Data was analyzed with FlowJo (version 10

Data was analyzed with FlowJo (version 10.0.6, Tree Star, Ashland, OR) and Graphpad Prism (version 5, GraphPad Software, Inc., La Jolla, CA). Generation of cell lines T cell stimulator cells (short designation: TCS) were generated as described previously [54]. All accessory molecules tested were functional in our reporter system. Engagement of CTLA-4, PD-1, BTLA and TIGIT by their ligands significantly inhibited T cell activation, whereas binding of 2B4 by CD48 resulted in enhanced responses. Mutational analysis revealed intracellular motifs that are responsible for BTLA mediated T cell inhibition and demonstrates potent reporter inhibition by CTLA-4 independent of cytoplasmic signaling motifs. Moreover, considerably higher IC50 values were measured for the CTLA-4 blocker Ipilimumab compared to the PD-1 antibody Nivolumab. Our findings show that coinhibitory pathways can be evaluated in Jurkat-based 6-Quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)- transcriptional Rabbit Polyclonal to CRABP2 reporters and yield novel insights on their function. Results obtained from this robust reductionist system can complement more time consuming and complex studies of such pathways in primary T cells. assessment of therapeutics targeting immune checkpoints. However, some of the constraints described for the use of primary human cells also apply to mouse models, and moreover findings in murine model systems might not always accurately reflect the function of these molecules in human cells. Studies on transformed T cell lines have given valuable insights into signal transduction processes ensuing engagement of the TCR complex and costimulatory receptors [12-18]. The use of such T cell lines for studying coinhibitory pathways has a large potential to overcome impediments associated with primary human T cells. In particular numerous important aspects relating to human coinhibitory pathways become directly accessible to experimentation. Employing a robust T cell system will not only give rise to reproducible data but will also provide molecular and mechanistic insights into immune checkpoints. Results obtained in such a rather reductionist system are bound to complement observations made in primary human cells and pre-clinical animal models. Furthermore and most importantly, they could not only serve as a guiding principle for more intricate and time-consuming studies but may be easily implemented into a high throughput data platform to screen for agonists or antagonists to immune checkpoints. Here we have engineered fluorescence-based transcriptional reporters based on the human Jurkat T cell line to express CTLA-4, PD-1, BTLA, 2B4 or TIGIT. T cell stimulator cells expressing the respective ligands for these molecules were used to specifically and physiologically trigger these receptors during T cell receptor engagement. The results of this study demonstrate that our cell line-based platform is a powerful and versatile tool to investigate T cell coinhibitory pathways and reveal novel insight into the function of immune checkpoints. RESULTS Use of a transcriptional reporter T cell line for the assessment of PD-1 mediated coinhibition The human T cell line Jurkat E6.1 was transduced to express a transcriptional NF- B::eGFP reporter and a clone exerting high sensitivity towards stimulation with PMA/Ionomycin and immobilized anti-CD3 was selected for further use (Figure ?(Figure1A).1A). PD-1 was expressed in these Jurkat reporter cells and a cell clone that had high and homogenous PD-1 expression was selected for further studies (Figure ?(Figure1B).1B). PD-1 expressing reporter cells and control reporters were stimulated in the presence of immobilized immunoglobulin fusion proteins representing the extracellular domains of PD-L1 (PDL1-Ig). PDL1-Ig potently inhibited PD-1 reporter activation in a dose-dependent 6-Quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)- manner (Figure 1C, 1D). In a next set of experiments, T cell stimulator cells (TCS) that coexpress membrane-bound anti-CD3-scFv and high levels of PD-L1 were generated to trigger PD-1 signaling (Figure ?(Figure1E).1E). Importantly, the availability of reporters lacking PD-1 and TCS expressing membrane-bound anti-CD3 single chain antibody fragment but not PD-L1 allow to assess the effects of PD-1-PD-L1 6-Quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)- interaction in a well-controlled system (Figure ?(Figure1F).1F). Fluorescence microscopy revealed strongly reduced reporter gene expression in PD-1 reporter cells compared to that observed in control reporter cells stimulated in presence of PD-L1. In contrast, stimulation with TCS expressing CD80 greatly enhanced eGFP expression in both reporter cell lines (Figure ?(Figure1G).1G). Flow cytometric 6-Quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)- analysis confirmed that PD-1 reporter activation was strongly inhibited by the presence of PD-L1 and furthermore demonstrated that this effect was 6-Quinoxalinecarboxylic acid, 2,3-bis(bromomethyl)- fully reverted in the.