A cellular platform for the evaluation of immune checkpoint molecules

// Sabrina Jutz 1,* , Annika Hennig 1,* , Wolfgang Paster 1 , Omer Asrak 1 , Dejana Dijanovic 1 , Florian Kellner 2 , Winfried F. Pickl 3 , Johannes B. Huppa 2 , Judith Leitner 1 and Peter Steinberger 1 1 Division of Immune Receptors and T cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 2 Department of Molecular Immunology, Immune Recognition Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 3 Division of Cellular Immunology and Immunohematology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria * These authors have contributed equally to this manuscript and share first authorship Correspondence to: Peter Steinberger, email: // Keywords : T cell activation, NF-κB, reporter cell lines, Jurkat, immune checkpoints Received : November 14, 2016 Accepted : April 22, 2017 Published : May 04, 2017 Abstract Blockade of the T cell coinhibitory molecules CTLA-4 and PD-1 has clinical utility to strengthen T cell responses. In addition to these immune checkpoints an ever-growing number of molecules has been implicated in generating coinhibitory signals in T cells. However, investigating coinhibitory molecules in primary human cells is complicated by the restricted expression and promiscuity of both coinhibitory receptors and their ligands. Here we have evaluated the potential of fluorescence-based transcriptional reporters based on the human Jurkat T cell line in conjunction with engineered T cell stimulator cell lines for investigating coinhibitory pathways. CTLA-4, PD-1, TIGIT, BTLA and 2B4 expressing reporter cells were generated and activated with T cell stimulator cells expressing cognate ligands of these molecules. 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 transcriptional 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.