Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

Human epidermal Langerhans cells lack functional mannose receptors and a fully developed endosomal/lysosomal compartment for loading of HLA class II molecules.

Langerhans cells (LC) represent the dendritic cell (DC) lineage in the epidermis. They capture and process antigens in the skin and subsequently migrate to the draining lymph nodes to activate naive T cells. Efficient uptake and processing of protein antigens by LC would, therefore, seem a prerequisite. We have now compared the capacity of human epidermal LC, blood-derived DC and peripheral blood mononuclear cells to endocytose and present (mannosylated) antigens to antigen-specific T cells. Moreover, we have determined the expression of mannose receptors, and the composition of the intracellular endosomal/lysosomal MHC class II-positive compartment. The results indicate that LC have poor endocytic capacity and do not exploit mannose receptor-mediated endocytosis pathways. Furthermore, the composition of the class II compartment in LC is distinct from that in other antigen-presenting cells and is characterized by the presence of relatively low levels of lysosomal markers. These results underscore the unique properties of LC and indicate that LC are relatively inefficient in antigen uptake, processing and presentation. This may serve to avoid hyper-responsiveness to harmless protein antigens that are likely to be frequently encountered in the skin due to (mechanical) skin damage.

Distribution of HLA class II molecules in epidermal Langerhans cells in situ.

We performed immunoelectron microscopic studies to investigate the expression of HLA class II molecules in Langerhans cells (LC). In the epidermis, LC expressed class II molecules on the plasma membrane of the dendrites, resulting in a class II positive reticulo-epithelial network, but not on the surface of the cell body. In contrast, isolated LC as well as activated LC in situ displayed an even and strong expression of class II molecules on their entire cell surface. Therefore, isolation and/or activation results in redistribution and up-regulation of class II molecules on the cell surface. In addition, double-labeling experiments were carried out with monoclonal antibodies to class II antigens and to LAMP-1, CD63 and alpha-glucosidase, specific markers for organelles of the endosomal/lysosomal system. The results show expression of class II molecules on intracellular, electron-dense vesicular structures, and co-localization of class II molecules and the markers for late endosomes and early lysosomes in human LC in situ. Expression of these markers was not found on Birbeck granules, an LC-specific organelle.

Cytokeratin patterns of tissues related to cholesteatoma pathogenesis.

Specimens of cholesteatoma matrix, meatal epidermis, and middle ear epithelium were removed during surgery, and immunohistochemical techniques were used to investigate cytokeratin expression. The use of five chain-specific anticytokeratin monoclonal antibodies and one broad specific anticytokeratin monoclonal antibody showed the divergent behavior of middle ear epithelium compared with the cytokeratin expression of the other two types of epithelium. Middle ear epithelium was characterized by the presence of cytokeratins 4, 8, 18, and 19, whereas in both cholesteatoma and meatal epidermis cytokeratin 10 predominated. Furthermore, cholesteatoma showed an infrequent focal presence of cytokeratins 4, 18, and 19. The similarity between cholesteatoma and meatal epidermis with respect to morphology, and the presence of cytokeratin 10 support an epidermal origin of cholesteatoma. However, a metaplastic origin cannot be excluded, because of the infrequent occurrence of a small amount of cytokeratins 4, 18, and 19 in cholesteatoma matrix that was not found in meatal epidermis but was a component of the cytokeratin pattern of middle ear epithelium.