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Gene Expression Profiling of Tumor-Induced Myeloid Suppressor Cells

Posted on: Thursday, 17 June 2004, 06:00 CDT

Novel cancer vaccines based on tumor-associated antigens are currently tested in tumor-bearing patients, but the first clinical trials have questioned the possibility of inducing an immune response powerful enough to eradicate the host tumor. The observation that both experimental and human cancers are able to elude the immune defences they themselves elicit represents one of the unsolved enigmas of tumor immunology that advice a careful reconsideration of host-tumor interaction. Tumors are known to use various strategies to escape immune attack, either through a direct effect of molecules produced by the tumor itself, or by enlisting myeloid cells that interfere with the functions of tumor-reactive lymphocytes. Mouse tumors, for example, release cytokines acting on bone marrow precursors (e.g. GM-CSF, VEGF), which cause the accumulation of CD11b^sup +^/Gr-l^sup +^ myeloid suppressor cells (MSC) in secondary lymphoid organs and tumor masses. Elimination of MSC, either in vitro or in vivo, was shown to reverse completely the suppression of CD8^sup +^ T cell responses in tumor-bearing mice. These findings suggest that abrogation of MSC activity in vivo may represent a critical approach to the immunotherapy of cancer. To investigate deeper the nature of these suppressor cells and the molecular mechanisms used to inhibit T lymphocyte activity, CD11b^sup +^/Gr-l^sup +^ cells isolated from the spleens of tumor- bearing mice were immortalized and cloned. The resulting cloned MSC lines express monocyte/macrophage markers, but differ from previously characterized macrophage lines in their ability to inhibit T lymphocyte activation and trigger their apoptosis. The MSC lines represented an important investigative tool since they were a stable source of genes and molecules. A genome-wide profiling approach was undertaken by means of Affymetrix technology to identify molecules relevant to the physiology of MSC, as well as biological markers to detect/isolate them in vivo, and factors responsible for the suppression of T lymphocyte function. Expression profiles derived from cloned MSC lines were then compared with those obtained from fresh MSC isolated by magnetic micro-beads conjugated with CD11b monoclonal antibodies (mAb) from the spleen and tumour of C26-GM tumor-bearing mice and from the spleen of healthy mice. Transcriptome analyses indicated that CD11b^sup +^ cells enriched from spleen of tumour-bearing mice had a peculiar granulocyte/ inflammatory signature. The same cells cultured in standard medium for 24 hours were found to exhibit gene signatures associated with spontaneous production of both IFN-Yand IL-4. Among the genes spontaneously upregulated during the 24 hour cultures, two enzymes, nitric oxide synthase 2 and arginase 1 were asspciated with the immunosuppressive activity of MSC. Since these genes are considered antithetic markers of classical (M1) versus alternative (M2) activation of myeloid cells, MSCs appear to possess properties that can be ascribed to both M1- and M2-type macrophages. To avoid the possible cell perturbing effects of anti-CD11b mAb treatment, MSCs were also enriched from spleen of healthy and tumourbearing mice by gradient fractionation. Four distinct Tractions were collected. Alt the fractions from tumour-bearing but none of those derived from healthy mice showed the ability to release both INF-[gamma] and IL- 4 in ELISA and ELISPOT assay. In addition to define a peculiar phenotype of myeloid cells, these results open novel possibilities to alleviate the immune dysfunctions induced by MSC in tumor- hearing hosts by administration of novel drugs affecting the L- arginine metabolism.

L. Dolcetti1, G. Gallina1, C. De Santo1, P. Seraflni1,1. Marigo1, S. Cingarlini1, S. Bicciato2, A. Luchini2, M. Mattioli3, A. Neri3, V. Bronte1, P. Zanovello1

1 Department of Oncology and Surgical Sciences, Padua University, Padua, Italy; 2 Department of Chemical Process Engineering, Padua University, Padua, Italy; 3 Laboratory of Experimental Hematology and Molecular Genetics, Ospedale Policlinico, IRCCS, University of Milan, School of Medicine, Milan, Italy

Copyright Edizioni Minerva Medica Mar 2004

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