Hiroya Kobayashi

Peptide epitope identification for tumor-reactive CD4 T cells

Because T lymphocytes have the capacity to recognize tumor cells, significant efforts are being devoted towards the development of T cell-based immunotherapy for cancer. Most of this work has centered in the induction of anti-tumor CD8 T cells, which exhibit cytolytic activity towards tumor cells expressing tumor-specific or tumor associated antigens. Unfortunately to this day, T cell-based immunotherapy for cancer remains suboptimal. One of the possible explanations is that these immunotherapies have ignored the role that CD4 T helper lymphocytes play in the generation and persistence of CD8 T cell responses. Thus, we believe that in order to obtain clinical benefits T cell-based immunotherapy must stimulate both CD8 and CD4 tumor-reactive T cell responses. During the past seven years our group has focused on the identification of CD4 T cell epitopes from tumor-associated and tumor-specific antigens that could be used to complement the already identified CD8 T cell epitopes to produce effective vaccination strategies against numerous tumor types. We will describe here the strategy we used that resulted in the identification and characterization of numerous CD4 T cell epitopes that are applicable to developing therapies against hematological malignancies and solid tumors.

Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

DAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-beta1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver.

Functional Analysis of Birch Pollen Allergen Bet v 1-Specific Regulatory T Cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1141–156 and Bet v 151–68). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1141–156 appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-β. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.

ANALYSIS OF ANCHOR RESIDUES IN A NATURALLY PROCESSED HLA-DR53 LIGAND

The peptide motif of the HLA-DR53 (DRB4*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Haradas syndrome, was determined by peptide binding assay using human L plastin p581–595 peptide and its substituted analogues. L plastin p581–595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1*0901/DRB4*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylate peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. binding peptides in combination with functional analyses, by alignment of sequenced endogeneous peptides, and by the use of an M13 display library (Rammensee et al. 1995; Hammer et al. 1993, 1992). No sequence information has been reported for naturally occurring HLA-DR53 (DRB4*0101)-associated peptides partly because their expression on the cell surface is relatively low for sequencing endogeneous self-peptides (Kinouchi et al. 1995). It has been shown that HLA-DR53 is positively associated with Vogt-Koyanagi-Haradas Syndrome in Japanese subjects (Moriuchi et al. 1979). The identification of a peptide motif for HLA-DR53 may help in understanding the mechanisms of this disease. We have previously reported that naturally processed peptides bound to HLA-DR9/DR53 molecules (Futaki et al. 1995). In this report, we determined the peptide which could bind to the HLA-DR53 molecule and we identified a putative HLA-DR53-specific binding motif by a peptide binding assay using L-cell transfectants expressing HLA-DR molecules.

Naturally processed HLA-DR9/DR53 (DRB1*0901/DRB4*0101)-bound peptides

It has been shown that HLA-DR9 (DRBI*0901) is positively associated with birch pollen allergy in Japanese subjects (Kumai et al. 1990) and myasthenia gravis of Japanese children (Matsuki et al. 1990; Nomura et al. 1993). The elucidation of the characteristics of naturally processed peptides bound to HLA-DR9 (DRBI*0901) molecules may help in understanding the pathogenesis of these diseases. Peptides were isolated and sequenced from HLA-DR9/ DR53 (DRBI*0901/DRB4*0101) molecules according to the method used by Van Bleek and Nathenson (1990) with minor modifications. Briefly, HLA-DR molecules were solubilized by Nonidet-P40 from 3 x 1010 cells of the EBVKY cell line (homozygous for DR9 (DRB 1 *0901) and DR53 (DRB4*0101; Murakami et al. 1986) and immunopurified by L243 antibody specific for HLA-DR (Deborah et al. 1979). The isolated HLA-DR molecules were treated with 10% acetic acid at 37 °C for 20 min and the released peptides were separated from HLA-DR molecules by filtration through Centricon 10 (Amicon, Denvers, MA). They were fractionated by reverse-phase high-pressure liquid chromatograpyh (RP-HPLC), using C2/C18 RPC column (Pharmacia, Uppsala, Sweden). The amino acid sequence of each peptide peak was determined by automated Edman microsequencing (477A; Applied Biosystems, Foster City, CA). The source proteins of peptides were identified by searching the PRF-SEQDB and the GenBank databases, using the JOIS-F sequence search system. Amino acid sequences of 19 peptides separated from DR9/DR53 molecules were determined. Eleven of them were consistent with parts of the known protein amino acid sequences. (Table 1). Those peptides from human L plastin, human apolipoprotein B-100, and unknown source protein differed in length at Nor C-termini. This finding is in accordance with reports by others and by our own describing peptides bound to HLA-class II molecules (Kinouchi et al. 1994). Each peptide may bind to class II molecules after

Six-Transmembrane Epithelial Antigen of the Prostate as an Immunotherapeutic Target for Renal Cell and Bladder Cancer

PURPOSE T-cell based immunotherapy for renal cell and bladder cancer is one of the most promising therapeutic approaches. STEAP is a novel cell surface protein that is over expressed in various cancers, including renal cell and bladder cancer. Recently we induced STEAP specific helper T lymphocytes that recognize the naturally processed STEAP peptide epitopes STEAP(102-116) and STEAP(192-206) arising from STEAP expressing tumor cells. Thus, STEAP may be a useful tumor associated antigen for designing T-cell based immunotherapy. We determined whether STEAP could induce anti-cellular immune responses to urological cancer. MATERIALS AND METHODS We selected 2 previously described STEAP derived epitope peptides, STEAP(102-116) and STEAP(192-206), and examined their ability to elicit helper T-lymphocyte responses by in vitro vaccination of CD4 T lymphocytes from healthy individuals and patients with cancer. RESULTS STEAP peptides induced helper T-lymphocyte responses using lymphocytes from healthy individuals that directly recognized STEAP expressing, DR positive renal cell and bladder cancer cells, and autologous dendritic cells pulsed with STEAP expressing tumor cell lysates in a major histocompatibility complex class II restricted manner. These peptides also stimulated T-cell responses in patients with renal cell or bladder cancer. Each STEAP peptides behaved as a promiscuous T-cell epitope, in that they stimulated T cells in the context of multiple major histocompatibility complex class II alleles. CONCLUSIONS Results show that STEAP helper T-lymphocyte epitopes could be used to optimize T-cell based immunotherapy against STEAP expressing renal cell and bladder cancer.

Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1)

Involucrin is one of the precursor proteins of keratinocyte cornified envelope that is formed beneath the inner surface of the cell membrane during terminal differentiation. Although involucrin is specifically expressed in the upper squamous cells of the epidermis, the precise regulatory mechanism of involucrin gene expression remains unknown. Transcriptional enhancer factor 1 (TEF-1), which binds to SV40 enhancer, is a nuclear protein expressed in various types of cells including keratinocytes. Immunohistochemical study has revealed that TEF-1 protein is highly expressed on the basal cell layer of the epidermis. To examine the possible regulatory mechanism of involucrin gene expression by TEF-1 protein, we analysed involucrin promoter activity of the INV-CAT vector, which was constructed by connecting the 5′ upstream region of the involucrin gene (−801 bp upstream from the transcription start site and downstream including the untranslated first exon) to the chloramphenicol acetyltransferase (CAT) reporter gene. The INV-CAT vector was transfected to SV40-transformed human keratinocytes (SVHK). Cotransfection of the TEF-1 expression vector significantly repressed INV-CAT promoter activity in a dose-dependent manner. The repression was also observed by transfection of the GAL4-TEF-1 vector, which was constructed by replacement of the TEF-1 DNA binding domain by the GAL4 activator domain. This suggests that TEF-1-induced repression is due to interference/squelching of a limiting transcriptional intermediary factor that is essential for involucrin expression. Analysis of the deleted INV-CAT vector suggested that the region from −599 to −495 of the involucrin gene, which contains two possible TEF-1 binding sites, was critical for the repression of the involucrin gene by TEF-1. By gel retardation analysis, the specific DNA binding of SVHK cell nuclear extracts and the recombinant TEF-1 protein was confirmed. TEF-1-dependent repression of involucrin gene expression might explain the suprabasal involucrin expression in the epidermis.

Promiscuous peptides on the nontypeable Haemophilus influenzae P6 outer membrane protein.

IntroductionP6 outer membrane protein is one of the candidates for a vaccine formulation against nontypeable Haemophilus influenzae (NTHi) infection. As otitis-prone children who have recurrent episodes of acute otitis media because of NTHi show an impaired immune response to P6, an innovative approach to vaccination is required to augment their immune response.Results and DiscussionWe previously identified human HLA-DR9-restricted T cell epitope peptide and highly immunogenic analog peptides on P6 for peptide vaccine candidates. To develop a vaccine formulation effective in the general population, we identified promiscuous T cell epitope peptides (p41–55, p71–85) on P6. In addition to stimulating with potentially promiscuous peptides (p30–44, p45–59) selected using a computer algorithm, we established peptide-specific T cell lines which respond to P6.ConclusionOur present results indicate that these peptides would be candidates for a widely applicable peptide vaccine formulation.

Focal adhesion kinase as an immunotherapeutic target

BackgroundFocal adhesion kinase (FAK) is a ubiquitously expressed non-receptor tyrosine kinase involved in cancer progression and metastasis that is found overexpressed in a large number of tumors such as breast, colon, prostate, melanoma, head and neck, lung and ovary. Thus, FAK could be an attractive tumor associated antigen (TAA) for developing immunotherapy against a broad type of malignancies. In this study, we determined whether predicted T cell epitopes from FAK would be able to induce anti-tumor immune cellular responses.MethodsTo validate FAK as a TAA recognized by CD4 helper T lymphocytes (HTL), we have combined the use of predictive peptide/MHC class II binding algorithms with in vitro vaccination of CD4 T lymphocytes from healthy individuals and melanoma patients.ResultsTwo synthetic peptides, FAK143–157 and FAK1,000–1,014, induced HTL responses that directly recognized FAK-expressing tumor cells and autologous dendritic cells pulsed with FAK-expressing tumor cell lysates in an HLA class II-restricted manner. Moreover, since the FAK peptides were recognized by melanoma patient’s CD4 T cells, this is indicative that T cell precursors reactive with FAK already exist in peripheral blood of these patients.ConclusionsOur results provide evidence that FAK functions as a TAA and describe peptide epitopes that may be used for designing T cell-based immunotherapy for FAK-expressing cancers, which could be used in combination with newly developed FAK inhibitors.

CD4^+ T-cells from peripheral blood of a patient with psoriasis recognize keratin 14 peptide but not 'homologous' streptococcal M-protein epitope

Psoriasis has been recognized as an immunologically mediated inflammatory skin disease that has been associated with group A, beta-haemolytic streptococcal infections. Notably cross-reactive autoimmune mechanism, which is mediated by T cells reacting to epitopes that are common to streptococcal M-protein and keratin, has been proposed in psoriasis. In order to investigate this possibility, peptides corresponding to M-protein and human epidermal keratin, which share some amino acid sequence between them, were synthesized and tested for their ability to stimulate T-cells of patients with psoriasis. Among five cases examined, we isolated a CD4(+) T-cell line that recognized the type I keratin (K14)(p168-181) when it was presented by the patients HLA-DR molecules from a single psoriatic patient, whose MHC allele was HLA-A2/A26, -B27/B16, -DR4/DR8, -DQ8. Further analysis disclosed that the critical peptide recognized by the T-cell line was 10-mer keratin(p171-180) (DLRNKILTAT). However, corresponding M6 protein with homology to K14 did not stimulate the T-cell response and no evidence for cross-reactivity was obtained. The K14-responsive T cell line produced IFN-gamma, but little IL-4 when stimulated with irradiated autologous PBMC pulsed with this peptide. Thus, the finding that human epidermal keratin peptide is immunogenic in a psoriasis patient may provide the evidence that T lymphocytes play an important role in the pathogenesis of psoriasis as an autoimmune disorder participated with Th1 like cells. However, the keratin-responsive T cell line was detected in only one of five cases of psoriasis examined, suggesting that such T cell line appears to be not so popular in psoriatic patients. No evidence for cross-reactivity to streptococcal M protein also suggests that the contribution of streptococci may simply be inducing proliferation of various repertoire of T cells (including K14-responsive T cells) possibly through a superantigen-dependent process.