A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ.
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A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion). (+info)
Expression of Fas (CD95/APO-1) ligand by human breast cancers: significance for tumor immune privilege.
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Breast cancers have been shown to elicit tumor-specific immune responses. As in other types of cancer, the antitumor immune response fails to contain breast tumor growth, and a reduction in both the quantity and cytotoxic effectiveness of tumor-infiltrating lymphocytes (TILs) is associated with a poorer prognosis. Fas ligand (FasL) induces apoptotic death of activated lymphocytes that express its cell surface receptor, FasR (CD95/APO-1). FasL-mediated apoptosis of activated lymphocytes contributes to normal immune downregulation through its roles in tolerance acquisition, immune response termination, and maintenance of immune privilege in the eye, testis, and fetus. In this report, we demonstrate that breast carcinomas express FasL. Using in situ hybridization and immunohistochemistry, we show that breast tumors constitutively express FasL at both the mRNA and protein levels, respectively. FasL expression is prevalent in breast cancer: 100% of breast tumors (17 of 17) were found to express FasL, and expression occurred over more than 50% of the tumor area in all cases. By immunohistochemistry, FasR was found to be coexpressed with FasL throughout large areas of all the breast tumors. This suggests that the tumor cells had acquired intracellular defects in FasL-mediated apoptotic signaling. FasL and FasR expression were independent of tumor type or infiltrative capacity. FasL expressed by tumor cells has previously been shown to kill Fas-sensitive lymphoid cells in vitro and has been associated with apoptosis of TILs in vivo. We conclude that mammary carcinomas express FasL in vivo as a potential inhibitor of the antitumor immune response. (+info)
Tumor infiltration by adoptively transferred T cells is independent of immunologic specificity but requires down-regulation of L-selectin expression.
(27/1358)
Adoptive immunotherapy with anti-CD3/IL-2 activated tumor-draining lymph node (LN) T cells is capable of eradicating tumor established at various histological sites. Tumor-specific effector lymphocytes have recently been identified to be LN T cells with down-regulated L-selectin (L-selectin-). Using fluorochrome labeling, the present study determined the early trafficking pattern of systemically transferred cells. In mice with 10-day established pulmonary 3-methylcholanthrene (MCA) 205 metastases, accumulation of cells in tumors was evident as early as 2 h after i.v. cell transfer, and, by 24 h, >50-fold higher numbers of cells were seen in metastases than in normal tissues. Similarly, transferred cells selectively infiltrated s.c. tumors, albeit at a lower rate. Analysis of the transferred cells isolated from recipient mice revealed that tumor-infiltrating cells were mostly L-selectin- (>95%). By contrast, only 24% and 58% L-selectin- cells were found in the LN and spleen, respectively. The ability of L-selectin- cells to accumulate at tumor sites was confirmed by the transfer of purified cell populations. Despite this selective tumor infiltration, the trafficking pattern did not reflect antigenetic specificity, and tumor regression occurred only after the transfer of tumor-specific effector cells. These results, thus, suggest that there are two distinct mechanisms operative in successful adoptive immunotherapy. Early infiltration of tumors by transferred cells is dictated by the physiological properties of cells and is independent on their immunologic specificity. Tumor regression, however, requires immunologically specific interactions at the site of tumor. (+info)
Increased frequency of antigen-specific CD8(+) cytotoxic T lymphocytes infiltrating an Epstein-Barr virus-associated gastric carcinoma.
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Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue. (+info)
Astrocytoma infiltrating lymphocytes include major T cell clonal expansions confined to the CD8 subset.
(29/1358)
Anaplastic astrocytoma and glioblastoma are frequent and malignant brain tumors that are infiltrated by T lymphocytes. Whether these cells result from non-specific inflammation following blood-brain barrier disruption or an antigen-driven specific immune response is unknown. In this study, an in-depth characterization of TCR diversity in tumor and blood RNA biopsies was performed in a series of 16 patients with malignant astrocytoma. Whilst there was no obvious restriction of the AV and BV gene segment usage, complementarity-determining region 3 size analysis and sequencing of amplified TCR transcripts revealed multiple T cell oligoclonal expansions in all astrocytomas analyzed. Unique T cell clones were present in different adjacent areas of a given tumor, but never detected in the blood. Quantification of the number of TCR clonal transcripts per microg of tumor RNA indicated that certain T cell clonal expansions may represent at least 300 cells/10(6) tumor cells. Furthermore, we demonstrated that the in vivo expanded clones were almost exclusively confined to the CD8(+) subset. Overall, these data suggest that spontaneous antigen-driven immune responses may be elicited against human astrocytoma despite the immunosuppressive microenvironment generated by the brain and the tumor itself. However, the ultimate failure of the immune system to control tumor growth could be the consequence of a deficient CD4 T(h) component of the response. This observation could have important consequences for the development of immunotherapies for astrocytoma patients. (+info)
Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial.
(30/1358)
We conducted a phase 1 trial of direct injection of an E1, E3-deleted adenovirus encoding interleukin-2 (AdCAIL-2) into subcutaneous deposits of melanoma or breast cancer. Twenty-three patients were injected at seven dose levels (10(7)-10(10) p.f.u). Local inflammation was observed at the site of injection in 60% of patients, but side-effects were otherwise minor. Incomplete local tumor regression occurred at the site of injection in 24% of patients, but no conventional clinical responses were seen. Circulating CD4 and CD8 counts fell significantly 24 h after injection. Post-injection biopsies demonstrated tumor necrosis and lymphocytic infiltration with the predominant tumor-infiltrating cells both CD3- and CD8-positive. Vector-derived sequences were detected in 14 of 18 biopsies examined 7 days after injection and vector-derived hIL-2 mRNA was detected in 80% of 7-day biopsies processed after injection of 10(8) p.f.u. of AdCAIL-2 or higher. While IL-2 was detectable by ELISA in tumor biopsies at 48 h, no protein was detectable in injected tumors after 7 days and no circulating IL-2 was detectable at any time-point. No Ad5E1 sequences were detected either before or after injection indicating absence of replication-competent virus or endogenous E1-like sequence; furthermore, only rare vector shedding was detected. Anti-adenovirus and neutralizing antibody titers were elevated 1 month after injection in all patients. This trial therefore confirms the safety of use of adenoviral vectors for gene delivery in humans and demonstrates successful transgene expression even in the face of pre-existing immunity to adenovirus. (+info)
Alpha/beta- and gamma/delta TCR(+) lymphocyte infiltration in necrotising choroidal melanomas.
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AIM: To detect specific tumour infiltrating T cells (TIL) carrying antigen specific MHC-I restricted receptor genes on necrotising and non-necrotising malignant melanomas and to correlate the findings with clinical data. METHODS: alpha/beta- and gamma/delta- TIL were determined by immunohistochemical staining in melanomas of patients with known follow up of more than 10 years. An antigen retrieval method was used to determine variable genes delta1 and gamma1 on TCR(+) cells by an anti-TCR Vdelta1 and anti-CrgammaM1, and of Valpha and Vbeta TCR(+) by an anti-pan-TCR(+) alpha/beta antibody. RESULTS: Intratumoral TIL were present in 86 of 113 (76.1%) necrotising melanomas (NMM) v 21 of 100 (21%) in non-necrotising melanomas (MM); of these, Valpha/beta- TCR(+) cells were present in 52 of 74 (70.3%) TIL harbouring NMM v four of 21 (19%) MM; Vgamma1 in 29 of 74 (39.2%) NMM v two of 21 (10%) MM; and Vdelta1 in 39 of 74 (52.7%) NMM v three of 21 (14%) MM. Extratumoral lymphocytic infiltration was seen in 86 (76.1%) NMM including Valpha/beta TCR(+) cells in 10 (11.6%) cases, v five (5%) MM cases with no Valpha/beta TCR(+) cells detected. Vgamma1 and Vdelta1 TCR(+) cells were not found in extratumoral infiltrates. CONCLUSIONS: In NMM, the median survival was 69.3 (range 6-237) months, 19 of 74 patients (25.7%) survived 5 years, and mortality was associated with advanced stage (p<0.001), patient age (p<0.023), and extent of necrosis (p<0.048). Survival was increased with evidence of Vgamma1 and Vdelta1 TCR(+) cells (p<0.026). (+info)
Increased activation of lymphocytes infiltrating primary colorectal cancers following immunisation with the anti-idiotypic monoclonal antibody 105AD7.
(32/1358)
BACKGROUND: The anti-idiotypic monoclonal antibody 105AD7 mimics the tumour associated antigen 791Tgp72, expressed on 70-80% of colorectal cancers. Phase I studies have shown that the vaccine is non-toxic, and a number of patients have been immunised prior to resection of their primary tumours. AIMS: To assess lymphocyte activation at the tumour site by measuring expression of the alpha subunit of the interleukin 2 receptor (CD25). METHODS: Nineteen patients with primary colorectal cancer were immunised with varying doses of 105AD7 prior to resection of their primary tumours. Samples of normal bowel and tumour edge/centre from 16 patients were available for immunohistochemical staining with a monoclonal antibody against CD25. Samples from a matched control group were also stained. Fresh tumours from 14 immunised patients and 31 unimmunised control patients were disaggregated, and the lymphocytes obtained labelled for CD25. Samples were analysed blindly by flow cytometry. RESULTS: Median infiltration of lymphocytes expressing CD25, measured immunohistochemically, was higher in trial patients, as was the ratio of tumour to normal bowel infiltration. Flow cytometric analysis of fresh tumour from immunised patients showed a significantly higher percentage of lymphocytes expressing CD25 tumour infiltrating lymphocytes than their matched and unmatched controls. DISCUSSION: The alpha subunit of the interleukin 2 receptor is increased on tumour infiltrating lymphocytes, in patients immunised with the colorectal cancer vaccine 105AD7. This suggests a population of activated lymphocytes capable of targeting 791Tgp72 expressing tumour cells, such as circulating micrometastases. 105AD7 may have a role as adjuvant therapy in early stage disease. (+info)