Ralf Geertsen

Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II and ICAM-1 molecules.

IL‐10 is a cytokine which shows various effects including inhibition of T‐cell proliferation or HLA‐dependent antigen presentation. In this study, we analysed the effects of exogenous or autocrine IL‐10 on proliferation and expression of immunocritical surface molecules. Fourteen cultures of human melanoma cells were established from primary melanomas, locoregional lymph‐node or distant metastases. In 5 melanoma cell cultures, proliferation in the presence of IL‐10, anti‐IL‐10 antibodies (Ab) or control Ab was assessed with colorimetric and [3H]thymidine uptake assays. Flow cytometric analysis was used to quantify the expression of human leukocyte antigen (HLA) class‐I, HLA class‐II and intercellular adhesion molecule (ICAM)‐I and the IL‐10 receptor (IL‐10R). IL‐10 production of melanoma cells was documented by RT‐PCR and IL‐10 protein was detected in the supernatants by means of ELISA. IL‐10 enhanced proliferation and prolonged survival of melanoma cells in 5 out of 5 cultures. Anti‐IL‐10 Ab decreased proliferation. IL‐10R expression was found in 12 out of 14 (86%) melanoma cell cultures. The expression of HLA‐I, HLA‐II and ICAM‐I on all melanoma cells that were positive for IL‐10R showed a reduction of 10‐60% by IL‐10, whereas the surface levels of HLA‐I, HLA‐II and ICAM‐I in 5 out of 5 cell cultures revealed an increase of 10‐170% by anti‐IL‐10 Ab. These findings suggest that IL‐10 is an autocrine growth factor with significant impact on immunocritical molecules in melanoma. IL‐10 effects have to be considered when planning therapeutic immunointerventions in melanoma patients. Int. J. Cancer 71:630‐637, 1997.

Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution

Monoclonal antibody T311 specifically detects tyrosinase protein expression. Tyrosinase‐derived peptides are recognized by CD8+ T‐celis and applied in immunotherapy. We examined formalin‐fixed paraffin‐embedded tissue of 50 melanoma (primary n=31, metastatic n=19) and 41 control cases (junctional, dermal, compound, Spitz, Reed, balloon‐cell nevi) by immunochemistry using the alkaline phosphatase‐anti‐alkaline phosphatase method after antigen retrieval. Staining with mAb T311 showed a sensitivity of 94% for melanoma with a very high specificity for melanocytic cells. Immunopositivity (94% of melanomas overall) correlated inversely with clinical stage: clinical stage I and stage II showed 100%, stage III and stage IV 86% immunoreactivity each. Staining changed from an exclusively homogeneous pattern in early stages to a more heterogeneous pattern in later stages. Melanocytic control tissue like nevi of different subtypes all showed weak to moderate, homogeneous immunoreactivity with polarity towards the epidermis. RT‐PCR ELISA analysis of short‐term melanoma cell cultures displayed mRNA expression in only half of the originally immunopositive tumors only, suggesting rapid mRNA expression loss in culture. mAb T311 allows detection of melanoma‐associated tyrosinase protein expression and thus profiling of melanomas using routine archival tissue suited for immunotherapy approaches involving tyrosinase derived epitopes.

Melan A/MART-1 immunoreactivity in formalin-fixed paraffin-embedded primary and metastatic melanoma: frequency and distribution

Monoclonal antibody (MAb) A103 specifically detects Melan A/MART-1 protein expression. Melan A/MART-1 -derived peptides are recognized by CD8+ T-cells and are used in immunotherapy. We examined formalin-fixed paraffin-embedded tissue from 57 melanomas (34 primary, 23 metastatic) and 39 control cases (junctional, dermal, compound, Spitz, Reed and balloon-cell naevi) using the alkaline phosphatase and anti-alkaline phosphatase immunochemical method after antigen retrieval. Immunoreactivity was rated as low, medium or high, and staining pattern as homogeneous or heterogeneous. Staining with MAb A103 showed a sensitivity of 88% for melanoma, with a very high specificity for melanocytic cells. Immunopositivity decreased along with clinical stage, with stage I showing 100%, stage II 88%, stage III 90% and stage IV 75% immunoreactivity. Staining changed from an exclusively homogeneous pattern in the early clinical stages to a more heterogeneous pattern in the later stages. Melanocytic control tissues consisting of naevi of different subtypes all showed weak to moderate homogeneous immunoreactivity, with polarity towards the epidermis. Analysis of short-term melanoma cell cultures using reverse transcription-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) demonstrated mRNA expression in only one third of the originally immunopositive tumours, suggesting rapid mRNA expression loss in culture. MAb A103 allows the detection of melanoma-associated Melan A/MART-1 protein expression in routine archival tissue and thus enables the profiling of melanomas suited for immunotherapy approaches involving Melan A/MART-1 derived epitopes.

IL-12 directly up-regulates the expression of HLA class I, HLA class II and ICAM-1 on human melanoma cells: a mechanism for its antitumor activity?

IL‐12 enhances cytolytic activity and proliferation of NK and T cells, and induces cytokines such as IFN‐γ. No direct effects on non‐hematopoietic cells have been shown. This study investigates the effects of IL‐12 on melanoma cells in vitro. We analyzed 15 melanoma cell cultures and 1 melanoma cell line. Out of 16 samples 13 expressed the β chain of the IL‐12 receptor (IL‐12Rβ). Preincubation with IL‐12 increased the surface levels of human leukocyte antigen (HLA) class I, HLA class II and intercellular adhesion molecule (ICAM)‐1 of those cultures with IL‐12Rβ expression. The effects of IL‐12 on HLA class I could be blocked by an IL‐12‐neutralizing monoclonal antibody (mAb), but not by an mAb against IFN‐γ. Melanoma cells transduced with IL‐12 expressed enhanced levels of HLA class I, HLA class II and ICAM‐1 compared to controls. Co‐incubation of the melanoma cells with allogeneic peripheral blood mononuclear cells (PBMC) resulted in enhanced proliferation and increased production of IL‐2 and IFN‐γ after pretreatment with IL‐12. IL‐12 pretreatment increased the susceptibility of melanoma cells to lysis by prestimulated autologous PBMC. Since IL‐12 induced immunocritical surface molecules on melanoma cells, it might be beneficial during immune interventions in melanoma patients.

SEZARY SYNDROME, T-HELPER 2 CYTOKINES AND ACCESSORY FACTOR-1 (AF-1)

Mycosis fungoides and the Sézary Syndrome are characterized by clonal accumulation of well differentiated T-helper memory cells in the skin and, in the case of the Sézary Syndrome, also in the blood and lymph nodes. Well known immunological abnormalities in patients with MF and SS include reduced delayed type hypersensitivity, decreased proliferation upon stimulation with mitogens of the peripheral blood mononuclear cells, elevated IgE or IgA serum levels and eosinophilia. These abnormalities can be explained by the predominance of T-helper 2 cytokines. Clonal T-lymphocytes purified from the peripheral blood of SS patients transcribe mainly IL-10 and IL-5. They can be CD7 positive or negative. These clonal T cells express the accessory factor-1 (AF-1) or Interferon gamma receptor beta-chain that is essential for Interferon gamma signalling. These results imply perspectives for new therapeutical approaches, such as IL-12 or chimeric fusion proteins.

Loss of single HLA Class I allospecificities in melanoma cells due to selective genomic abbreviations

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8+ T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA‐A2 peptides. To identify the reasons underlying the loss of single HLA‐A allospecificities, we searched for genomic alterations at the locus for HLA Class I α‐chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA‐A2 gene sequences as determined by polymerase chain reaction‐sequence specific primers (PCR‐SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group‐specific primer‐mixes spanning the 5′‐flanking region of the HLA‐A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA‐A2‐restricted cytotoxic T‐cell clones were performed in HLA‐A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA‐A2 negative cells. We could document the occurrence of selective HLA‐A2 deficiencies in cultured advanced‐stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA‐A gene locus.

Impact of interferons on the expression of melanoma-associated antigens in melanoma short-term cell cultures.

Some immunotherapeutic approaches based on melanoma-associated antigens rely on in vitro cultivation of melanoma cells. A beneficial effect of interferons has been shown in melanoma. This study aimed to determine whether stimulation of patient-derived melanoma short-term cell cultures using interferon-α and -γ changes the expression pattern of melanoma-associated antigens. Lymph node, skin and brain metastases were cultivated for up to 3 weeks and treated with interferon-α, interferon-γ or mock stimulation. Expression of the melanoma-associated antigens MAGE-3, MelanA/MART-1 and tyrosinase was determined by flow cytometry and compared with the expression pattern of HLA class I molecules. We found consistently enhanced expression of HLA class I molecules, whereas the melanoma-associated antigens showed mixed responses, with moderate induction, suppression or no visible effect. The reaction to interferon stimulation was similar for all the antigens examined within a single melanoma cell culture. In contrast to the HLA class I molecules, which showed induced expression with interferon, the melanoma-associated antigens showed a varied response to interferon stimulation. Differential reaction to interferon stimulation is of importance to immunotherapeutic modalities and might influence progression of the disease. We therefore suggest that evaluation of variation in melanoma-associated antigen expression in the clinical setting may help to identify patients who would profit from adjuvant interferon therapy.

Inverse regulation of neuronal cellular adhesion molecule (NCAM) by IFN-γ in melanoma cell cultures established from CNS lesions

In advanced stages of malignant melanoma (MM), metastases to the CNS are frequently observed. Few results are available on trophic factors and immunological features involved in the process of invasion and adhesion of circulating metastatic cells into the CNS. A direct comparison of remote metastases found in different locations of the same patient might help to identify such properties. For this purpose, we screened a panel of MM cell cultures, which had been established from patients with surgically removed MM lesions of the CNS, for expression and regulation of immunorelevant molecules. The results were compared with standard controls and cultures established from non‐CNS metastatic lesions of the same patients. No significant differences were observed for expression of HLA‐I, HLA‐II, ICAM‐1 and the melanoma‐associated antigens Mage‐3, MelanA and tyrosinase. Constitutive expression of the neuronal cell adhesion molecule (NCAM) was found in all CNS‐derived samples and in fewer than 50% of non‐CNS derived cultures. IFN‐γ was found to have a weak up‐regulating effect in all non‐CNS‐derived cultures, except normal melanocytes. However, in 6/7 CNS‐derived cultures, pre‐treatment with IFN‐γ reduced expression of NCAM to 28% to 77% of the level in untreated cultures. The presence and regulation of NCAM differs between MM cells derived from CNS metastases and non‐CNS‐derived melanocytic cells. Thus, NCAM might be a candidate immunoregulating molecule involved in the formation of CNS metastases of MM. Int. J. Cancer 83:135–140, 1999.

Upregulation of interleukin-12 receptor on peripheral blood mononuclear cells and HLA class I, HLA class II or ICAM-1 on melanoma cells by B7.1 and interleukin-12: a mechanism for immunostimulatory impact of melanoma cells adenovirally transfected with B7.1 and IL12?

Melanoma is an immunogenic tumour and may express both HLA class I and class II molecules. These can be recognized by cytotoxic T-cells. Melanoma cells can evade immunosurveillance due to the lack of co-stimulatory molecules such as B7.1 or B7.2. Interleukin-12 (IL12) exerts antitumour effects, and B7.1 and IL12 synergistically induce effective antitumour immunity. We investigated the immunostimulatory potential of melanoma cells adenovirally transduced with B7.1, IL12 or B7.1 plus IL12. We observed that: (i) melanoma cells transduced with B7.1 plus IL12 can elicit a strong proliferative response from peripheral blood mononuclear cells (PBMCs); (ii) a high level of TH1 cytokine production from PBMCs was induced by melanoma cells transduced with Adv-B7.1 plus Adv-IL12; (iii) the expression of HLA class I antigens, HLA class II antigens or ICAM-1 antigens was higher on melanoma cells transduced with Adv-IL12 or Adv-B7.1 plus IL12 than those transduced with Adv-LacZ or wild-type melanoma cells; and (iv) the expression of IL12 receptors on PBMCs was upregulated by melanoma cells transfected with Adv-IL12 or Adv-B7.1 plus IL12. Thus, melanoma cells transduced with both Adv-IL12 and B7.1 may represent another clinical approach for antimelanoma gene therapy.

Interleukin-10 Inhibits the Immune Stimulatory Potential of Melanoma Cells Retrovirally Transduced with Human B7.1 or B7.2

Melanoma is an antigenic tumor [1]. Several proteins have been identified that are presented as peptides in the grove of the HLA-I complex. These proteins include members of the MAGE family [2], gp 100 [3] or MelanA [4]. Melanoma presents various specific antigens associated with HLA-I but it does not induce an immune response resulting in elimination of malignant cells. It is speculated that melanoma cells induce tolerance instead of activation by presenting antigens without the respective costimulatory signals [5]. These costimulatory signals can be provided by B7.1 (CD80) or B7.2 (CD86), two molecules signalling to T cells via CD 28 [5–7]. The immunogenicity of tumor cells is increased if the cells are transfected with human B7.1 [8]. Expression of B7.1 on melanoma cells has been shown to induce rejection of murine melanoma in vivo [9]. Treatment of mice bearing an eight-day established melanoma by intraperitoneal injection of B7+ tumor cells resulted in complete tumor regression [10]. In human melanomas, B7.1 expression was found only in regressing lesions [11]. There are human gene therapy protocols for melanoma that apply melanoma cells genetically engineered to express B7.1 [12].