摘要:

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummarySmall peptides, 8–10 aminoacids long, derived from degradation of cytoplasmic proteins by a proteasome–proteinase complex, are usually presented and recognized by CD8+cytolytic T lymphocytes (CTLs) associated with major histocompatibility complex (MHC) class I molecules. Recently synthetic peptides were used for the in vitro induction of tumor-specific CTLs, offering another strategy in the study of the immune-response repertoire and providing a new tool in cancer vaccination and immunotherapy. Peptides derived from otherwise normal proteins, overexpressed in many tumors as products of the protooncogene, may represent a target for an immune response. This is the case ofHER-2/neugene (also known asErbB-2), encoding a cysteine-rich glycoprotein transmembrane receptor with tyrosine kinase activity (gp185neu). Recent data, demonstrating that HLA-A2.1–related peptides are able to stimulate in vitro CD8+lymphocytes, prompted us to study the binding to HLA-A2.1 molecules of several gp185 synthetic peptides containing a cystein residue and to define the relevance of this amino acid residue in the reduced or oxidated form of the sulfhydryl group. We found that monomers and their homodimers, linked by a disulfide bridge, bind to HLA-A2.1 molecules with overlapping affinity. These results suggest that additional amino acids of the nonapeptide do not prevent the binding and the HLA refolding through chemical or sterical interactions. This might be of particular relevance for the in vivo processing of cysteine-rich proteins. BecauseErbB-2 molecules, as tumor-differentiation antigens in melanoma, are cysteine-rich molecules, it may be relevant to evaluate the possible role of the cystine residues interacting with the T-cell receptor. The recognition of these heterodimers by CD8+lymphocytes will require functional in vivo studies.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummarySeveral investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was sub-cloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/106cells/24 h), adult fibroblasts (625 units/106cells/24 h), and embryonic fibroblasts (3,975 units/106cells/24 h) were 150− to 1,000-fold higher than that secreted by the activated PBMC (4 units/106cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/106cells/24 h pLXSN-iIL2 = 375–625 vs. pLXSN-tIL2 = 90–440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7–11% by day 7, 0–29% by day 14, and 25–50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis ofSacI-digested genomic DNA from the LXSN-tIL2 producer cell line andSacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryA recombinant vaccinia virus encoding the gene for granulocyte-macrophage colony-stimulating factor (rV-GM-CSF) was used to infect the poorly immunogenic murine colon adenocarcinoma cell line, MC-38. Infection of MC-38 tumor cells with rV-GM-CSF completely suppressed the growth of the MC-38 primary tumors, whereas progressively growing tumors were formed in mice injected with MC-38 cells infected with wild type V-Wyeth. Irradiation of the recipient B6 mice before implantation of rV-GM-CSF-infected tumor cells resulted in the development of progressively growing tumors. Moreover, in vivo T-cell depletion studies revealed that growth suppression of the rV-GM-CSF-infected tumor cells was dependent on the presence of both CD4+and CD8+T-cell subsets. Subsequent studies established that this immunity was long-lasting and antigen specific, as demonstrated by the protection of rV-GM-CSF-immunized mice from MC-38 tumor challenge but not from challenge with another syngeneic tumor cell type. No such effects were observed when MC-38 tumor cells were infected with recombinant vaccinia viruses expressing interleukin (IL)-2 or IL-6. The results demonstrate that paracrine release of biologically active murine GM-CSF by tumor cells infected with rV-GM-CSF enhances the intrinsic immunogenicity of a poorly immunogenic murine tumor. Presumably the augmentation of tumor immunogenicity induces an antigen-specific T-cell-dependent antitumor response that prevents the formation of primary tumors and protects mice from tumor challenge. Thus in this experimental model, GM-CSF functions as a highly effective vaccine adjuvant.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryTwenty-eight primary and 29 metastatic melanoma lesions and 18 pigmented nevi lesions were analyzed by using the immunoperoxidase reaction with anti-MART-1 and anti-gp100 monoclonal antibodies (mAbs). The MART-1 was expressed in 28, 29, and 18, and gp100 was expressed in 27, 28, and eight of these lesions, respectively. Intensity and percentage of stained cells with anti-MART-1 mAb were stronger and higher than those with anti-gp100 mAb. MART-1 was expressed homogeneously in primary melanoma and pigmented nevi, whereas it was heterogeneously expressed in metastatic melanoma lesions. The level of expression of MART-1 in primary melanoma lesions did not correlate with any clinicopathologic parameters. These results suggest that anti-MART-1 mAb is a useful tool for immunohistochemical analysis of melanocytic lesions and also is useful for patient's selection and monitoring of antigen-loss variants in clinical trials with the MART-1-based immunotherapy.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryThe human immune repertoire appears to be capable of recognizing normal antigens expressed by tumor cells. Among these antigens, those of differentiation, characterized by a restricted tissue expression, could be of clinical interest since they may represent a target for immunotherapeutic protocols. In this context we have evaluated, in benign and malignant lesions of the melanocytic lineage, the expression of the Melan-A/MART-1 antigen, which has been shown to be recognized by T cells, of HLA-A2 melanoma patients. The immunohistochemical analysis conducted with a Melan-A/MART-1 monoclonal antibody demonstrated that the antigen expression does not correlate with transformation or tumor progression. At variable levels Melan-A/MART-1, differently from other differentiation antigens, is homogeneously expressed by multiple autologous metastases and by melanoma metastases at different body sites. This tissue distribution adds further biological support to the ongoing use of Melan-A/MART-1-related peptides in active immunotherapy.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryThe immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR β-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several β-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryGranzyme B is a protein thought to play a pivotal role in the cytolytic functions of T cells. In view of this, the inducibility of this gene in freshly isolated T cells (T-TILs) infiltrating human renal cell carcinoma (RCC) in vitro was examined by using the reverse transcriptase-polymerase chain reaction (RT-PCR). A reduction in granzyme B messenger RNA (mRNA) expression in stimulated T-TILs from five of nine patients with RCC compared with autologous peripheral blood T cells was noted. The reduced expression was observed after multiple stimuli including anti-CD3 antibody, interleukin-2 (IL-2), and phytohemagglutinin (PHA). Because CD8+T cells represent the predominant cytotoxic population, the ability of this cell population to express granzyme B mRNA after stimulation also was examined. When compared with CD8+peripheral blood lymphocytes (T-PBLs) from patients with RCC and normal donors, the induction of granzyme B mRNA was reduced in CD8+T-TILs. CD8+T-TILs also had lower non-major histocompatibility complex (MHC)-restricted cytotoxic activity than did CD8+T-PBLs against both Daudi cells and allogeneic RCC cell lines. These results show that in a subset of patients with RCC, depressed lytic activity of CD8+TILs compared with CD8+PBLs is present. Reduced granzyme B mRNA expression also was noted in selected patients.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryBiopsies from 12 patients with progressive metastatic melanoma were excised during chemoimmunotherapy to evaluate and characterize the local immune response in situ in the metastases. These findings were compared with the distribution of lymphocyte subsets in the peripheral blood and correlated with the clinical data. The biopsy specimens were prepared for microscopic procedures, and the fields for analyses were chosen to involve a section of both the stroma and the tumor area. The number of each lymphocyte subset was calculated and compared with the number of melanoma cells in the field, allowing quantitative characterization of the immune reaction in different samples. Comparison of the lymphocyte subsets of peripheral blood and metastatic lesions revealed equal relative amounts of CD4+(helper) and CD8+(suppressor/cytotoxic) cells in both tissues, but 10− to 20-fold fewer CD56+(natural killer, NK) cells, and a total absence of CD20+(B) cells in the metastatic lesions. The prognosis of patients was viewed at different stages of the disease. The median survival from the primary diagnosis of patients with a tumor CD4+/CD8+ratio above the median was 4.4 years compared with 2.4 years for those with a ratio below the median (Logrank, p = 0.02). In the multivariate analysis, the only statistically significant prognostic factors were the CD4+/CD8+ratio of the tumor (p = 0.010) and of the peripheral blood (p = 0.020). Monitoring of CD4+and CD8+cells may thus provide valuable information about the state of host defense, with a high CD4+/CD8+ratio indicating more favorable prognosis.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

SummaryThe bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial responses. Most patients developed human anti-murine immunoglobulin antibodies (HAMA), which can inhibit OC/TR mAb-mediated lysis. We generated a chimeric version of the OC/TR mAb to decrease the immunogenicity of the OC/TR mAb and to allow more extended treatment schedules. Sp2/0 myeloma cells were transfected with chimeric heavy- and light-chain genes encoding the anti-CD3 mAb and the MOv18 mAb, respectively, which are reactive with FBP. The resulting cell line produced 80 μg/ml of total immunoglobulin G (IgG), of which 11.5% was the functionally active chimeric OC/TR mAb. Chimeric OC/TR F(ab')2fragments mediated lysis of IGROV-1 ovarian carcinoma cells by human T cells at antibody concentrations of ≥ 1 pg/ml. Specific lysis was still detectable at an effector-to-target cell ratio as low as 0.4. Two patients with ovarian carcinoma treated with F(ab')2fragments of the murine OC/TR developed distinct HAMA titers, which were mainly anti-idiotypic and only partly directed against the murine antibody constant regions. However, of the two patients that were treated with the F(ab')2fragments of the chimeric OC/TR mAb, only one developed a low transient HAMA response just above background level. In conclusion, the generation of chimeric OC/TR may allow more extended clinical studies of bispecific mAb-mediated immunotherapy of ovarian carcinoma.

ISSN:1524-9557    

出版商:OVID    

年代:1997

数据来源: OVID