Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo.

Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8-10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-beta-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-beta-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

Clonal T cell responses in tumor infiltrating lymphocytes from both regressive and progressive regions of primary human malignant melanoma.

The T cell receptor (TCR) BV variable (V) gene repertoire of tumor infiltrating lymphocytes (TIL) found in progressive and regressive regions of the same primary human melanomas were characterized by reverse transcription coupled polymerase chain reaction (RT-PCR). After surgery, the tumors were divided into different parts which were judged as regressive or progressive regions by visual inspection. Subsequently this diagnosis was confirmed by histology. From a total of four primary melanomas analyzed, 2 were drawn to be HLA-A2+. Only relatively few BV-gene families were expressed at significant levels in each of the samples. Comparison of the BV-expression in regressive versus progressive regions of the same tumor revealed major differences in all cases examined. Direct sequencing of RT-PCR products indicated that highly expressed BV-gene families were of clonal origin in both the regressive and progressive regions. Together, these data strongly suggest the occurrence of clonal T cell responses in both regressive and progressive areas of the same primary tumor. The differences in expression of certain BV-genes may correlate with the functional activity of certain populations of tumor-infiltrating T cells.

Human lymphoma-lymphoma hybrids and lymphoma-leukemia hybrids. II. Epstein-Barr virus induction patterns.

In addition to the spontaneous expression of markers of the early and late phases of the viral cycle [Epstein-Barr virus (EBV), early viral cycle antigens (EA), and viral capsid antigen (VCA)] by a Panel of hybrid cell lines derived by fusion of human hematopoietic cell lines, the induction of these markers by three chemical inducers [5-iodo-2'-deoxyuridine, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), and sodium butyrate] were analyzed. A variant of the prototype producer of lytic EBV, P3HR-1 (PICAT) was observed to show consistent low spontaneous and induced production of EA and VCA as compared to the P3HR-1 line from which it was derived. An "autohybrid" (PICATPO-1) between these two lines showed a low production of VCA. Hybrids between Raji and P3HR-1 sublines (RUD-PICAT-1 and RUDPUT-2) showed reduced expression of EBV antigens. Both hybrids were capable of VCA expression, in contrast to the Raji parent that expressed EA but not VCA. A new Raji-Daudi hybrid (DITRUD-1) expressed spontaneous and induced EA and VCA at levels intermediate between its two parents. A different type of hybrid was derived from a Daudi subline and the K562 leukemia cell line (DUTKO-1) and was found to be capable of spontaneous as well as induced EA and VCA expression. Both DUTKO-1 and DITRUD-1 were similar to Daudi in their induction profile in respect to a very low response to TPA.

Human lymphoma-lymphoma hybrids and lymphoma-leukemia hybrids. I. Isolation, characterization, cell surface markers, and B-cell markers.

Four new somatic cell hybrids were obtained by fusion of various Burkitt's lymphoma (BL)-derived cell lines that had different selective markers: Raji-P3HR-1, Daudi-Raji, and a P3HR-1-P3HR-1 "autohybrid" derived from two P3HR-1 sublines. In addition, a hybrid was obtained between the Daudi (BL) line and the human leukemia cell line K562. The hybrids were extensively characterized by means of chromosome, isozyme, and HLA surface markers. The phenotypic differences between the parent cell lines allowed some conclusions with respect to the expression of latent Epstein-Barr virus (EBV) genomes, C3 and EBV receptors, and of immunoglobulin and beta 2-microglobulin-HLA expression as well as the influence of the leukemia cell (K562) genome on B-cell properties in the Daudi-K562 hybrid. B-cell and differentiated markers of these hybrids were characterized. High-level expression dominated for the marker C3 and EBV receptors, which showed a good correlation coefficient of 0.84, as was true for Fc receptors and surface immunoglobulin. The Daudi-K562 hybrid showed loss of all B-cell markers but retention of the leukemia cell markers (e.g., hemoglobin synthesis).

Hybridization of a myeloid leukemia-derived human cell line (K562) with a human Burkitt's lymphoma line (P3HR-1).

The myeloid leukemia-derived Epstein-Barr virus (EBV)-negative human lymphoid cell line K562 was successfully hybridized with the EBV-carrying Burkitt's lymphoma line P3HR-1. Authenticity of the hybrid PUTKO-1 was established by chromosome and isoenzyme studies. A virtually complete hybrid PUTKO-1 carried the EBV genome derived from the lymphoma parent. It averaged 26 EBV DNA copies per cell and was 100% positive for Epstein-Barr virus-associated nuclear antigen (EBNA). In most respects, the hybrid resembled the K562 parent: It had a high Fc receptor concentration, high sensitivity to natural killer cells, absence of EBV C3 receptors, and deficiency of membrane-associated beta 2-microglobulin (beta 2M) and HLA, in parallel with intracellular synthesis and secretion of beta 2M to the medium. Unlike the P3HR-1 parent, the hybrid was completely nonpermissive for antigens of the EBV cycle, early antigen, and viral capsid antigen. None of the 3 inducing agents, 5-lodo-2'-deoxyuridine, 12-O-tetradecanoyl-phorbol 13-acetate, or sodium butyrate, caused any viral antigen synthesis in PUTKO-1 in contrast to the good inducibility of the parental P3HR-1 subline. Thus the myeloid parent restricted expression of EBV antigens except EBNA. This exception further supports the concept that EBNA is an autonomous function of the viral genome, independent of host cell control that regulates expression of antigens related to the viral cycle. On the contrary, extinction of viral antigens in this hybrid between 2 cell lineages supports our previous concept that the ability to produce viral antigens is similar to a differentiated B-cell property.

Human-human hybridomas and human monoclonal antibodies obtained by fusion of lymph node lymphocytes from breast cancer patients.

Lymphocytes from lymph nodes obtained from breast cancer patients undergoing mastectomy were fused with the 0467.3, UC729HF2, or KR-12 human cell lines, totaling 42 fusions with lymphocytes from 23 patients. A total of 1696 human-human hybridomas were generated, 675 (39.8%) of which produced human IgG and/or IgM. Seventy-three human hybridomas produced antibodies binding to autologous malignant breast tissue and/or MCF-7 cells, as assayed by immunohistology or by cell-binding enzyme-linked immunosorbent assay. Twelve of these hybridomas, all reacting with malignant breast tissue, were subcloned to stabilize the production of human immunoglobulin. The reaction patterns of these 12 human monoclonal antibodies were investigated further by immunohistology on formalin-fixed, paraffin-embedded tissues. The reaction patterns of the various antibodies showed substantial variation and the antibodies reacted with a varying frequency with antigens expressed by different malignant breast tumors. One of these antibodies, MAC 40/43 (IgM), reacted with malignant breast and colon carcinomas and other epithelial derived neoplasms but did not react with normal breast tissue or with other normal and malignant tissues tested, except for a weak reaction with certain normal epithelial tissues. The antigen defined by MAC 40/43 was identified as a Mr approximately equal to 47,000 glycoprotein.

Blood group ABO-related glycosylation of urothelial cell lines: immunocytological, enzymatic, and genetic characterization.

Three immortalized, human urothelial cell lines were characterized with respect to their ABO-related carbohydrate phenotypes using a panel of monoclonal antibodies directed to a series of carbohydrate epitopes (Lac, sialylated Lac, Le(a), sialylated Le(a), Le(x), sialylated Le(x), H types I and II, Ley, Leb, A monofucosylated types I and II, ALey, Aleb, and A type III). The glycosyltransferases forming some of these epitopes (beta 1-3/4 galactosyltransferase, alpha 1-2 fucosyltransferase, alpha 1-3 galactosyltransferase, and alpha 1-3-N-acetyl-galactosaminyltransferase) were determined by enzyme assays. The ABO gene complex was analyzed by Southern blotting, Northern blotting, and polymerase chain reaction across the O deletion and across base differences between the A and B alleles. The immunocytochemical stainings showed marked differences between the three cell lines; the high grade (tumorigenic, metastatic) cell line showed difucosylated types I and II structures, and the low grade (nontumorigenic, nonmetastatic) cell lines showed monofucosylated types I and II structures. Polymerase chain reaction genotyping of the cell lines indicated that one was OO, one was AA, and one was A plus a mutated allele. Northern blotting showed RNA encoding the A transferase. However, even though both of the A cell lines seemed to have an intact gene, which could produce A transferase and transcribed RNA, none of them showed any activity of the A gene encoded enzyme or any A-structures at the cell surface. In contrast, the three other examined glycosyltransferases were active. The three urothelial cell lines reflect in vivo findings in humans. They represent a competent system for in vitro studies of the different carbohydrate transferase genes responsible for the carbohydrate structures expressed on the cell surface in bladder tumors.

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma.

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.

Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3.

Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3. Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-gamma receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2(+)-binding protein annexin II, and proteins from the hsp70 family. Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides. These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy.

The p16-cyclin D/Cdk4-pRb pathway as a functional unit frequently altered in melanoma pathogenesis.

The p16Ink4/CDKN2, D-type cyclins, their partners Cdk4/Cdk6, and pRb constitute a G1 regulatory pathway commonly targeted in tumorigenesis. Genetic, immunochemical, and functional cell cycle analyses showed abnormalities of this pathway in each of 22 human melanoma cell lines examined. Normal melanocytes and all melanoma lines expressed Cdk4, Cdk6, and cyclins D1 and D3. The tumor suppressors p16Ink4/CDKN2 and pRb were lost in 17 and 4 cases, respectively, due to various genetic mechanisms, including transcriptional block of p16 and nonsense mutations of RB1. Ectopic expression of p16 prevented S-phase entry of Rb+/p16- but not Rb-deficient melanoma lines. The SK29-MEL-1 cell line harboring an R24C mutation in Cdk4 expressed wild-type pRb and overabundant p16, the latter preventing endogenous Cdk6 but not Cdk4 from associating with cyclin D1. Microinjection of cyclin D1-neutralizing antibody arrested the SK29-MEL-1 cells in G1, whereas pl6 did not, indicating that the cyclin D1/Cdk4-R24C complex is required for G1 progression, and the resistance of the complex to p16 in vivo. These data strongly support the candidacy of Cdk4 as a novel proto-oncogene, provide further evidence for the p16-cyclin D/Cdk-pRb pathway as a functional unit, and suggest that deregulation of this checkpoint may represent a common step in the multistep progression of sporadic malignant melanomas.

Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum.

Recently, the ganglioside FucGM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]-Gal beta 1-4Glc beta 1-1 Cer) was identified as a small cell lung cancer (SCLC) marker both in chemical and histochemical studies. In order to further determine whether the FucGM1 ganglioside is shed from the tumor site and consequently is present in the serum of SCLC patients, we produced a series of new monoclonal antibodies raised against FucGM1 and related glycolipids. Shedding of the FucGM1 ganglioside was studied both in vitro and in vivo using SCLC cell lines and nude mice xenografts of SCLC cells as model systems, and finally immunochemical analyses were performed on serum samples from patients with SCLC. High-performance thin-layer chromatography immunostaining demonstrated the presence of FucGM1 in conditioned culture media obtained from FucGM1-positive SCLC cell lines. Furthermore, tumor extracts of SCLC cell line xenografts in nude mice were positive for the FucGM1 marker, and more importantly the marker was also present in serum samples from these mice. Twenty serum samples were obtained from patients with histologically verified SCLC. Eight patients had localized disease, and the remaining patients had disseminated cancer involving metastases to other organ sites. Sera from 4 of these patients were clearly positive, and 2 additional cases were found to be weakly positive. The positive patient sera were all from patients with extensive disease. Sera from 12 patients with non-SCLC and 20 healthy individuals were all found to be negative. These results clearly establish the FucGM1 glycolipid as a potential serum marker of SCLC for which a sensitive immunoassay should be developed and tested using a larger series of serum samples.

Recombinant interleukin-2 and lymphokine-activated killer cell treatment of advanced bladder cancer: clinical results and immunological effects.

The purpose of this study was to evaluate the efficacy of treatment with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells in patients with advanced bladder cancer and to study the induced changes in the distribution of leukocyte subsets in blood and tumor. Nine patients with metastatic transitional cell cancer of the bladder were treated with a continuous infusion of rIL-2 combined with lymphocytes stimulated in vitro with rIL-2. None of the patients responded to the therapy despite substantial changes observed in the immunological cells, both in tumor and blood. The rIL-2 infusion induced migration of leukocytes to the tumors, which was related to increased expression of the adhesion molecule VLA-1 on both peripheral blood mononuclear cells and the endothelial cells of small tumor vessels. Only T-cells, predominately expressing IL-2 receptors, and macrophages infiltrated the tumors. Natural killer cells remained few or absent in the tumors, even though the natural killer cells in peripheral blood were activated by the treatment. This study shows that the present technique of rIL-2 and lymphokine-activated killer cell therapy is able to induce substantial changes in the immune system of patients with metastatic bladder cancer. However, this treatment did not induce tumor regression, which may be due to the advanced stage of disease.

Somatic mutation of the Peutz-Jeghers syndrome gene, LKB1/STK11, in malignant melanoma.

Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identified as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identified in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identified, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) affecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.

Aberrant p27Kip1 promoter methylation in malignant melanoma.

p27Kip1 is a regulator of the mammalian cell cycle and a putative tumor suppressor. Distinct altered patterns of p27Kip1 protein expression are found in a variety of human carcinomas, and p27Kip1 expression levels usually correlate directly with disease-free survival. The mechanism(s) by which p27Kip1 expression is reduced or lost during tumorigenesis remains unclear. Specific alterations of the p27Kip1 gene, including mutations and homozygous deletions, are exceedingly rare in human cancers. We have used methylation-specific PCR and bisulfite genomic sequencing to examine the methylation status of p27Kip1 in 61 primary and metastatic tumors and 35 cell lines from patients with malignant melanoma. Dense methylation of a CpG island in the promoter region of p27Kip1 was detected in four of 45 metastatic tumors (9%) and three of the cell lines (9%), including two cell lines established from two different metastases from the same patient. Examination of a naturally occurring, allele-specific sequence variant demonstrated that p27Kip1 methylation is associated with transcriptional silencing in situ. Cell lines with p27Kip1 methylation showed retention of the wild-type allele and detectable p27Kip1 protein whose abundance was reduced compared with normal melanocytes. Collectively, our data suggest that DNA methylation may be a cause of monoallelic p27Kip1 silencing in malignant melanoma, which would support a role for p27Kip1 haploinsufficiency in human cancer.

Peptides spanning the junctional region of both the abl/bcr and the bcr/abl fusion proteins bind common HLA class I molecules.

The Philadelphia (Ph) chromosome, resulting from the t(9;22) translocation, is characteristic of chronic myeloid leukemia (CML). As a result of this translocation, two novel chimeric genes are generated and the bcr/abl and abl/bcr fusion proteins expressed. The bcr/abl fusion mRNA is present in all CML patients, whereas the reciprocal abl/bcr fusion mRNA is detectable in about 80% of the Ph+ CML patients. These fusion proteins may undergo enzymatic degradation in the cytosol and give rise to MHC class I restricted peptide epitopes originating from the junctional regions of the translocation products, which thus may serve as novel tumor specific antigens. Previously, other groups have tested peptides corresponding to the junctional region of the bcr/abl protein for their binding capacity to HLA class I molecules and have identified a few candidate epitopes. Peptides originating from the abl/bcr fusion protein have on the other hand so far been neglected, for no apparent reason. We have now extended these studies to include also the reciprocal abl/bcr translocation product by testing a large panel of synthetic peptides corresponding to the junctional regions of both the abl/bcr and the bcr/abl fusion proteins for their ability to stabilize HLA class I molecules. We find that the abl/bcr translocation product may be an even more important source of CML specific peptide antigens and together the junctional sequences of both these proteins contain peptide sequences which bind efficiently to a number of HLA molecules (HLA-A1, -A2, -A3, -A11, -B7, -B27, -B35) and thus may serve as candidate CML specific tumor antigens.

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb.

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.

Concurrent disruption of p16INK4a and the ARF-p53 pathway predicts poor prognosis in aggressive non-Hodgkin's lymphoma.

The INK4a/ARF locus at chromosome 9p21 encodes two structurally and functionally distinct molecules with tumor-suppressive properties. p16INK4a controls cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein (Rb), while ARF prevents MDM2-mediated degradation of p53. By using a panel of PCR-based methods, we have examined the status of the p16INK4a, ARF and p53 genes in 123 cases of non-Hodgkin's lymphoma (NHL) at diagnosis. Alterations of one or more of these genes were detected in seven of 36 (19%) cases with low- to intermediate-grade histology, and in 35 of 87 (40%) cases with aggressive histology. For the aggressive lymphomas, the Kaplan-Meier estimate of overall survival for cases with disruption of either p16INK4a or the ARF-p53 pathway was not different from cases with retention of both pathways (5 year survival 45% vs 35%; P= 0.85), suggesting that selective inactivation of one of the pathways does not significantly influence overall survival. By contrast, the 5-year survival was only 7% for cases with concurrent disruption of p16INK4a and the ARF-p53 pathway vs 38% for cases with retention of one or both pathways (P = 0.005). Similar results were obtained when the analysis was confined to diffuse large B cell lymphomas (P= 0.019). On stepwise multivariate regression analysis including factors from the international prognostic index, concurrent disruption of p16INK4a and the ARF-p53 pathway was an independent negative prognostic factor in NHL with aggressive histology (P = 0.006). Our results suggest that the compound status of the p16INK4a and ARF-p53 pathways is a major determinant of outcome in NHL.

Mutation and allelic loss of the PTEN/MMAC1 gene in primary and metastatic melanoma biopsies.

The PTEN/MMAC1 gene on chromosome 10q23 encodes a lipid phosphatase with tumor-suppressive properties. Germline PTEN/MMAC1 mutations have been implicated as the predisposing factor in Cowden disease and other hamartoma syndromes, and somatic mutations and deletions have been identified in a wide range of human cancers, including 30-40% of metastatic melanoma cell lines. To study further the possible role of PTEN/MMAC1 in the pathogenesis and progression of malignant melanoma, we examined uncultured specimens from 16 primary and 61 metastatic tumors from 67 patients. Denaturing gradient gel electrophoresis was used to analyze systematically the coding region of PTEN/MMAC1 and revealed mutations in four of the metastatic samples (7%). Sequence analysis of the mutants identified a 1 bp frameshift insertion, a 2 bp frameshift deletion, an 11 bp frameshift deletion, and a single base substitution resulting in the generation of a premature stop codon. Analysis of two intragenic polymorphisms showed allelic loss in three of eight informative primary tumors (38%) and in 18 of 31 metastatic tumors (58%). One of the mutant cases showed allelic loss, suggesting that both PTEN/MMAC1 alleles were inactivated in this tumor. Altogether, these results suggest that mutation and deletion of PTEN/MMAC1 may contribute to the development and progression of malignant melanoma.

Expression of CD94/NKG2 subtypes on tumor-infiltrating lymphocytes in primary and metastatic melanoma.

Natural killer receptors are expressed both on natural killer populations and subpopulations of T cells, mainly alpha/beta TCR+CD8+ T cells. We have characterized the expression of the C-type lectin natural killer receptor CD94/NKG2 on tumor-infiltrating lymphocytes in primary and metastatic melanoma lesions. By immunohistochemistry, 5-10% of the tumor-infiltrating lymphocytes, both in primary and metastatic lesions, expressed CD94. More than 95% of these CD94+ cells coexpressed CD8 and the percentage of CD94 expression within the CD8+ cell population ranged from 5 to 20% with a higher expression in metastatic lesions. CD94/NKG2 exists both in an inhibitory and an activating form; thus, it was necessary to determine whether the inhibitory CD94/NKG2-A/B, the activating CD94/NKG2-C/E, or both were expressed on tumor-infiltrating lymphocytes. Reverse transcription-polymerase chain reaction using specific primers for NKG2-A/B and C/E mRNA revealed the presence of NKG2-C/E in all primary and metastatic lesions. In contrast, the inhibitory NKG2-A/B was only present in 50% of primary tumors whereas 80% of tumor-infiltrating lymphocytes in metastatic lesions expressed these transcripts. In healthy humans, the mean number of inhibitory natural killer receptors is higher than that of activating receptors, but the opposite was true for tumor-infiltrating lymphocytes in melanoma. The reversal of the ratio of inhibitory to activating natural killer receptors among tumor-infiltrating lymphocytes suggests a regulated event due to either specific factors within the tumor microenvironment, preferential homing of T cell subsets, or certain stages of T cell activation.

Accumulation of identical T cells in melanoma and vitiligo-like leukoderma.

The cloning of genes encoding melanoma antigens has opened new possibilities for the treatment of patients with cancer; however, most tumor rejection antigens recognized by tumor infiltrating lymphocytes are the products of genes that are also expressed by normal melanocytes. Hence, a large set of antigenic determinants of the self have not induced self-tolerance and these peptide determinants furnish target structures for immune responses directed against tumors. The notion that the immunotherapeutic targets involved in cancer regression comprise normal differentiation antigens is stressed by the association between vitiligo-like leukoderma, due to destruction of normal melanocytes, and melanoma regression, due to destruction of cancer cells. Nevertheless, this is the first report to demonstrate by means of a new technique based on reverse transcription polymerase chain reaction and denaturing gradient gel electrophoresis, the presence of clonally expanded T cells with identical BV regions in areas of destruction of both normal and neoplastic cells.