Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients
2012; Elsevier BV; Volume: 11; Issue: 11 Linguagem: Inglês
10.1074/mcp.m112.019232
ISSN1535-9484
AutoresOlesya Chornoguz, Alexei Gapeev, Michael C. O’Neill, Suzanne Ostrand‐Rosenberg,
Tópico(s)T-cell and B-cell Immunology
ResumoThe major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4+ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii− cells may present peptides not presented by Ii+ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii+ cells, but will not be tolerant to novel peptides presented by Ii− cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii− tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii+ and Ii− MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii− cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii− and Ii+ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii− cells are not presented by Ii+ cells, and are derived from source proteins not used by Ii+ cells. Peptides from Ii− cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7+ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients. The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4+ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii− cells may present peptides not presented by Ii+ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii+ cells, but will not be tolerant to novel peptides presented by Ii− cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii− tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii+ and Ii− MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii− cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii− and Ii+ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii− cells are not presented by Ii+ cells, and are derived from source proteins not used by Ii+ cells. Peptides from Ii− cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7+ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients. Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC) 1The abbreviations used are:ANNartificial neural net analysisAPCsAntigen presenting cellsCIITAMHC class II transactivator/CIITAMCF10 cells transfected with CIITA/CIITA/CD80CIITA cells transfected with and expressing CD80/CIITA/Ii siRNA/CIITA cells down-regulated for Ii by RNA interferenceCLIP MHCclass II-associated invariant chain peptide/DR7MCF10 cells transfected with and expressing HLA-DR7/DR7/CD80/DR7 cells transfected with and expressing CD80HELHen egg lysozymeHLAHuman leukocyte antigenHLA-DR7MHC class II DR7 alleleIFNγinterferon gammaIiinvariant chainmAbMonoclonal antibodyMCF10malignant human breast cancer cellsMCF10Anonmalignant human breast cancer cellsMHCmajor histocompatibility complexPBMCperipheral blood mononuclear cellsPEphycoerythrinPE-Cy5-Cy7 PE coupled to cyanin −5, −7Th1Type 1 CD4+ T helper lymphocytes.1The abbreviations used are:ANNartificial neural net analysisAPCsAntigen presenting cellsCIITAMHC class II transactivator/CIITAMCF10 cells transfected with CIITA/CIITA/CD80CIITA cells transfected with and expressing CD80/CIITA/Ii siRNA/CIITA cells down-regulated for Ii by RNA interferenceCLIP MHCclass II-associated invariant chain peptide/DR7MCF10 cells transfected with and expressing HLA-DR7/DR7/CD80/DR7 cells transfected with and expressing CD80HELHen egg lysozymeHLAHuman leukocyte antigenHLA-DR7MHC class II DR7 alleleIFNγinterferon gammaIiinvariant chainmAbMonoclonal antibodyMCF10malignant human breast cancer cellsMCF10Anonmalignant human breast cancer cellsMHCmajor histocompatibility complexPBMCperipheral blood mononuclear cellsPEphycoerythrinPE-Cy5-Cy7 PE coupled to cyanin −5, −7Th1Type 1 CD4+ T helper lymphocytes. class I, molecules capable of activating CD8+ T-cells that directly kill tumor cells (1Fang L. Lonsdorf A.S. Hwang S.T. Immunotherapy for advanced melanoma.J. Invest. Dermatol. 2008; 128: 2596-2605Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 2Mellman I. Coukos G. Dranoff G. Cancer immunotherapy comes of age.Nature. 2011; 480: 480-489Crossref PubMed Scopus (2533) Google Scholar). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4+ T-cells despite compelling evidence that Type 1 CD4+ T helper cells facilitate the optimal activation of CD8+ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease. artificial neural net analysis Antigen presenting cells MHC class II transactivator MCF10 cells transfected with CIITA CIITA cells transfected with and expressing CD80 /CIITA cells down-regulated for Ii by RNA interference class II-associated invariant chain peptide MCF10 cells transfected with and expressing HLA-DR7 /DR7 cells transfected with and expressing CD80 Hen egg lysozyme Human leukocyte antigen MHC class II DR7 allele interferon gamma invariant chain Monoclonal antibody malignant human breast cancer cells nonmalignant human breast cancer cells major histocompatibility complex peripheral blood mononuclear cells phycoerythrin -Cy7 PE coupled to cyanin −5, −7 Type 1 CD4+ T helper lymphocytes. artificial neural net analysis Antigen presenting cells MHC class II transactivator MCF10 cells transfected with CIITA CIITA cells transfected with and expressing CD80 /CIITA cells down-regulated for Ii by RNA interference class II-associated invariant chain peptide MCF10 cells transfected with and expressing HLA-DR7 /DR7 cells transfected with and expressing CD80 Hen egg lysozyme Human leukocyte antigen MHC class II DR7 allele interferon gamma invariant chain Monoclonal antibody malignant human breast cancer cells nonmalignant human breast cancer cells major histocompatibility complex peripheral blood mononuclear cells phycoerythrin -Cy7 PE coupled to cyanin −5, −7 Type 1 CD4+ T helper lymphocytes. Activation of CD4+ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4+ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3Neefjes J. Jongsma M..L. Paul P. Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation.Nat. Rev. Immunol. 2011; 11: 823-836Crossref PubMed Scopus (1112) Google Scholar, 4Germain R.N. Uncovering the role of invariant chain in controlling MHC class II antigen capture.J. Immunol. 2011; 187: 1073-1075Crossref PubMed Scopus (11) Google Scholar). As a result, tumor-reactive CD4+ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated "MHC II cancer vaccines" that consist of Ii− tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II+Ii−CD80+ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4+ T cells produce IFNγ and provide help to CD8+ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4+ Th1 cells that facilitate antitumor immunity. In vitro (5Armstrong T.D. Clements V.K. Martin B.K. Ting J.P. Ostrand-Rosenberg S. Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6886-6891Crossref PubMed Scopus (140) Google Scholar) and in vivo (5Armstrong T.D. Clements V.K. Martin B.K. Ting J.P. Ostrand-Rosenberg S. Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6886-6891Crossref PubMed Scopus (140) Google Scholar, 6Humphreys R.E. Hillman G.G. von Hofe E. Xu M. Forcing tumor cells to present their own tumor antigens to the immune system: a necessary design for an efficient tumor immunotherapy.Cell. Mol. Immunol. 2004; 1: 180-185PubMed Google Scholar, 7Baskar S. Glimcher L. Nabavi N. Jones R.T. Ostrand-Rosenberg S. Major histocompatibility complex class II+B7–1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice.J. Exp. Med. 1995; 181: 619-629Crossref PubMed Scopus (195) Google Scholar) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4+ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4+ T cells with a distinct repertoire of T cell receptors (8Bosch J.J. Thompson J.A. Srivastava M.K. Iheagwara U.K. Murray T.G. Lotem M. Ksander B.R. Ostrand-Rosenberg S. MHC class II-transduced tumor cells originating in the immune-privileged eye prime and boost CD4(+) T lymphocytes that cross-react with primary and metastatic uveal melanoma cells.Cancer Res. 2007; 67: 4499-4506Crossref PubMed Scopus (32) Google Scholar, 9Dissanayake S.K. Thompson J.A. Bosch J.J. Clements V.K. Chen P.W. Ksander B.R. Ostrand-Rosenberg S. Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy.Cancer Res. 2004; 64: 1867-1874Crossref PubMed Scopus (43) Google Scholar, 10Srivastava M.K. Bosch J.J. Thompson J.A. Ksander B.R. Edelman M.J. Ostrand-Rosenberg S. Lung cancer patients' CD4(+) T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells.Cancer Immunol. Immunother. 2008; 57: 1493-1504Crossref PubMed Scopus (93) Google Scholar, 11Thompson J.A. Dissanayake S.K. Ksander B.R. Knutson K.L. Disis M.L. Ostrand-Rosenberg S. Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain.Cancer Res. 2006; 66: 1147-1154Crossref PubMed Scopus (42) Google Scholar, 12Thompson J.A. Srivastava M.K. Bosch J.J. Clements V.K. Ksander B.R. Ostrand-Rosenberg S. The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T lymphocytes.Cancer Immunol. Immunother. 2008; 57: 389-398Crossref PubMed Scopus (29) Google Scholar). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13Chamuleau M.E. Souwer Y. Van Ham S.M. Zevenbergen A. Westers T.M. Berkhof J. Meijer C.J. van de Loosdrecht A.A. Ossenkoppele G.J. Class II-associated invariant chain peptide expression on myeloid leukemic blasts predicts poor clinical outcome.Cancer Res. 2004; 64: 5546-5550Crossref PubMed Scopus (54) Google Scholar, 14van Luijn M.M. Chamuleau M.E. Thompson J.A. Ostrand-Rosenberg S. Westers T.M. Souwer Y. Ossenkoppele G.J. van Ham S.M. van de Loosdrecht A.A. Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses.Haematologica. 2010; 95: 485-493Crossref PubMed Scopus (20) Google Scholar), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4+ T cells (14van Luijn M.M. Chamuleau M.E. Thompson J.A. Ostrand-Rosenberg S. Westers T.M. Souwer Y. Ossenkoppele G.J. van Ham S.M. van de Loosdrecht A.A. Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses.Haematologica. 2010; 95: 485-493Crossref PubMed Scopus (20) Google Scholar, 15van Luijn M.M. van den Ancker W. Chamuleau M.E. Zevenbergen A. Westers T.M. Ossenkoppele G.J. van Ham S.M. van de Loosdrecht A.A. Absence of class II-associated invariant chain peptide on leukemic blasts of patients promotes activation of autologous leukemia-reactive CD4+ T cells.Cancer Res. 2011; 71: 2507-2517Crossref PubMed Scopus (19) Google Scholar). We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II+Ii− and MHC II+Ii+ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7+ breast cancer cells are unique to Ii− cells and are derived from source proteins not used by Ii+ cells. Ii− peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II+Ii− tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients. Human breast cancer cell line MCF10CA1 (hereafter called MCF10), its nonmalignant counterpart MCF10A (16Pauley R.J. Soule H.D. Tait L. Miller F.R. Wolman S.R. Dawson P.J. Heppner G.H. The MCF10 family of spontaneously immortalized human breast epithelial cell lines: models of neoplastic progression.Eur. J. Cancer Prev. 1993; 2: 67-76Crossref PubMed Google Scholar), MCF10 transductants (MCF10/DR7/CD80, MCF10/DR7/CD80/Ii, MCF10/CIITA/CD80, and MCF10/CIITA/CD80/Ii siRNA32; hereafter called /DR7, /DR7/Ii, /CIITA, and/CIITA/Ii siRNA) were cultured and/or generated as described (11Thompson J.A. Dissanayake S.K. Ksander B.R. Knutson K.L. Disis M.L. Ostrand-Rosenberg S. Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain.Cancer Res. 2006; 66: 1147-1154Crossref PubMed Scopus (42) Google Scholar, 12Thompson J.A. Srivastava M.K. Bosch J.J. Clements V.K. Ksander B.R. Ostrand-Rosenberg S. The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T lymphocytes.Cancer Immunol. Immunother. 2008; 57: 389-398Crossref PubMed Scopus (29) Google Scholar). Cells were expanded to ∼1 × 109 cells/line using Hyperflask tissue culture flasks (Corning, Corning, NY). MCF10 cells are: HLA-DRβ1*0401, DRβ1*0701. Peripheral blood mononuclear cells (PBMC) from healthy human donors and from breast cancer patients were obtained from the University of Maryland Medical School. Healthy donor BC100206 is HLA-DRβ1*0401and *0701; healthy donors BC100306, BC061505, and BC051505 are DRβ1*0701. Breast cancer patients 3 and 10 are HLA-DRβ1*0701. Breast cancer patient 3 is stage III, ER+/PR+, HER-2/neu−; patient 10 is stage II, ER+/PR+, HER-2/neu−. Patients were bled into ACD (citrate) tubes. Within 24 h of collection PBMC were isolated on Ficoll gradients, immediately cryopreserved at a controlled freeze rate of 1 °C/min, and stored in the vapor phase of liquid nitrogen until used. PBMC were >90% viable upon thawing. Use of human materials was approved by the UMBC IRB. Chemicals were purchased form Sigma Aldrich unless otherwise noted. Monoclonal antibodies L243-FITC (HLA-DR-specific), CD80-PE, Ii, CD3-FITC, CD8-PE, CD45RO-allophycocyanin, CD25-PE-Cy7, and CD56-PE-Cy7 were purchased from BD Pharmingen. HLA-A,B,C-PE-Cy5, CD4-Pacific Blue and CD4-eFluor 460 were purchased from Biolegend (San Diego, CA). Cell surface and intracellular staining for flow cytometry was performed as described (9Dissanayake S.K. Thompson J.A. Bosch J.J. Clements V.K. Chen P.W. Ksander B.R. Ostrand-Rosenberg S. Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy.Cancer Res. 2004; 64: 1867-1874Crossref PubMed Scopus (43) Google Scholar, 12Thompson J.A. Srivastava M.K. Bosch J.J. Clements V.K. Ksander B.R. Ostrand-Rosenberg S. The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T lymphocytes.Cancer Immunol. Immunother. 2008; 57: 389-398Crossref PubMed Scopus (29) Google Scholar). Stained cells were analyzed using a Cyan ADP flow cytometer and Summit analysis software, v2.1 (Beckman/Coulter). Cells were incubated at 37 °C for 48 h with 200 units IFNγ/ml. Washed cells were stained with mAbs to HLA-DR to ascertain HLA-DR expression. Approximately 1 × 109 cultured cells per cell line were harvested and lysed on ice for 1 h in lysis buffer (20 mm Tris-HCl, pH 8.0; 150 mm NaCl,1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; (17Depontieu F.R. Qian J. Zarling A.L. McMiller T.L. Salay T.M. Norris A. English A.M. Shabanowitz J. Engelhard V.H. Hunt D.F. Topalian S.L. Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12073-12078Crossref PubMed Scopus (84) Google Scholar) containing a Complete Mini Protease Inhibitor Mixture tablet (Roche). Lysates were ultra centrifuged at 129,888 × g for 1 h at 4 °C in a SW40Ti swinging bucket rotor and the supernatants harvested and stored at −80 °C. MHC II peptides were obtained by HPLC (Biologic HR, BioRad, Hercules, CA) as follows: Thawed supernatants were pre-cleared on a 5 ml Protein G Sepharose column equilibrated with lysis buffer and then applied to a 2 ml in-house generated L243 mAb (pan HLA-DR) Sepharose column (9Dissanayake S.K. Thompson J.A. Bosch J.J. Clements V.K. Chen P.W. Ksander B.R. Ostrand-Rosenberg S. Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy.Cancer Res. 2004; 64: 1867-1874Crossref PubMed Scopus (43) Google Scholar) equilibrated with two column volumes of lysis buffer. The loaded column was sequentially washed with 20 column volumes of 20 mm Tris-HCl, 150 mm NaCl, pH 8.0; 20 mm Tris-HCl, 1 m NaCl, pH 8.0, followed by 20 column volumes of 20 mm Tris-HCl, pH 8.0. MHC II-peptide complexes were eluted with five column volumes of 0.2 N acetic acid, and eluates were lyophilized and stored at −20 °C. Lyophilized material was resuspended in 50 μl water and acidified to ∼pH3 with glacial acetic acid to dissociate peptides from MHC II molecules. The resulting material was analyzed on a LTQ XL (ThermoFisher) mass spectrometer interfaced with a UltiMate 3000 nanoLC (Dionex). Peptides were concentrated on FACET biocompatible strong cation exchange (SCX) trapping columns (Protea Biosciences, Morgantown, WV) and separated on Acclaim PepMap 100 columns (C18,5 μm, 100 Å, 75 μm i.d. × 15 cm, Dionex) at 300 nl/min using a 70 min. gradient program. Mobile phases were: 0.1% formic acid in water (A), 80% acetonitrile, 20% water with addition of 0.1% formic acid (B), and 2 m ammonium bicarbonate in water (C). Peptide samples were loaded onto the SCX trapping column and washed with 4% B, 96% A for 0.75 min, then eluted onto an analytical column over 10 min at 15% C, 4% B. Peptides were separated by a gradient of mobile phase B, which was 15, 45, 100, and 4% at min. 20, 40, 50, and 55, respectively. Eluted peptides were ionized on a Thermo Scientific (nanospray ionization) NSI source, equipped with a dynamic NSI probe. The electrospray capillary was at 200 °C temperature and under 1.7 kV voltage. For each cell line two affinity purifications were performed, and for each affinity purification two LC-MS/MS runs were conducted. Mass spectrometric analysis was performed using unattended data-dependent acquisition mode, in which the mass spectrometer automatically switched between acquiring a survey mass spectrum (full MS) and consecutive CID of up to four most abundant ions (MS/MS). To facilitate identification of a broad range of peptides, dynamic exclusion for MS/MS was used. Individual precursor ions were selected no more than twice over the duration of 20 s, and were then placed in the exclusion list for 120 s. The m/z tolerance window for dynamic exclusion was 1.5 Da. Spectra were searched against the International Protein Index (IPI) human database, (version 3.26, 67665 entries) on BioWorks 3.3.1 SP1 platform (ThermoFisher), using SEQUEST and the following search parameters: peptide mass tolerance: 2 Da; fragment mass tolerance: 1 Da, no enzyme specificity. Results of the SEQUEST searches (.out files) were converted into mzXML files and analyzed on Peptide Prophet using Trans-Proteomic Pipeline version 4.3 revision 1 (18Deutsch E.W. Mendoza L. Shteynberg D. Farrah T. Lam H. Tasman N. Sun Z. Nilsson E. Pratt B. Prazen B. Eng J.K. Martin D.B. Nesvizhskii A.I. Aebersold R. A guided tour of the Trans-Proteomic Pipeline.Proteomics. 2008; 10: 1150-1159Crossref Scopus (601) Google Scholar). Peptides that were identified in both affinity purifications and both LC-MS/MS runs were considered as reliable identifications. These peptides had a minimal Xcorr score of 1.5 for the charge state of 1, a minimal Xcorr score of 2.0 for the charge state of 2, a minimal Xcorr score of 2.5 for the charge state of 3, and a minimal Peptide Prophet probability of 0.07. Peptides with Peptide Prophet probability greater than 0.05 were subjected to Artificial Neural Network (ANN) analysis. We purposefully included all peptides identified with a more relaxed filtering criterion to facilitate MHC II peptide binding prediction accuracy. Subcellular localization of source proteins for peptides was determined using LOCATE subcellular localization database (http://locate.imb.uq.edu.au/) and WOLF Psort (http://wolfpsort.org/). Two hundred and sixty seven published HLA-DR7-restricted peptides of 9–25 amino acids in length (MHCBN database, version 4.0; http://www.imtech.res.in/raghava/mhcbn/index.html) were used to train an ANN. The network was a three layer back propagation network, trained to produce four output classes, representing highest to lowest binding affinities. The amino acid strings were initially coded by amino acid names. A "1" in a specific position of the 20 node input corresponded to the particular amino acid in the ordered list of 20 amino acids; the other positions being set to "0." The binding region was assumed to be nine amino acids in length. Therefore, larger peptides were broken into (n-9 + 1) 9-mers prior to coding. This produced 1318 9-mer sequences. A 9-mer was coded by 9 lines of 20 input nodes and 4 output nodes representing the affinity levels. Members of the smaller affinity classes were duplicated as necessary to provide roughly equal representation in the training set. Training was stopped when the root mean square error reached 0.30. Nine-mers whose highest score was in the expected affinity group were chosen for continued study. The network was thus used to index relevant 9-mers within the larger peptide sequence and produced 122, 106, 12, and 27 peptides in high (H), medium (M), low, and nonbinding groups, respectively. A new neural network was then trained using members from each binding affinity group. Each amino acid was represented by its Borstnick-Hofacker (B-H) distance (19Borstnik B. Hofacker G.L. Functional aspects of the neutral patterns in protein evolution. Academic Press, Guiderland1985Google Scholar) to each of the other 20 amino acids. These distances were divided into 16 bins, spanning the range of B-H distances from 0.1 to 1.6 units. Thus each amino acid was represented by 20 lines of 16 elements each. The order of the 20 lines was Leu, Ser, Arg, Ala, Val, Pro, Thr, Gly, Ile, Tyr, Asn, Lys, Asp, Glu, Cys, Phe, Trp, and Met. If Leu was coded, the first line would have all zeros, and a "1" somewhere representing the B-H distance between Leu and the particular amino acid. For example, val for line 5 had a "1" in the first bin on the right for a 0.1 B-H distance (rounded from 0.14). Coding a nine amino acid sequence therefore required 9 blocks of 20 lines of 16 elements each, or 2709 zeros and 171 ones. Coding the sequences in this way revealed patterns involving near-neighbor substitutions in forming classifications. The network had 2880 input neurons, 10 hidden neurons, and four output neurons. It was trained on 174 examples drawn from the four affinity classes and tested on 33 and 53 additional H and M class examples. A test example was judged correct if its highest score occurred in the expected class and the score was 0.5 or higher. The network correctly identified 29 of 32 high affinity peptides as such and generated a single false positive in 103 test peptides. This network was then used to test the peptides isolated from HLA-DR7 molecules of Ii+ and Ii− MCF10 cell lines. Peptides >9 amino acids were analyzed for all possible contiguous 9-mer sequences in the larger peptide sequence. The resultant 9-mer sequences were then coded in the same manner as those used for training the network. Five peptides unique to Ii− cells and two peptides present in both Ii− and Ii+ cells scored >0.92 (H binding class). Seven MS-identified peptides and Her2/neu peptide 776 (20Sotiriadou R. Perez S.A. Gritzapis A.D. Sotiropoulou P.A. Echner H. Heinzel S. Mamalaki A. Pawelec G. Voelter W. Baxevanis C.N. Papamichail M. Peptide HER2(776–788) represents a naturally processed broad MHC class II-restricted T cell epitope.Br. J. Cancer. 2001; 85: 1527-1534Crossref PubMed Scopus (57) Google Scholar, 21Salazar L.G. Fikes J. Southwood S. Ishioka G. Knutson K.L. Gooley T.A. Schiffman K. Disis M.L. Immunization of cancer patients with HER-2/neu-derived peptides demonstrating high-affinity binding to multiple class II alleles.Clin. Cancer Res. 2003; 9: 5559-5565PubMed Google Scholar) were synthesized in the University of Maryland, Baltimore biopolymer facility. Peptides were assessed for their ability to activate T cells as previously described (9Dissanayake S.K. Thompson J.A. Bosch J.J. Clements V.K. Chen P.W. Ksander B.R. Ostrand-Rosenberg S. Activation of tumor-specific CD4(+) T lymphocytes by major histocompatibility complex class II tumor cell vaccines: a novel cell-based immunotherapy.Cancer Res. 2004; 64: 1867-1874Crossref PubMed Scopus (43) Google Scholar, 11Thompson J.A. Dissanayake S.K. Ksander B.R. Knutson K.L. Disis M.L. Ostrand-Rosenberg S. Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain.Cancer Res. 2006; 66: 1147-1154Crossref PubMed Scopus (42) Google Scholar, 12Thompson J.A. Srivastava M.K. Bosch J.J. Clements V.K. Ksander B.R. Ostrand-Rosenberg S. The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T
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