A cellular platform for the evaluation of immune checkpoint molecules (1)
Immune checkpoints diminish desired T cell responses and blocking coinhibitory receptors like PD-1 or CTLA-4 was shown to be effective in cancer treatments. The considerable potential of T cell coinhibitory pathways in cancer immunotherapy has fuelled intense efforts to reveal the molecular modus operandi of T cell-expressed receptors like BTLA, TIGIT, CD96, TIM-3, 2B4 and LAG-3 which have all been implicated in negatively regulating T cell responses upon engagement of their cognate ligands. The use of primary APCs, like dendritic cells (DC), for studying T cell inhibitory pathways is often hampered by low ligand expression and consequently insufficient receptor engagement. Fluorescence-based transcriptional reporters based on the human Jurkat T cell line to express CTLA-4, PD-1, BTLA, 2B4 or TIGIT. Reporter cells (5×10^4 cells/well) were cocultivated with T cell stimulator cells (TCS) (1-1.5×10^4 cells/well) for 24 hours for testing.
The human T cell line Jurkat E6.1 was transduced to express a transcriptional NF- κB::eGFP reporter and a clone exerting high sensitivity towards stimulation with PMA/Ionomycin and immobilized anti-CD3 was selected for further use. The availability of reporters lacking PD-1 and TCS expressing membrane-bound anti-CD3 single chain antibody fragments but not PD-L1 allow to assess the effects of PD-1-PD-L1 interaction in a well-controlled system.
The costimulatory CD226 (DNAM-1) and the coinhibitory receptor TIGIT (WUCAM) bear similarities with CD28 and CTLA-4 and they also share two ligands, CD112 (Poliovirus receptor-related 2; PVRL2 also known as nectin-2) and CD155 (Poliovirus receptor, PVR). The Jurkat cells endogenously express moderate levels of CD226. The low binding signal obtained upon analysis of CD113 expression is likely due to a weak reactivity of the antibody employed. CD113 and CD155 are both functional ligands for the inhibitory TIGIT.
The presence of CD48 led to a significantly higher stimulation of CD244 expressing reporter cells, demonstrating a costimulatory effect of the CD244 – CD48 axis in our reporter system.
In a set of TCS expressing either murine PD-L1 or HVEM, as well as NF-κB::eGFP reporter cells expressing murine PD-1 or murine BTLA, inhibition of NF-κB activation upon engagement of murine PD-1 or BTLA by their respective ligand, indicating the human Jurkat T cell line has utility for assessing murine immune checkpoint molecules.
Reporters expressing PD-1 with TCS-PD-L1 in the presence of different concentrations of a commercial PD-1 antibody (EH12.2H7) and the therapeutic Nivolumab also targeting PD-1 were stimulated, similarly, the reporter cells expressing full length CTLA-4 and CTLA-4 Δcyt in presence of the therapeutic anti CTLA-4 antibody Ipilimumab were stimulated by TCS-CD80. IC50 calculations indicated a slightly higher efficacy of the therapeutic antibody Nivolumab compared to the commercial anti-PD-1 EH12.2H7, and IC50 values calculated for the CTLA-4 antibody Ipilimumab were considerably higher than those calculated for the PD-1 antibody Nivolumab, respectively.
Jurkat T cells are widely used for investigating T cell activation and signaling processes and numerous landmark studies are based on results generated in this cell line as a reductionist model for human T cells. However, expression of coinhibitory receptors is low or absent on Jurkat cells even after activation. Interestingly the extracellular domain of CTLA-4 is sufficient to potently suppress T cell activation in a ligand dependent manner. However, these results do not exclude that engagement of CTLA-4 by its natural ligands generates inhibitory signals in T cells.