Jesper Zeuthen

Prognostic value of the CD4+/CD8+ ratio of tumour infiltrating lymphocytes in colorectal cancer and HLA-DR expression on tumour cells

The purpose of this study was to clarify whether HLA-DR expression of colorectal tumour cells or the CD4+/CD8+ ratio of the tumour infiltrating lymphocytes is significantly associated with the prognosis of colorectal cancer. Using flow cytometry, we studied the tumour cell expression of the HLA class II in 70 enzymatically dissociated colorectal cancers and the phenotype of tumour infiltrating lymphocytes (TILs) in 41 cases. There was no trend in 5-year survival between three levels (low, medium, high) of HLA-DR expression on the tumour cells. Patients with low CD4+/CD8+ ratios had a better clinical course, with significantly higher 5-year survival, p=0.046, independent of the Dukes stage and age. Our results have implications for tumour immunology; colorectal cancer cells might be a target for cytotoxic T-lymphocytes, however the tumour cells are not able to initiate an immune response. Stimulation of the immune system could possible be obtained using dendritic cells cultured in vitro and loaded with tumour antigens.

Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and Annexin II eluted from melanoma cells

Abstract In a search for potentially tumour-specific MHC-class-II-restricted antigens, the immunogenicity of endogenous peptides that had been eluted from HLA-DR molecules of the human melanoma cell line FM3 (HLA-DRB1*02x, DRB1*0401) was tested in vitro. Two 16-mers representing gp100 positions 44–59, and annexin II positions 208–223 bound well to isolated DRB1*0401 molecules and are discussed here. HLA-DR-matched normal donors T cells were cultured with peptide-pulsed artificial antigen-presenting cells (CHO cells cotransfected with genes for HLA-DRB1*0401 and CD80 and coexpressing high levels of both human molecules). Specific sensitization was achieved against both peptides, as measured in assays of autocrine proliferation and interleukin-2 secretion. Moreover, responses to native autologous melanoma cells but not to autologous B cells were also observed. In view of the expression of fas by the activated T cells and of fas ligand by the melanoma cells, blockade of potential fas/fas-ligand interactions was undertaken using monoclonal antibodies (mAb). The antagonistic fas-specific mAb M3, but not the fas agonist M33, caused a markedly enhanced T cell response to FM3 cells. These results demonstrate that synthetic peptide antigens are able to sensitize T cells in vitro for effective MHC-class-II-restricted recognition of melanoma cells.

In situ T cells in melanoma

Abstract During the past decade new insights have been gained into the role of T lymphocytes in the hosts immune response to cancer in general and to melanoma in particular. Several melanoma-associated antigens (MAA) recognized by T cells have been characterized, and a number of HLA class I- and class II-restricted peptides have been identified. These antigens can be divided into three different groups: tumor-associated testis-specific antigens, melanocyte differentiation antigens, and mutated or aberrantly expressed antigens. These proteins give rise to several antigenic peptides. The number of known melanoma-associated peptides that can induce killing by cytotoxic T-lymphocytes (CTL) exceeds 30 and is still increasing. In line with these findings, clinical data indicate that the immune system is essential in the control of tumor growth. A brisk infiltration of lymphocytes is associated with a favorable prognosis, and complete or partial regression of primary melanoma occurs quite frequently. Furthermore, immunomodulatory therapies have accomplished complete or partial tumor regression in a number of patients. However, the immune response is in most cases inadequate to control tumor growth as tumor progression often occurs. Hence, the coexistence of a cellular immune response in melanoma lesions, demonstrated by the presence of clonally expanded T cells, remains a major paradox of tumor immunology. In the present paper we review current knowledge regarding tumor infiltrating lymphocytes (TIL) in melanoma and discuss possible mechanisms of escape from immune surveillance.

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.

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.

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

Abstractu2003Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens u200a–u200a present also in normal melanocytes u200a–u200a and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells.

Comparative delineation of T cell clonotypes in coexisting syngeneic B16 melanoma

Abstractu2002B16 is a murine melanoma of C57Bl/6 origin, which rapidly develops as a tumor when inoculated into syngeneic immunocompetent hosts. Nevertheless, B16 tumors are considered to be immunogenic since tumor regression can be induced by means of immunotherapeutic intervention. Furthermore, B16 melanoma cells express several melanoma-associated antigens that may serve as targets for autologous T cells. To study the in vivo T cell response against B16, with particular emphasis on diversity and systemic involvement, we ex- amined the spectra of T cell clonotypes in coexisting B16 melanoma lesions in C57Bl/6 mice. Three tumors from each animal (nu2009=u20098) were examined for the presence of clonotypic T cells using the highly sensitive T cell receptor (TCR) clonotype mapping technology. Systematic analysis of the TCRB variable regions 1–16 revealed from 19 to more than 30 clonotypic TCR transcripts in each tumor. To study intra- and inter-individual variations in the T cell response further, more than 600 clono-typic TCR transcripts were compared for sequence identity. Overall, approximately 2% of the T cell clonotypes were detected in more than one tumor from the same animal. Furthermore, none of the detected clonotypes was present in more than one animal, arguing against recurrent or “public” T cell responses against B16 melanoma. Our data strongly suggest that anti-melanoma T cell responses in this murine model encompass mainly localized T cells, and that systemic involvement is limited.

Detection of circulating tumor lysate–reactive CD4+ T cells in melanoma patients

Purpose: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which expresses high amounts of melanoma antigens. Methods: Fresh peripheral blood was incubated with and without lysate for 6xa0h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-γ+ cells in the CD4+ population, the response to lysate was calculated as the difference between the number of activated IFN-γ-producing CD4+ cells in the lysate-stimulated and the nonstimulated sample. Results: An immune response to lysate was observed in blood samples from 11 of 15 patients (73%) with metastatic melanoma. A weak response was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially in donors with high background levels of spontaneous IFN-γ production, indicating an inhibitory effect of the lysate. Conclusions: This method for detection of a peripheral T-cell immune response in melanoma patients has several advantages for clinical use. The tumor lysate preparations may contain large numbers of stimulating antigens (known, as well as unknown) and are easily prepared and handled. Potentially, the assay might be useful as a diagnostic tool, a marker of residual or recurrent disease, a prognostic factor, or a predictor or monitor of the effect of antineoplastic therapy including immune-modulating therapy.

Immunohistochemical characterisation of the local immune response in azoxymethane-induced colon tumours in the BDIX inbred rat strain

The aim of the present study was to characterise the local immune response in a chemically induced colon tumour model in the rat. Elucidating the character of the immune reaction may contribute to optimizing immunotherapeutic regimens for colon carcinoma in this model. Colon cancer was induced by four weekly subcutaneous azoxymethane injections in inbred rats of the BDIX/OrlIco strain in two separate studies. Azoxymethane‐induced tumours show many similarities to spontaneously occurring human colon carcinomas with respect to histopathological appearance. In our studies, the overall inflammatory reaction of the submucosa below the tumour was evaluated in haematoxylin‐eosin‐stained tissue sections. Phenotypic characterization of leukocyte infiltration in the tumour tissue was performed by immunohistochemical staining using antibodies detecting various leukocyte subsets, i.e. T cells, natural killer cells, macrophages/monocytes, and dendritic cells. The results showed that the azoxymethane‐induced colon tumours were strongly infiltrated by macrophages. Furthermore, the tumours showed a moderate degree of infiltrating CD4‐positive cells. Very few natural killer, CD8‐positive T cells and dendritic cells (identified by the OX62 antibody) were seen in the tumour tissue. Virtually no CD25‐positive cells were found. This immunohistochemical characterisation of the tumour‐infiltrating immune response in this rat model could form the basis for studies aimed at developing new immunotherapeutic regimens for human colon cancer.

Monoclonal Antibodies Reactive with Components in Serum-Free Conditioned Medium from a Human Breast Cancer Cell Line (MCF-7)

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.