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.

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 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response to lysate was calculated as the difference between the number of activated IFN-gamma-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-gamma 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.

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.

Cancer/testis antigens: structural and immunobiological properties.

Characterization of tumor-associated antigens recognized by cytotoxic T lymphocytes which has evolved during recent years opens new possibilities for specific anti-cancer immunotherapy. Among different groups of tumor-associated antigens, cancer/testis (CT) antigens (expressed in many tumors and among normal tissues only in testes) represent the most perspective antigens for immunotherapy because of their broad tumor-specific expression. More than 50 CT antigens have been described so far and, for many of them, epitopes recognized by T lymphocytes have been identified. The most studied group of CT antigens is the MAGE proteins, which form the so-called MAGE superfamily, together with some MAGE-like proteins that have a different distribution than classical CT antigens. The MAGE superfamily includes five families: MAGE-A, MAGE-B, MAGE-C, MAGE-D, and necdin. Comparison of the structure of members of MAGE superfamily points to the existence of a domain organization of these proteins. The central, core domain (second domain) is highly conservative. The first domain is homologous among MAGE family members with a CT expression, but unique for each member of the MAGE-D and necdin families. In addition to the homology of the central domain, the third domain is also homologous among all members of MAGE superfamily, but to a much lesser extent. The MAGE-D proteins contain an additional, fourth domain, which in the case of MAGE-D3 coincides with trophinin, a separate molecule described previously as an adhesion molecule that participates in embryo implantation. The structural classification of the members of MAGE superfamily might help in the future to understand the biological function of MAGE proteins. One important property of the CT antigens is the up-regulation of their expression by DNA demethylating agents, indicating a possible mechanism for their re-expression in tumors. One of the implications of this particular property could be that a combination of immunotherapy targeting CT antigens with chemotherapy inducing up-regulation of CT antigens might result in more efficient tumor eradication.

Human teratocarcinoma cell lines. A review.

A review is given of studies on human teratocarcinoma cell lines in culture. A human ovarian teratocarcinoma line (PA-1) has been characterized in detail (Zeuthen et al. 1980) and has been shown to exhibit some properties of human teratocarcinoma stem-cells. This line forms embryoid bodies with a distinct outer cell layer analogous to endodermal cells formed by some mouse teratocarcinoma cell lines, and also is capable of a certain limited differentiation in culture as well as upon transplantation to nude mice. A panel of human testicular teratocarcinoma cell lines has been studied by Andrews et al. (1980): While these cells also share some embryonic characteristics, their differentiation in culture is more limited. Studies of various markers suggest that these lines are equivalent to a pre-blastocyst stage derivation. The PA-1 cell line and possibly two other testicular teratocarcinoma lines could possibly be derived at a stage equivalent to a later stage of development.