Costimulatory receptors belong to different molecule-families and consequently they can induce signaling events that are distinct from the pathways induced by CD28 ligation. CD28, CD2, LFA-1, ICOS or 4-1BB. Mouse monoclonal to CRTC3 The mean inhibitory concentrations (IC50) for Aza and CsA were LY3039478 determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC50 for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation. 1.?Introduction T cells have important roles in allograft rejection, graft versus host diseases and autoimmune pathologies. In most cases the clinical management of these conditions requires the extensive use of immunosuppressive agents to control aberrant T cell responses. Such drugs limit and down-modulate T cell activation by targeting different cellular processes. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like Cyclosporine A (CsA) more specifically target T cells by interfering with signaling pathways in these cells. Since many different signals can contribute to T cell activation processes, the interplay between such signals and immunosuppressive agents might have differential effects on the outcome of T cell responses. Especially costimulatory signals generated by interaction of antigen presenting cells (APC)-expressed ligands with their T cell-expressed receptors have a crucial role in the efficient activation of T cells that recognize antigen. The interaction of CD80/CD86 with CD28 is generally regarded as the primary T cell costimulatory pathway [1]. However, there are many alternative costimulatory ligand-receptor pairs that potently enhance the proliferation, differentiation and cytokine production of T cells that recognize antigens [2C4]. Among these the CD58 C CD2, 4-1BBL C 4-1BB, ICOS-L C ICOS and CD54 C LFA-1 (CD11a/CD18) pathways are well documented to generate strong and consistent costimulatory effects in human T cells [2,5]. Costimulatory receptors belong to different molecule-families and consequently they can induce signaling events that are distinct from the pathways induced by CD28 ligation. Previous studies have shown that engagement of the CD28 costimulatory pathway greatly reduces the sensitivity of T cells to the immunosuppressive effect of CsA [6,7]. By contrast, it is not known whether triggering alternative costimulatory receptors has similar effects. Furthermore, currently there is limited knowledge how different costimulatory signals affect the immunosuppressive effects of other drugs in clinical use. We have previously developed a cellular system termed T cell stimulator cells that allows analyzing the effect of different costimulatory signals on human T cells [5,8,9]. This system is based on cell lines engineered to express membrane-bound anti-human-CD3 antibody-fragments that trigger the TCR-complex on human T cells upon co-culture. By expressing high levels of human costimulatory ligands of interest on the T cell stimulator cells it is possible to analyze and compare human T cells that receive distinct costimulatory signals. In this study we used T cell stimulator lines expressing CD80, CD58, 4-1BBL, ICOS-L. CD54 and T cell stimulator lines expressing anti-CD3 antibody-fragments but no costimulatory molecules to activate T cells purified from healthy individuals. Using this system we determined the mean inhibitory concentrations (IC50) for CsA and Aza LY3039478 for the proliferation of human T cells receiving different costimulatory signals. 2.?Material and methods 2.1. Antibodies, cell culture and FACS staining 293T cells and the mouse thymoma cell line Bw5147 (short designation within this work Bw) were cultured as described [9]. The ethical review board of the General Hospital and the Medical University of Vienna approved the human studies performed within this work and informed consent was obtained from the donors. PBMC were isolated from heparinised whole blood of healthy volunteer donors by standard density centrifugation with Ficoll-Paque (Amersham Bioscience, Roosendaal, Netherlands). Untouched human T cells were obtained through depletion of CD11b, CD14, CD16, CD19, CD33 and MHC-class II bearing cells with LY3039478 the respective mAbs by MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). Untouched CD8+ and CD4+ T cells were isolated from human T cells using MACS in conjunction with antibodies to CD8 or CD4. The mAbs to CD4 (VIT4), CD8 (VIT8), CD11b (VIM12), CD14 (VIM13), CD33 (4D3), MHC-class II (1/47), CD80 (7-480), CD58 (1-456) and CD54 (5-216) were produced at our Institute. The mAb to CD14 (MEM-18) was purchased from An der Grub (Kaumberg, Austria), CD19 mAb (BU12) from Ancell (Bayport, MN), 4-1BBL from Biolegend (San Diego, CA) and ICOS-L (2D3/B7H2) from.