T cell stimulator cells expressing find more membrane-bound anti-CD3 antibodies at a high density induced moderate proliferation in human T cells even in the absence of human costimulatory molecules and as expected T cells activated with stimulator cells harbouring high levels of anti-CD3 in combination with human CD80 showed the highest proliferative response (Fig. 1C). To visualize the interaction of human T cells and stimulator cells, we performed co-culture experiments using CFSE-labeled T cells and CMTMR-labeled stimulator cells. Large clusters of T cells and stimulator cells expressing

CD80 can be observed whereas much smaller clusters are formed when T cells were activated by stimulator cells expressing anti-CD3 but no human costimulatory molecule

(Fig. 1D). T cell stimulator cells transduced to express different costimulatory molecules are excellent tools to compare these ligands regarding Epigenetics inhibitor their capacity to activate human T cells. We have generated stimulator cell lines retrovirally expressing different costimulatory molecules at high levels (Fig. 2). The resultant cell lines were used to stimulate purified T cells isolated from different healthy donors and T cell proliferation was assessed. As shown in Fig. 2B stimulation of human T cells in the presence of the costimulatory molecules used in this study (CD80, ICOSL, CD58, CD54 and 4-1BBL) significantly enhanced T cell proliferation compared to T cells co-cultured with stimulator cells expressing no human costimulatory molecule. Furthermore, our data show that CD80 was

the strongest costimulatory ligand tested in these experiments and demonstrate that among the other molecules analyzed CD58 is the most potent inducer of T cell proliferation. There is an increasing number of immunosuppressive and immunomodulatory drugs for treatment of patients suffering from autoimmune diseases and recipients of hematopoietic stem cells or solid organs. Many of these drugs target fast dividing cells whereas others specifically suppress T cells or counteract inflammatory processes. Antibodies or receptor fusion proteins that block the cytokine TNF-α are successfully used in patients suffering from psoriasis, rheumatoid Methisazone arthritis and various other autoimmune diseases (Aringer and Smolen, 2008, Bosani et al., 2009 and Taylor and Feldmann, 2009). TNF-α is a pleiotrophic cytokine and the beneficial effects of TNF-α blockade are mainly ascribed to its capacity to prevent and down-modulate proinflammatory processes. Whereas other members of the TNF-family have been shown to act as potent costimulatory molecules, few studies have addressed the ability of TNF-α to directly contribute to T cell activation processes. We found that expressing TNF-α on T cell stimulator cells enhances their ability to induce proliferation in purified human T cells (Fig. 3A).