WP 12. Technologies – cell sorting, gene expression and cytometric profiling

This area will be lead by Partner 3 (Berlin) with input from Partner 1 (KI), Partner 2 (Leiden), Partner 4 (Vienna), Partner 6 (Amsterdam), Partner 8 (Nijmegen), Partner 10 (Zurich), Partner 11 (Lund), Partner 15 (Genmab), Partner 19 (Warsaw), Partner 24 (Biovitrum) and Partner 26 (Oligene)

Objective

To develop, standardise and provide cell sorting strategies for functional and gene expression studies on defined cell populations from autoimmune patients

Workplan

The cytometry and cell sorting platforms will combine the complementing expertise of existing cytometric laboratories within the consortium:

•  Berlin-DRFZ (Partner 3): high-sensitivity cytometry and high-throughput sorting of human leukocytes, combinatorial cell sorting (MACS and FACS), functional cytometry and development of new cell sorting applications. The focus will be on functional subpopulations of T and B lymphocyte populations, e.g. memory, naïve, antigen-specific, or subpopulations secreting certain cytokines. Gene expression profiling facilities are also established and analyses will be performed together with Partner 3 (Berlin-Charité) and WP 14.

•  Leiden University Medical Center (Partner 2) applies high-sensitivity and high-throughput cytometry for separation of defined cell populations. This group will focus on cell sorting procedures for subpopulations of monocytes and macrophages from blood and tissues. Moreover detailed analysis of gene expression in isolated cell populations will be performed to better define subpopulations of macrophages.

•  University of Amsterdam , Academic Medical Center , (Partner 6) has specialised on the isolation and characterisation of fibroblast-like synoviocytes (FLS). One of the goals will be to establish defined purification protocols based on immunolabelling and to define a useful marker for these cells using proteomics approaches and immunisation of mice with arthritis FLS. Candidate genes can also be used for further characterisation of subpopulations of these cells and for the generation of tissue-specific knock-out mice for functional gene analysis.

•  Nijmegen University , Research Laboratory Rheumatology (Partner 8) will concentrate on isolation of monocytes using FACS technologies and establish defined culture systems to generate macrophages or mature and immature dendritic cells.

•  As a second major issue, this group will contribute laser dissection technologies to isolate synovial lining cells (only layers of 1–3 cells) from synovial tissue samples, using frozen sections. The final goal will be to use this technology for the isolation of defined macrophage and DC populations directly from tissues under conditions which prevent shifts in gene expression. Gene expression analysis can then be done after controlled amplification.

•  University of Zürich , Department of Rheumatology (Partner 10) has a focus on the isolation and functional characterisation of synovial fibroblasts. They will concentrate on the separation of these cells and their use for functional analysis, e.g. in vitro analysis of the interaction of synoviocytes with purified B/T cells and the SCID-transplantation model. Functional analyses on purified cell populations will be performed by using anti-sense technologies and si-RNA for specific gene silencing in functional assays. Gene array techniques are also available.

•  Karolinska Institutet, Stockholm (Partner 1). The Centre for Molecular Medicine in collaboration with the Department of Biotechnology at the Royal College of Technology in Stockholm, will focus on three objectives: (i) to develop optimal ways for handling of very small biopsy materials from synovial tissue and muscle in order to optimise expression array studies; this will be done in close collaboration with the group of Prof Joakim Lundeberg (Royal College of Technology) who is developing several new techniques for expression analysis in extremely small specimens. (ii) Proteomics analysis using both MALDI-ToF and CELLDI-ToF equipment and by means of customised antibody chips which are currently under development within the group of Prof Mathias Uhlén and Ass Prof Jacob Odeberg at the Royal College of Technology; (iii) Use of laser-capture technologies to dissect tissues and cell for further use with the ultra sensitive methods developed within the Royal College of Technology and in other parts of the consortium.

•  Partner 19 (Warsaw) has specialized on the isolation, culture and characterization of cells participating in chronic inflammatory responses (fibroblast-like synoviocytes-FLS, monocytes/macrophages, T and B cells) in synovial tissue, peripheral blood and bone marrow. Gene profile of purified cell subpopulations from these sources will be compared in collaboration with Partner 3 (Charité-DRFZ) and/or Partner 10( Zurich ).

Expected achievements at 18 months

•  Generation of standardised expression profiles of highly purified cell populations relevant for AUTOCURE (from blood and tissues).

•  determination of interindividual variance in gene expression between defined cell populations of normal healthy individuals.

•  standardised separation protocols and quality controls for cell populations from blood and tissue.

•  assistance for establishing sort protocols for cell populations relevant for AUTOCURE members.

•  one core facility for cell separations for AUTOCURE projects, which require specialised equipment or experience will be established.

•  an organisation will be established among the centres to provide expertise and technical assistance to improve and to establish new techniques for separation of certain cell populations.