Biomedical subjects

M Sensi

Publications and source records attributed to M Sensi.

At least 37 records · Page 2Linked to original sources

PubMed1998

Immunohistochemical analysis of the T-cell receptor beta-chain variable regions expressed by T lymphocytes infiltrating primary human melanoma.

The T-cell receptor beta-chain variable (TCRBV) region repertoire expressed by tumor-infiltrating lymphocytes was characterized by immunohistochemical analysis using a panel of 18 monoclonal antibodies on cryosectioned specimens of 14 primary vertical growth phase (VGP) melanomas with a T-cell infiltrate histopathologically defined as brisk or nonbrisk. T lymphocytes present in the VGP of all patients displayed a restricted T-cell receptor usage, with a pattern of reactivity similar in brisk versus nonbrisk infiltrates. No evidence of restriction was found in the extra-VGP lymphocytic infiltrates, when available, within the same specimen. Furthermore, the repertoire of TCRBV expressed in nodal metastases was similar to that of the corresponding primary melanomas in the two cases tested. The results obtained by this in situ analysis indicate that the TCRBV repertoire in VGP is determined by a preferential migration of T lymphocytes, possibly indicative of an immune response to melanoma-associated antigens.

Adult↗

PubMed1997

TCR beta-chain variable region-driven selection and massive expansion of HLA-class I-restricted antitumor CTL lines from HLA-A*0201+ melanoma patients.

Recognition of a given melanoma Ag involves a limited array of T cell clones bearing a structurally defined TCR. The aim of this study was to verify whether this information can be used to isolate and expand such anti-tumor effectors from fresh lymphocyte populations. We found that one to three different TCR beta-chain variable (TCRBV) regions were significantly expanded in 4-wk mixed lymphocyte-tumor cultures (MLTC) from six HLA-A*0201+ melanoma patients, and that the T cells expressing the expanded TCRBV regions were involved in HLA class I-restricted lysis of the tumor. T cell activation by mAbs to MLTC-selected TCRBV region and CD28 resulted in large scale expansion (1-10 x 10(9) cells) of T cell lines, highly enriched for the expression of a single TCRBV region and for CD8+ T cells. The TCRBV-driven selection was equally effective when applied to patients' or healthy donors' lymphocytes, and the T cell lines isolated from melanoma patients exerted HLA class I-restricted lysis of the autologous tumor. MLTC and TCRBV-selected lines recognized allogeneic melanomas sharing HLA-A and -B alleles with the autologous tumor, but only two of the HLA-A2-restricted lines were directed to a known peptide from melanoma-associated Ags. Single-strand conformation polymorphism analysis indicated a polyclonal composition of both MLTC and TCRBV-selected lines, but expansion of clonotypes with identical CDR3 length was observed only in the MLTC lines. Thus, TCRBV-driven selection can be exploited to obtain large scale expansion of antitumor CTL lines from melanoma patients.

Antibodies, Monoclonal↗

PubMed1997

Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine.

Metastatic melanoma patients treated with an autologous DNP-modified tumor cell vaccine develop inflammatory responses in metastatic tumors characterized by infiltration of CD8+ T cells. To further define this immune response, we analyzed T cell receptor beta-chain variable (TCRBV) region repertoire in biopsy specimens and peripheral blood lymphocytes of six patients. After administration of DNP vaccine, a restricted set of TCRBV gene families was found to be expanded compared with prevaccine metastases. In several postvaccine lesions of one patient, obtained over a 2-yr period, TCRBV14+ T cells were clonally expanded and identical T cell clonotypes could be detected. Two major recurring clones were biased toward the use of TCRBJ1S5. Furthermore, T cell lines derived from two such infiltrated skin lesions and, enriched in TCRBV14+ T cells, displayed HLA-class I-restricted lysis of the autologous melanoma cells. Clonal expansion of T cells was demonstrated in the T cell-infiltrated, postvaccine metastasis of a second patient as well. These results indicate that vaccination with autologous, DNP-modified melanoma cells can expand selected clones of T cells at the tumor site and that such clones are potentially destructive to the tumor.

Cancer Vaccines↗

PubMed1997

Levels of advanced glycosylation end-products (AGE) in sera of pregnant diabetic women: comparison between type 1, type 2 and gestational diabetes mellitus.

The chronic hyperglycemia can lead to an increase of the advanced glycosylation end-products (AGE) levels on proteins and macromolecules. Abnormal levels of AGE in several tissues has been associated with the pathogenesis of late diabetic complications. In diabetic pregnant women, high AGE levels might influence the delicate maternal-fetal balance and therefore alter the pregnancy outcome. In this preliminary study, we have measured the AGE in sera of 44 diabetic women in two trimester. Sixteen sera from non diabetic pregnant women have been used as controls. The AGE have been analyzed by means of an ELISA method with an antiserum anti-RNAse-AGE, produced in the Laboratory of Clinical Biochemistry of the Istituto Superiore di Sanità. Diabetic patients type 1 and type 2, in good metabolic control, showed normal AGE levels at both trimester. Patients with gestational diabetes showed significantly high serum AGE levels (p < 0.05). A more extended study will give better insight on the association between AGE levels and a physiopathology of diabetic pregnancy.

Adult↗

PubMed1996

Interaction with fibronectin regulates cytokine gene expression in human melanoma cells.

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Base Sequence↗

PubMed1996

Conserved TCR usage by HLA-Cw* 1601-restricted T cell clones recognizing melanoma antigens.

In this study we determined TCR alpha and beta chain nucleotide sequences of HLA-Cw* 1601-restricted cytoxic T lymphocyte (CTL) clones obtained from the peripheral blood lymphocytes (PBL) of a melanoma patient. These clones were previously shown to be involved in the recognition of melanoma-associated antigenic epitopes SAYGEPRKL and AARAVFLAL encoded by gene MAGE-1 and BAGE respectively. All (3/3) anti-MAGE-1 CTL clones displayed TCRBV5 usage and one clonotype was found twice, > 1 year apart, in patient's PBL. Two out of three anti-BAGE CTL clones showed the same TCRAV/AJ and TCRBV/BJ combinations and differed in the alpha chain CDR3 for two residues and in the beta chain CDR3 for a single nucleotide which, however, did not change translation. These results suggest a pattern of TCR conservation in CTL selected for recognition of MAGE-1 or BAGE peptides on the autoiogous melanoma.

Amino Acid Sequence↗

PubMed1996

Peripheral, but not central, nervous system abnormalities are reversed by pancreatic islet transplantation in diabetic Lewis rats.

Neuroelectrophysiological recordings represent a non-invasive and reproducible method of detecting central and peripheral nervous system alterations in diabetes mellitus. In order to evaluate whether the normalization of metabolic control obtained by pancreatic islet transplantation could reverse diabetic neuroelectrophysiological alterations, or prevent further deterioration, we used an experimental model in which pancreatic islets (n = 1200) were injected into the portal vein of inbred Lewis rats (used as islet donors as well as recipients). Islets were injected 4 months after diabetes induction, since previous work had shown functional but not morphological damage at the nervous tissue level at this stage of the disease. Visual (V), brainstem auditory (BA) and somatosensory (S) evoked potentials (EPs) were measured in streptozotocin-induced, islet-recipient diabetic rats (n = 7), streptozotocin-induced diabetic rats (n = 16) and non-diabetic control rats (n = 12). Metabolic parameters and electrophysiological recordings were evaluated before diabetes induction, before transplantation and 4 months later. After transplantation, glycaemic levels returned to normal values within 1 week and remained so until the end of the study, as confirmed by a normal oral glucose tolerance test and by an increase in body weight. Electrophysiological recordings were altered in diabetic animals before transplantation. Four months after transplantation EP recordings improved, with a detectable gradient from the peripheral to the central structures. SEPs were significantly improved in the peripheral tarsus-L6 tract and the L6-cortex tract (P < 0.005 and P < 0.01 versus diabetic rats) and were ameliorated without achieving statistical significance in the central L6-cortex tract. BAEP latency values tended to improve in transplanted rats, but the differences versus non-transplanted diabetic animals failed to reach significance. VEP values remained clearly pathological and even deteriorated after transplantation. These results show that normalization of metabolic control by pancreatic islet transplantation can reverse some of the already established neuroelectrophysiological alterations at the peripheral nervous system level, but does not affect other alterations at the central nervous system level.

Animals↗

PubMed1995

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Amino Acid Sequence↗

PubMed1995

Analysis of TCR usage in human tumors: a new tool for assessing tumor-specific immune responses.

Tumor-infiltrating lymphocytes (TILs), through displaying a T-cell receptor (TCR) repertoire as heterogeneous as that of normal peripheral blood T cells, show overexpression of TCR variable-gene segments that include clonally expanded TCR sequences. Here, Marialuisa Sensi and Giorgio Parmiani analyze the available information on TCR usage by T cells present in the infiltrate of histologically different tumors and suggest that the analysis of TCR sequences represents a powerful new tool to assess the in vivo immune response to growing tumors. Ultimately, this strategy may lead to the identification and manipulation of T-cell populations endowed with antitumor reactivity.

Amino Acid Sequence↗

PubMed1995

Role of advanced glycation end-products (AGE) in late diabetic complications.

To evaluate accumulation of advanced glycation end-products (AGE) in diabetes and its possible correlation with late diabetic complications, AGE levels were measured by spectrofluorimetry in eye lens and sciatic nerve proteins and isolated tail tendon collagen of rats with experimental diabetes of 3- and 6-month duration. The values obtained were compared to those from age-matched control rats and correlated with cataract presence and somatosensory evoked potential (SEP) alterations. Diabetic animals had increased AGE levels in all tissues at both times; cataract developed in 29% of diabetic rats at 3 months and in 57% at 6 months; SEP conduction velocity was reduced in diabetic animals both at 3 (54.5 +/- 1.8 S.E.M. m/s vs. 73.9 +/- 1.0, P < 0.0001) and 6 months (59.5 +/- 1.4 vs. 71.5 +/- 1.6, P < 0.0001) from diabetes induction. No eye lens AGE level differences were observed when cataract presence was considered. Interestingly, in diabetic rats, increased sciatic nerve AGE levels were associated with reduced SEP. These data show that: (1) AGE levels are increased as early as 3 months from development of hyperglycemia; (2) other factors, in addition to an enhanced rate of fluorescent AGE formation, might play important roles in the pathogenesis of diabetic cataract; (3) increased peripheral nerve AGE levels are associated with SEP alterations.

Animals↗

PubMed1994

H-2Kb and H-2Db gene transfections in B16 melanoma differently affect non-immunological properties relevant to the metastatic process. Involvement of integrin molecules.

Modification of non-immunological cell adhesion properties plays a major role in the decrease in metastatic ability observed after transfection of the H-2Kb gene in H-2-negative B16-derived melanoma clone cells. To investigate the role played by different class-I major histocompatibility complex (MHC) genes on non-immunological properties relevant for metastasis, transfection with the H-2Db gene alone or in conjunction with the H-2Kb gene was performed. H-2Db gene transfection did not modify either metastatic potential or non-immunological cell adhesion properties. Double KbDb transfectants showed a decreased metastatic ability when compared to control clones and to Db transfectants; a decrease in homotypic adhesive ability was also observed, even though not in all clones studied: therefore expression of the Db gene is also relevant. The mechanisms of homotypic cell adhesion were studied and found to be dependent upon temperature and divalent cations. Adhesion was partially inhibited by an antiserum directed against the beta 1 integrin subunit, whereas anti-alpha IIb beta 3 was ineffective. Cell pre-treatment with anti-beta 1 serum reduced metastatic ability. A decreased expression of alpha 4 and alpha 6 integrin subunits was observed in Kb clones, whereas no difference in the levels of some homophilic cell adhesion molecules, such as N-CAM and alpha IIb beta 3, was found. Adhesion required the activity of tyrosine kinases, as suggested by the decreased adhesive properties and impaired metastatic ability of cells pre-treated with the tyrosine-kinase-inhibitor genistein. These results are compatible with involvement of integrin molecules of the beta 1 family in the adhesive ability of these cells. Our data show that: (a) immunological and non-immunological effects of MHC transfection are correlated and depend on the class-I gene used, suggesting that MHC gene therapy can be highly successful only if appropriate MHC genes are transfected; (b) non-immunological cell-adhesion properties modified after MHC transfection could be related to an impairment of integrin-mediated adhesive interactions.

Animals↗

PubMed1994

Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene.

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.

Base Sequence↗

PubMed1994

Cytotoxic T lymphocytes recognize tumor antigens of a murine colonic carcinoma by using different T-cell receptors.

To see whether different antigens expressed by the same tumor are recognized by distinct T-cell receptors (TCR), we used cytotoxic T-lymphocyte (CTL) lines known to lyse in vitro the syngeneic BALB/c adenocarcinoma C-26. Four of these CD3+ CD8+ lines showed 4 different patterns of lysis on a panel of MHC-class-I-compatible targets. The activity was H-2d-restricted and could be blocked by anti-CD3 and anti-TCR-alpha/beta monoclonal antibodies (MAbs). The CTL lines were also effective, although to a different extent, in adoptive immunotherapy of mice bearing lung metastases. Phenotypic analysis revealed in all the lines a high frequency of cells positive for CD45, asialo GM1 (ASGM1), lymphocyte-function-associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1) and CD44, but negligible expression of L-selectin (LAM-1) and very late antigen-4 (VLA-4); 2 lines expressed the vitronectin-receptor (VN-R). Analysis of TCR V beta-chains used by the 4 lines showed selective presence of V beta 6, V beta 8.2, V beta 8.3 and of V beta 13 chains. MAbs directed to these V beta chains blocked their lytic activity in vitro. V alpha-chain transcripts of the lines were identified by polymerase chain reaction (PCR) as V alpha ITT11 and V alpha 52 in 2 lines, while one could not be identified. Analysis of V beta s in mixed lymphocyte-tumor-cell cultures (MLTC) of T cells deriving from tumor-infiltrating lymphocytes (TIL) or from spleen of C-26 tumor-bearing or immune animals indicated that the TCR of the CTL lines were representatives of the TCR repertoire recognizing C-26 tumor, since their V beta s were shown to be selectively expanded in MLTC.

Amino Acid Sequence↗

PubMed1994

Effect of protein kinase C inhibitors on invasiveness of human melanoma clones expressing different levels of protein kinase C isoenzymes.

The involvement of protein kinase C (PKC) in the mechanism of chemotaxis and invasiveness of human melanoma has been studied in 6 clones of 665/2 cell line characterized by a different integrin profile, differentiation grade and in vitro invasive ability. The levels of total protein kinase C activity revealed a direct correlation with the chemotactic and invasive ability of these clones. Protein kinase C inhibitors, sphingosine and staurosporine, reduced chemotaxis and invasiveness of the highly invasive clone 2/60, while 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) was ineffective. Immunofluorescence analysis revealed high levels of protein kinase C alpha in clone 2/60, while the less invasive clone 2/21 expressed low levels of protein kinase C alpha and beta, but surprisingly appreciable levels of protein kinase C gamma. Downregulation with phorbol 12-myristate 13-acetate (TPA) did not affect invasiveness of clone 2/60 unless the compound was present during the assay. H7 strongly increased invasiveness of clone 2/21 and was able to reverse the inhibitory effect of TPA on clone 2/60. Preliminary experiments showed higher levels of diacylglycerol in clones with lower protein kinase C, suggesting a constitutive downregulation of the enzyme in low invasive clones. Our results support a role for protein kinase C in the invasion process, but point out the complexity of the mechanism which might involve the proteolytic fragment of the enzyme, protein kinase M.

Alkaloids↗

PubMed1994

Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw*1601.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, which codes for antigen MZ2-E which is presented by HLA-A1. Gene MAGE-1 is expressed in many tumors of several types but not in normal tissues except for testis. We show here that gene MAGE-1 directs the expression of another antigen recognized by CTL on the MZ2-MEL cells. This antigen, which was named MZ2-Bb, consists of MAGE-1-encoded peptide SAYGEPRKL bound to major histocompatibility molecule HLA-Cw*1601. The HLA-Cw*1601 allele was found to be expressed by 7 out of 99 individuals from a Caucasian population. Our results extend the range of tumor patients who could be eligible for immunization against MAGE antigens.

Amino Acid Sequence↗

PubMed1994

Characteristics of proteinuria in experimental diabetes mellitus.

An impairment of protein charge selectivity has been invoked to explain the initial anionic proteinuria in diabetic nephropathy. The aims of this work were to investigate charge and size protein perm-selectivity abnormalities in experimental diabetes and to monitor these changes over time after diabetes induction. Diabetes was induced in 56 Sprague-Dawley male rats by streptozotocin; the control group was represented by 38 normal rats. Blood glucose, body weight, urine volumes, and proteinuria in 24-h urine collections were evaluated at 3, 6, 9, and 12 months of diabetes. The Bradford method and mono- and bidimensional gel electrophoresis were used to determine and characterize proteinuria. Body weight increase was lower (P < 0.05, P < 0.0001, P < 0.05 at 3, 6, and 12 months, respectively), urine volumes were greater (P < 0.001, P < 0.005, P < 0.05 at 6, 9, and 12 months, respectively) and the proteinuria was significantly increased (P < 0.05 at 3 months, P < 0.001 at 6 months, P < 0.01 at 9 months, and P < 0.05 at 12 months) in diabetic rats compared with the control group. When the charge and the size of urine proteins were considered, small (30 kDa) and anionic proteins were found to be mainly excreted in diabetic rats, at 3 months of the disease; at 6 months, higher amounts of albumin and cationic proteins with higher molecular weight (50 kDa) were also found in the urine; at 9 and 12 months the changes previously described were associated with an excretion of proteins weighing about 75 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Albuminuria↗

PubMed1994

N-RAS mutations and susceptibility to lymphokine-activated killer (LAK) cells in human melanoma.

RAS oncogene expression has been reported to affect several biological features of rodent tumours cells, including lysability by activated natural killer cells. In order to examine whether expression of mutated RAS genes in human melanoma cells alters their susceptibility to lysis by LAK cells, seven melanoma lines were assessed for the presence of Ki- and N-RAS genes bearing all possible mutations at codons 12, 13 and 61. A panel of 21 clones deriving from the metastatic lesion Me665/2, which had a Gln-->Arg substitution at codon 61 of N-RAS (N-RAS/61+), were also examined. Melanoma cells and clones were used as targets of allogeneic LAK in a 4-h 51Cr-release assay. LAK showed a higher lysis on melanoma lines and clones harbouring a mutated RAS compared with counterparts bearing no RAS mutations. In addition, LAK-mediated lysis drastically decreased on Me665/2 sublines progressively selected by exposure to LAK. This loss was paralleled by a reduction or even disappearance of N-RAS/61+ mRNA signal in Me665/2 sublines. To evaluate whether N-RAS could directly modulate LAK susceptibility to lysis, N-RAS/61+ gene was transfected in two N-RAS wild type (N-RAS/61-) 665/2 melanoma clones by a cosmid vector. In contrast to the high lysability of melanoma cells constitutively expressing the mutationally active N-RAS oncogene, N-RAS/61+ transfectants did not show a consistent high lysability by LAK, compared with some control pSV2neo transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.

Amino Acid Sequence↗