Biomedical subjects

Y Kawakami

Publications and source records attributed to Y Kawakami.

At least 19 recordsLinked to original sources

PubMed1996

Loss of functional beta 2-microglobulin in metastatic melanomas from five patients receiving immunotherapy.

BACKGROUND: In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8+ T cells) can cause the regression of even large tumors. These antitumor CD8+ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the alpha chain and a noncovalently associated, soluble protein called beta2-microglobulin (beta 2m). Loss of beta 2m generally eliminates antigen recognition by antitumor CD8+ T cells. PURPOSE: We studied the loss of beta 2m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. METHODS: We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 40 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. RESULTS: After one melanoma cell line (1074mel) was found not to express functional beta 2m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of alpha-chain mRNA but variable levels of beta 2m mRNA. In addition, no immunoreactive beta 2m protein was detected by western blot analysis. When human beta 2m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be beta 2m positive. This result was consistent with the hypothesis that loss of beta 2m resulted from immunotherapy. CONCLUSIONS: These data suggest that the loss of beta 2m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.

Adult↗

PubMed1996

Clinical observation of spontaneous anginal attacks and multivessel spasm in variant angina pectoris with normal coronary arteries: evaluation by 24-hour 12-lead electrocardiography with computer analysis.

OBJECTIVES: Using a new, computerized 24-h 12-lead electrocardiographic (ECG) recording and analysis system (the EAGLE system), we sought to evaluate the clinical manifestations of ischemic episodes in patients with variant angina and normal coronary arteries. BACKGROUND: Although the prognosis of variant angina without significant organic stenosis is generally good, the incidence of multivessel spasm, a major prognostic factor, is surprisingly high in provocation tests. METHODS: A total of 122 patients with suspected variant or unstable angina underwent 24-h examination with the EAGLE system and two-channel Holter monitoring. Thirty patients in this group were diagnosed as having variant angina with normal or nearly normal coronary arteries. Twenty-two (73%) of these 30 patients developed anginal attacks with ST segment elevation during monitoring and were enrolled in the study. RESULTS: The 22 patients had a total of 138 episodes of transient ST segment elevation and 13 episodes of ST segment depression. No arrhythmias were observed during ST segment depression, but 26 episodes of ST segment elevation (19%) were associated with arrhythmias: 7 with premature ventricular contractions, 3 with ventricular bigeminy, 3 with complete atrioventricular (AV) block, 1 with complete AV block and couplets of premature ventricular contractions and 12 with marked sinus bradycardia (< 45 beats/min). Ten (45%) of the 22 patients had multivessel spasm. We observed three different patterns of multivessel spasm: 1) spasm at a different site on different occasions (migratory spasm); 2) spasm that sequentially affected two different sites; 3) simultaneous spasm at more than one site. The duration of ST segment elevation was much longer in patients with sequential and simultaneous spasm than in those with single-vessel spasm, and arrhythmias were more frequent during these two types of multivessel spasm. CONCLUSIONS: Although the prognosis of multivessel spasm is believed to be poor, this may not necessarily be so. Anginal attacks due to sequential and simultaneous multivessel spasm seem to be more dangerous than those involving single-vessel spasm or migratory multivessel spasm.

Aged↗

PubMed1995

Recognition of multiple epitopes in the human melanoma antigen gp100 by peripheral blood lymphocytes stimulated in vitro with synthetic peptides.

gp100 is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gp100 epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10(476) (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9(280) (Y-L-E-P-G-P-V-T-A), G9(154) (K-T-W-G-Q-Y-W-Q-V), or G9(209) (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gp100+ established melanoma cell lines. G10(476)- and G9(280)-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimulating factor and tumor necrosis factor alpha in response to T2 cells pulsed with relevant peptide, as well as to gp100+ melanoma cell lines. These results demonstrate that several peptides derived from the gp100 protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gp100 peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.

Amino Acid Sequence↗

PubMed1995

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells.

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.

Animals↗

PubMed1995

Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes.

A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA-A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti-peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24+ B cell lines pulsed with the peptide and HLA-A24+ tyrosinase+ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24+ melanoma patients.

Amino Acid Sequence↗

PubMed1995

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes.

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Alleles↗

PubMed1995

Isolation and characterization of a peptide isomerase from funnel web spider venom.

A novel peptide isomerase was purified from the venom of funnel web spider, Agelenopsis aperta. The complete primary structure of the isomerase has been established by sequence analyses of polypeptide chains, assignments of disulfide bridges, carbohydrate analyses, and mass spectrometry of sugar chains. The isomerase was found to be a 29-kDa polypeptide that consists of an 18-residue light chain and a 243-residue heavy chain connected by a single disulfide bridge. The heavy chain contains three intramolecular disulfide bridges and one N-linked oligosaccharide chain with a simple trimannosyl core structure. A sequence homology search showed a significant similarity of the enzyme with serine proteases, particularly around a putative catalytic triad of the isomerase. The isomerase specifically interconverts the configuration of Ser46 of a 48-amino-acid peptide, omega-agatoxin-TK, and the conversion rate from L-Ser to D-Ser was approximately two times faster than the reverse reaction.

Amino Acid Isomerases↗

PubMed1995

Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.

Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp 100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor-infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp 100 protein. Apart from the peptide corresponding to gp 100 amino acids 457-466, we identified the gp 100 peptide 154-162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp 100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp 100 epitope for TIL 1200. Together with the recently described gp 100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp 100, all presented in the context of HLA-A2.1. Therefore, gp 100 is an attractive target antigen in the development of immuno-therapeutic protocols against melanoma.

Amino Acid Sequence↗

PubMed1995

Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes.

The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor-infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24-restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and an Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290.

Amino Acid Sequence↗

PubMed1995

Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.

Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.

Amino Acid Sequence↗

PubMed1995

Inhibition of proliferation by L-myc antisense DNA for the translational initiation site in human small cell lung cancer.

We evaluated the antiproliferative effect of L-myc antisense DNA in NCI-H209, a human small cell lung cancer (SCLC) cell line overexpressing the L-myc gene. The synthetic DNA used in the present study was oligodeoxynucleoside phosphorothioate, which showed rapid incorporation into NCI-H209 cells and localized mainly in the cell nucleus and weakly in the cytoplasm. The exposure of this cell line to L-myc antisense DNA covering the translational initiation site of L-myc proteins inhibited the cell proliferation in a dose-dependent sequence-specific manner. Furthermore, the growth inhibition by this antisense DNA was correlated with the level of L-myc expression in three SCLC cell lines, NCI-H209, NCI-H510, and NCI-H82. In Western blot analysis, expression of the L-myc proteins was down-regulated in the antisense-treated cells compared with control-treated cells in NCI-H209. Together with unique characteristics of the L-myc gene, including: (a) a frequently amplified and overexpressed state in SCLC; and (b) very restricted and low-level expression in human adult tissues, the present data indicate that L-myc is a good candidate for the target gene for antisense DNA therapy based on molecular biological diagnosis in SCLC.

Base Sequence↗

PubMed1995

Purification of a major tyrosine kinase from RBL-2H3 cells phosphorylating Fc epsilon RI gamma-cytoplasmic domain and identification as the Btk tyrosine kinase.

Immunoglobulin E high affinity receptor-mediated signal transduction in mast cells results in a number of protein tyrosine kinases being activated as very early events in the process leading to degranulation. Some of these, such as the src kinases and the syk kinase, are known to be involved in this receptor-associated activation. In this paper we describe the search for other activation-associated tyrosine kinases by the ability to phosphorylate a cytoplasmic domain peptide of the Fc epsilon RI gamma-subunit. In utilizing a purification step previously used to isolate the 72 kDa syk kinase, we detected another kinase of molecular weight 79 kDa which we designated cd gamma kinase. The kinase was purified to near homogeneity by Heparin-agarose, Mono Q, and CM Sepharose chromatographies. The yield of enzyme was approx. 200 micrograms/10(9) cells. We characterized this kinase by its ability to phosphorylate both the cd gamma peptide (Km = 0.2 mM) and the cytoplasmic fragment of the Band III protein. The cd gamma kinase was distinguished from syk by inability to be precipitated by anti-syk antiserum and by partial peptide mapping. Cd gamma kinase was also distinguished from syk by cd gamma peptide and Band III substrate specificity. We identified the cd gamma kinase by Western blotting and by partial phosphopeptide mapping as Btk, the B-cell tyrosine kinase found to be defective in X-linked agammaglobulinemia.

Amino Acid Sequence↗

PubMed1995

Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

Amino Acid Sequence↗

PubMed1995

Molecular characterization of defective antigen processing in human prostate cancer.

BACKGROUND: Gene-modified tumor cell vaccines have shown efficacy in animal models of malignancy, including prostate cancer. Class I major histocompatibility complex (MHC) assembly and function in the cellular targets of such therapies is pivotal in determining the efficacy of specific cytokine-secreting tumor vaccines. PURPOSE: To help guide development of genetically engineered vaccine therapy for human prostate cancer, potential immune resistance pathways were evaluated by analysis of class I MHC assembly in prostate cancer cells. METHOD: Class I MHC assembly in metastasis-derived human prostate cancer cell lines (LNCaP, PPC-1, DU-145, PC-3, and TSU) and a normal prostate-derived cell line (TP-2) were characterized by phenotypic, molecular, and functional assays. Assembled class I MHC and antigen was measured by flow cytometry; mRNA levels of assembly components (class I MHC heavy chain, beta 2-microglobulin, and the antigen transporter gene product TAP-2) were determined; and antigen processing was measured with a chimeric reconstituted system using vaccinia vectors. Restoration of antigen processing was attempted by interferon gamma stimulation and by transfection with mouse class I MHC heavy-chain cDNA. RESULTS: Assembled class I MHC was underexpressed in two (LNCaP and PPC-1) of five prostate cancer cell lines compared with normal prostate-derived controls. PPC-1 cells underexpressed TAP-2 mRNA despite abundant class I MHC and beta 2-microglobulin message. Induction of TAP-2 by interferon gamma indicated that coding sequences for TAP-2 message were present in PPC-1. Resistance to cytotoxic T lymphocytes (CTL) lysis showed a functional defect in antigen transport by PPC-1 cells; reversal of the molecular defect with interferon gamma led to restoration of functional antigen processing. In contrast, LNCaP cells had competent antigen transport but deficient class I MHC heavy-chain function despite abundant class I MHC RNA; though refractory to stimulation by interferon gamma, this defect responded to transfection of class I MHC heavy-chain cDNA. CONCLUSIONS: Metastatic prostate cancer cells can escape T-cell recognition via divergent mechanisms of defective class I MHC assembly. The specific underexpression of TAP-2 gene product in PPC-1 cells contrasts with prior studies of TAP gene underexpression in lung cancer (which concurrently underexpressed class I MHC heavy chain) and provides evidence for a regulatory pathway controlling TAP-2 gene expression in human cancers that may not affect class I MHC heavy-chain expression. IMPLICATIONS: In clinical application of gene therapy for prostate cancer, these findings provide a rationale for focusing on strategies that can circumvent sole reliance on class I MHC-mediated tumor cell recognition by CTL.

Antigen Presentation↗

PubMed1995

Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen.

T cells can play a central role in the immune response to cancer, with tumor-specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) alpha and beta chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen-specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length alpha and beta T-cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor beta chain-specific mAb. Jurkat clone 5 TCR+ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T-cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR+ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy.

Amino Acid Sequence↗

PubMed1995

Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes.

The availability of antitumor cytotoxic T lymphocytes which can be generated from either peripheral blood lymphocytes after stimulation in vitro or tumor infiltrating lymphocytes (TIL) has made it possible to identify a number of melanoma antigens presented by major histocompatibility complex class I molecules. The present and previous studies indicated that TIL586 recognized an antigen expressed on most melanoma and normal melanocytes in the context of the HLA-A31 molecule. We report here the cloning of a cDNA that directs the expression of the shared melanoma antigen recognized by this TIL. The DNA sequence analysis revealed that the cDNA was almost identical to the gene encoding tyrosinase-related protein 1 or glycoprotein gp75 which was originally identified by serum antibodies in a patient with melanoma. The gene was found to be expressed only in melanoma, normal melanocyte cell lines, and retina, but not in other normal tissues tested. The gp75 antigen presented by HLA-A31 may therefore constitute a useful immune target for specific treatment of patients with melanoma, since both antibody- and T cell-mediated immune responses can be generated against this antigen.

Antigens, Neoplasm↗

PubMed1995

Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines. Fine specificity and MART-1 melanoma antigen recognition.

PBLs were isolated from 13 patients with metastatic melanoma. Mixed lymphocyte tumor cell cultures (ML TCs) were established (15 times) by using irradiated HLA-matched (one class I locus) allogeneic melanoma tumor cell lines (13 times) or autologous melanoma tumor cell lines (two times) in medium containing 120 IU/ml IL-2 and 100 IU/ml IL-4. PBLs grew to levels that could be assessed for functional reactivity 9 of 15 times. In seven of nine cases, CD3+CD8+ CTLs grew from MLTCs that were tumor specific; five were restricted by HLA-A2 and two were restricted by HLA-A24. Four of the tumor-specific CTL lines lysed autologous fresh tumor cells. Tumor-specific CTLs from two of three patients had cytolytic activity identical with tumor-infiltrating lymphocytes (TIL) derived from tumor biopsies removed earlier and grown in high concentrations (6000 IU/ml) of IL-2. Three of the HLA-A2-restricted tumor-specific CTLs were shown to recognize 293 cells transfected with HLA-A2.1 cDNA and the gene encoding the melanoma Ag, MART-1. In addition, these CTLs recognized the T2 cell line pulsed exogenously with the peptide MART-1(27-35), which is the nine-amino acid immunodominant epitope of the MART-1 Ag recognized on melanoma tumor cells by nearly all HLA-A2-restricted TIL. Thus, we have demonstrated the ability to generate tumor-specific CTLs from PBLs that are similar in their reactivity to TIL. This technique obviates the need for autologous tumor tissue and suggests that PBLs contain sufficient CTL precursors for use in generating antitumor CTLs for cellular immunotherapy trials.

Amino Acid Sequence↗

PubMed1995

Involvement of the Sonic hedgehog gene in chick feather formation.

To elucidate the molecular mechanisms of chick feather formation, we observed expression patterns of the Sonic hedgehog (Shh) gene, which is one of the vertebrate homologs of the Drosophila segment polarity gene, hedgehog, and encodes a signaling molecule functioning in limb pattern formation and motor neuron induction. We found that the Shh gene is also expressed in the apical region of the feather placodes and then in nine to eleven longitudinal stripes along feather filaments. The stripe was found to correspond to one of the outer marginal zones of each barb ridge, termed the zone of Shh expression. No significant expression signal was detected in the scale bud of developing legs. Thus, Shh is likely to function as an epithelial signaling molecule in epithelio-mesenchymal interaction during feather formation. Furthermore, since genes of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-4 (FGF-4) are coexpressed with Shh during feather formation as observed in limb morphogenesis, interactions among FGF-4, Shh and BMP-2 may be involved in formation of feather filaments and barbs in a similar fashion as elucidated in limb pattern formation.

Animals↗