WP 15. Technologies - cell biology for evaluation of targeted therapy

This area of work will be lead by Partner 7 (London) with input from Partner 1 (KI), Partner 2 (Leiden), Partner 3 (Berlin-Charité), Partner 4 (Vienna), Partner 9 (Montpellier), Partner 14 (Manchester), Partner 23 (Future Partner), Partner 24 (Biovitrum), Partner 25 (BMD), Partner 26 (Oligene) and Partner 27 (Arthrogen)

Detailed plan of investigation

1. Analysis of peripheral blood and synovial joint effector T cells ex vivo

We will initially study PBLs from well characterized patients with established, chronic, severe RA to define precisely the molecular fingerprint of chronically activated T cells in vivo. Samples will be obtained from our inflammatory arthritis clinic, but also from cohorts defined in WP3. This will include PBLs acquired both before and at disease onset, as well as samples obtained pre- and post therapy. Initial experiments will focus on comparing genotype-phenotype correlates of TCR z bright and TCR z dim T cell subsets from within PBL or synovial cell populations. Cell sorting will be used to obtain at high purity the desired cell populations for subsequent analysis (in cooperation with WP 12 – cytometry and cell sorting). Phenotyping will be undertaken by flow cytometry, proliferation assays, cytokine expression by intracellular staining, and assays of effector function including cell contact dependent effector responses and B cell help. Gene expression profiling will be undertaken on purified populations using Affymetrix gene chips (through Imperial College core genomics facility and in association with Partners 3 and 16) and data analysis and management coordinated through WP14.

2. Analysis of human and murine effector T cells generated in vitro

Stimulation of fresh PBL with cocktails of cytokines, including IL-2, IL-6 and TNF, or IL-15, generate a phenotype of effector cell which resembles RA synovial T cells in terms of the capacity to activate monocytes to produce proinflammatory cytokines in a cell contact dependent fashion. The cells generated after cytokine stimulation (T ck ) which includes CD4 + T cells, but also NK cells, will be used for analysis as above. Using cell surface biotinylation and affinity capture, protein mass spectrometric analysis will be used to compare the cell surface protein fingerprints of subsets of effector cells.

3 . DC mediated immunotherapy

To induce T cell tolerance we will use DC pulsed with a panel of human autoantigens defined in WP 13, in which the NF- k B pathways have been attenuated using dominant negative mutants of NIK and IKK2, or using siRNA technology. This approach will aim to selectively inhibit maturation of DC (by targeting the NF- k B/RelB and CD40 pathways). These experiments will form the basis of approaches to modify T cell responses in vivo in animal models of arthritis (WP 9).