μg/ml. Serum dilutions of immune animals or controls (1:100 dilutions) were incubated in peptide-coated microtiter plates for 2 h at room temperature. Plates were washed and then incubated with anti-mouse IgG-alkaline phosphatase and p-nitrophenyl phosphate substrate. Immunofluorescence Staining—Mice were immunized on day 0 with peptide in CFA, boosted on day 14 with peptide in IFA, and then at day 21 day were injected 1 × 104 B16F10 cells subcutaneously in the flank. Tumor-challenged mice were examined for palpable subcutaneous tumor growth, and tumors were removed 20-30 days after B16F10 injection. Tumors were excised, cut across the center, and fixed in 0.7% paraformaldehyde, 75 mml-lysine, and 10 mm NaIO4 in 0.1 m phosphate buffer, pH 7.4, for overnight. The tumors were then incubated in 30% sucrose in 0.1 m phosphate buffer, pH 7.4, until the tumors sank to the bottom of the tube. Tumors were then embedded in OCT compound, and 5-μm cryostat sections cut and stored at -80 °C. For CD8 staining, sections were brought to room temperature, rehydrated in PBS, and blocked with 5% goat serum. Slides were incubated with 1:50 dilution of rat anti-mouse CD8 mAb (Pharmingen) for 1 h at room temperature. Slides were rinsed in PBS and then incubated with biotinylated goat anti-mouse IgG (1 μg/ml; Pharmingen) for 30 min at room temperature. Slides were rinsed again, followed by 30 min of incubation with a 1:5,000 dilution of Alexa Fluor 594 (Molecular Probes, Eugene, OR), rinsed, and mounted using Prolong® Gold anti-fading agent (Molecular Probes, Eugene, OR). Negative controls, included staining sections with secondary antibody only, and positive controls were sections of C57BL/6 mouse spleen incubated with anti-CD8 mAb for hematoxylin and eosin staining; sections were rehydrated in water, followed by staining in Harris hematoxylin for 5 min (Fisher). Cells were washed in water and placed in differentiation solution (70% ethanol plus 0.25% HCl) for 1 min. The sections were then placed in Scott's Tap water/bluing solution (5.0 ml of NH4OH in 1 liter of distilled water), washed with 95% ethanol for 1 min, and placed in alcoholic eosin Y (Sigma) for 1 min. The sections were washed in 100% ethanol, cleared in three changes of SubX (Surigipath, Richmond, IL), air-dried, and mounted with Permount (Fisher). To assess the immunogenicity of Asp TRP-2-(181-188) and iso-Asp TRP-2-(181-188), we immunized C57BL/6 mice with each peptide emulsified in CFA. As seen in Fig. 2A, T cells from mice immunized with iso-Asp TRP-2-(181-188) not only proliferated in response to iso-Asp TRP-2-(181-188) but also proliferated in response to the unmodified Asp TRP-2-(181-188). The introduction of the iso-Asp residue into the TRP-2 peptide not only generated T cells that recognized this form of the peptide, but also T cells that were cross-reactive with the unmodified (Asp) form of TRP-2 peptide. In contrast, T cells from mice immunized with Asp TRP-2-(181-188) had little or no response to either form of the TRP-2 peptide (Fig. 2B). Because TRP-2 is a self-antigen, it is not surprising that there is only a minimal immune response to the endogenous aspartyl form of the TRP-2 peptide. As CD8+ T cells are generally regarded as the main effector cells generated in anti-tumor immune responses, we next wanted to determine the percentage of Asp TRP-2-(181-188)- or iso-Asp TRP-2-(181-188)-specific CD8+ T cells that were generated after immunization with each peptide. Significant populations of iso-Asp TRP-2-(181-188)-specific CD8+ T cells were generated upon immunization with iso-Asp TRP-2-(181-188) (1%) followed by a remarkable expansion with iso-Asp peptide in vitro (28.6%) (Fig. 3). A smaller percentage (13.6%) of iso-Asp TRP-2-(181-188)-specific CD8+ T cells also stained with the Asp TRP-2-(181-188). Nonspecific binding was minimal as demonstrated by tetramer staining with an irrelevant peptide (SIINFEKL, 0.037%). Immunization with Asp TRP-2-(181-188) did not generate a significant population of either Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188)-specific CD8+ T cells after expansion with peptide in vitro, as the percentages of tetramer-positive populations were lower than those staining with the SIINFEKL tetramer (Fig. 3). The presence of iso-Asp TRP-2-(181-188)-specific CD8+ T cells after immunization with iso-Asp TRP-2-(181-188) suggested these cells could function as CTL. Lymph nodes from either Asp TRP-2-(181-188)- or iso-Asp TRP-2-(181-188)-immunized mice were cultured for 5 days in the presence of the immunizing antigen before use in a CTL assay. As syngeneic target cells, EL-4 cells were pulsed with either Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188). As seen in Fig. 4A, iso-Asp TRP-2-(181-188) immunization induced an iso-Asp TRP-2-(181-188)-specific CTL response (42% lysis at 40:1 E:T ratio). These same lymphocytes also lysed EL-4 cells pulsed with Asp TRP-2-(181-188), albeit to a lesser extent (20% lysis at 40:1 E:T ratio). Immunization with Asp TRP-2-(181-188) did not generate any appreciable CTL response against EL-4 cells pulsed with either Asp TRP-2-(181-188) or the iso-Asp form of Asp TRP-2-(181-188) (Fig. 4B). We next examined the ability of CTL responses against iso-Asp TRP-2-(181-188) and Asp TRP-2-(181-188) to kill target cells in vivo. Syngeneic splenocytes were pulsed in vitro with either the Asp TRP-2 or iso-Asp TRP-2 peptide and then injected into mice immunized with Asp TRP-2 or iso-Asp TRP-2. Again, mice immunized with iso-Asp TRP-2-(181-188) lysed the Asp- and iso-Asp-pulsed splenocytes (57 and 46%, respectively) to a much greater extent than mice immunized with Asp TRP-2 (8 and 17% respectively; Table 1.)TABLE 1Iso-Asp TRP-2-(181-188) immunization induces in vivo killing of iso-Asp TRP-2-(181-188) and Asp TRP-2-(181-188) peptide-pulsed target cellsImmunizing peptideMouseTransferred splenocytesSpecific killingAverage specific killing%Asp TRP-21Unpulsed +42Asp TRP-2-(181-188) pulsed783124Unpulsed +115Iso-Asp TRP-2-(181-188) pulsed1817624Iso-Asp TRP-21Unpulsed +442Asp TRP-2-(181-188) pulsed60463344Unpulsed +505Iso-Asp TRP-2-(181-188) pulsed6057654 Open table in a new tab Antibodies have also been shown to play an important role in the immune response against some tumors. Serum was collected from Asp TRP-2-(181-188)- or iso-Asp TRP-2-(181-188)-immunized mice 28 days after immunization. B16F10 cells were incubated with sera, followed by incubation with a secondary antibody and subsequent analysis by FACS. Fig. 5 demonstrates that mice immunized with iso-Asp TRP-2-(181-188) developed a higher titer of antibodies against B16F10, as evidenced by increased antibody binding (18.9%) and mean fluorescence intensity (of 23.7) than sera from mice immunized with Asp TRP-2-(181-188) (14% cells stained positive; mean fluorescence intensity of 17). Background antibody staining was about 5% using serum from naive C57BL/6 mice (data not shown). Although TRP-2 is an intracellular protein, these data demonstrate that antibody binds to melanoma cells, a finding that has been reported previously by others in both mouse models of melanoma and human melanoma (15Jones E. Golgher D. Simon A.K. Dahm-Vicker M. Screaton G. Elliott T. Gallimore A. Novartis Found. Symp. 2004; 256: 147-169Google Scholar, 16Takechi Y. Hara I. Naftzger C. Xu Y. Houghton A.N. Clin. Cancer Res. 1996; 2: 1837-1842PubMed Google Scholar, 17Okamoto T. Irie R.F. Fujii S. Huang S.K. Nizze A.J. Morton D.L. Hoon D.S. J. Investig. Dermatol. 1998; 111: 1034-1039Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Peptide-based enzyme-linked immunosorbent assay studies also demonstrated the production of antibodies only from iso-Asp TRP-2-(181-188) immune animals with binding to both the iso-Asp and Asp form of TRP-2-(181-188) (Table 2). No significant binding to control SIINFEKL peptide was observed. The presence of antibody populations after immunization with post-translationally modified peptide illustrates the ability to break B cell tolerance that is established to the unmodified peptide.TABLE 2Anti-peptide antibody response from Asp TRP-2-(181-188)- or iso-Asp TRP-2-(181-188)-immunized mice obtained at day 28 (examined by conventional peptide enzyme-linked immunosorbent assay)ImmunizationDetecting peptide antigenTRP-2Iso-Asp TRP-2Control BSAaThe negative antigen control listed below were wells blocked with 1% bovine serum albumin (BSA).Asp TRP-2-(181-188)0.140.210.09Iso-Asp TRP-2-(181-188)0.851.180.16Normal mouse serum0.210.090.22a The negative antigen control listed below were wells blocked with 1% bovine serum albumin (BSA). Open table in a new tab Finally, we wanted to determine whether immunization with iso-Asp TRP-2-(181-188) induced the influx of CD8+ T cells into tumors in mice. Immunized mice were injected subcutaneously with 104 B16F10 cells and the tumors removed ∼20-30 days later. Histological examination of tumors from iso-Asp TRP-2-(181-188) immunized mice had spaces in the tissue (Fig. 6, lower panel). This type of morphology was absent in the tumors from nonimmune mice or TRP-2-(181-188)-immunized mice (Fig. 6, upper and middle panels). When examining CD8+ T cell infiltration in these tumors, little or no CD8+ T cell staining was observed in nonimmune mice (Fig. 7A) or in Asp TRP-2-(181-188)-immunized mice (Fig. 7B). However, tumors examined from mice immunized with iso-Asp TRP-2-(181-188) had a significant number of CD8+ T cells (Fig. 7, C and D).FIGURE 7Cellular infiltration into tumors from iso-Asp TRP-2-(181-188)-immunized mice. Nonimmune mice and mice vaccinated with Asp TRP-2-(181-188) or iso-Asp TRP-2-(181-188) were injected subcutaneously with 104 B16F10 cells. Tumors were excised, and frozen sections were stained for CD8+ T cells. A, tumor from a nonimmune mouse; B, tumor from Asp TRP-2-(181-188)-immunized mouse; C and D, tumors from two separate iso-Asp TRP-2-(181-188)-immunized mice. Magnification at ×40 with ×1 zoom.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has become apparent that the post-translational modification of self-antigens can break immune tolerance, frequently making otherwise immunologically ignored proteins immunogenic (6Mamula M.J. Gee R.J. Elliot J.I. Sette A. Southwood S. Jones P. Blier P.R. J. Biol. Chem. 1999; 274: 22321-22327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 18Doyle H.A. Mamula M.J. Trends Immunol. 2001; 22: 443-449Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The rationale of this study is to apply this concept to develop "autoimmunity" to tumor tissues. Tumor-associated antigens are often self-antigens that do not elicit immune responses, despite the presence of T cells that could react with these tumor antigens. The immune response to melanoma is weak or absent demonstrating that immune tolerance to these antigens remains intact (19Romero P. Valmori D. Pittet M.J. Zippelius A. Rimoldi D. Levy F. Dutoit V. Ayyoub M. Rubio-Godoy V. Michielin O. Guillaume P. Bastard P. Luescher I.F. Lejeune F. Lienard D. Rufer N. Dietrich P. Speiser D.E. Cerottini J. Immunol. Rev. 2002; 188: 81-96Crossref PubMed Scopus (133) Google Scholar, 20Lewis J.J. Janetzki S. Schaed S. Panageas K.S. Wang S. Williams L. Meyers M. Butterworth L. Livingston P.O. Chapman P.B. Houghton A.N. Int. J. Cancer. 2000; 87: 391-398Crossref PubMed Scopus (99) Google Scholar). In this study, we post-translationally modified the aspartic acid at position 183 in the melanoma tumor peptide antigen TRP-2-(181-188) to an isoaspartic acid to generate an immune response to TRP-2. Introduction of the iso-Asp residue into the TRP-2 peptide sequence generated an effective anti-iso-Asp TRP-2-(181-188) T cell response, as evidenced by both tetramer staining and CTL activity in vitro and in vivo. The CD8+ T cell immune response was also cross-reactive with the Asp form of TRP-2 peptide. The important implication of this finding is that the iso-Asp-modified TRP-2 peptide breaks immune tolerance that can then be driven or perpetuated by the cross-reactive unmodified protein. Immunization with iso-Asp TRP-2-(181-188) not only generated an effective T lymphocyte response but also induced antibodies capable of binding B16F10 melanoma cells. Our prior studies with modified autoantigenic targets of lupus erythematosus demonstrate that the immune response undergoes amplification or "epitope spreading" after tolerance is broken by iso-Asp-modified peptides (6Mamula M.J. Gee R.J. Elliot J.I. Sette A. Southwood S. Jones P. Blier P.R. J. Biol. Chem. 1999; 274: 22321-22327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Investigations are underway to determine whether epitope spreading in this model system occurs in a similar manner. Although epitope spreading is assumed to accelerate auto-immune pathology in diseases such as systemic lupus erythematosus and multiple sclerosis, this response would likely have a beneficial outcome in the immunologic clearance of tumors or even pathogens. Histologically, tumors from iso-Asp TRP-2 peptide immunized mice displayed a significant amount of CD8+ T cell infiltration that was not limited to the margins of the tumor. Histological examination of melanoma tumors from humans has often shown that T cell infiltration can occur at both the tumor margin and within the tumor itself (21Bernsen M.R. Diepstra J.H. van Mil P. Punt C.J. Figdor C.G. van Muijen G.N. Adema G.J. Ruiter D.J. J. Pathol. 2004; 202: 70-79Crossref PubMed Scopus (18) Google Scholar). Our data suggest that immunization with the iso-Asp TRP-2 peptide elicits an immune response that is effective in recruiting CD8+ T cells to the tumor site. The tumors from the iso-Asp TRP-2 peptide-immunized mice also displayed spaces within the tissue, a feature that was never observed in tumors from the other two immunization groups. Human melanoma tumors often display this type of appearance, thought to be the result of decreased angiogenesis and coagulative necrosis (22Anichini A. Vegetti C. Mortarini R. Cancer Immunol. Immunother. 2004; 53: 855-864Crossref PubMed Scopus (71) Google Scholar, 23Anichini A. Molla A. Mortarini R. Tragni G. Bersani I. Di Nicola M. Gianni A.M. Pilotti S. Dunbar R. Cerundolo V. Parmiani G. J. Exp. Med. 1999; 190: 651-667Crossref PubMed Scopus (164) Google Scholar). The mechanism behind the immunogenicity of the iso-Asp TRP-2-(181-188) peptide is most likely because of direct T cell recognition. Previous studies in our laboratory have demonstrated that the iso-Asp peptides bind with the same affinity to MHC class II as the Asp peptides (6Mamula M.J. Gee R.J. Elliot J.I. Sette A. Southwood S. Jones P. Blier P.R. J. Biol. Chem. 1999; 274: 22321-22327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In this study, iso-Asp and Asp forms of TRP-2-(181-188) bind MHC class I in a virtually identical manner (data not shown). Our work conceptually resembles other studies performed with tumor antigens, in that changing one amino acid within the sequence of a tumor antigen or using a xenogeneic peptide, generates an anti-tumor response (10Overwijk W.W. Tsung A. Irvine K.R. Parkhurst M.R. Goletz T.J. Tsung K. Carroll M.W. Liu C. Moss B. Rosenberg S.A. Restifo N.P. J. Exp. Med. 1998; 188: 277-286Crossref PubMed Scopus (402) Google Scholar, 24Browne W.B. Srinivasan R. Wolchok J.D. Hawkins W.G. Blachere H.E. Dyall R. Lewis J.J. Houghton A.N. J. Exp. 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The introduction of the isoaspartate residue within the TRP-2 peptide sequence makes this post-translationally modified peptide an ideal candidate for breaking immune tolerance to "self" tumor proteins and potentially for inclusion in other combined immunotherapeutic schemes. Taken together, the data demonstrate that the introduction of an iso-Asp post-translational peptide modification within the TRP-2-(181-188) peptide induces a strong anti-tumor immune response not found by immunization with the native peptide. Further studies with the iso-Asp TRP-2 peptide alone and in combination with immunomodulatory regimes should prove it to be an important component in immunotherapies against melanoma. We thank Promega, Inc. for providing Isoquant kits for the analysis isoaspartyl modifications in our studies.