A novel two-ligand system for investigation of signal integration by non-catalytic tyrosine-phosphorylated receptors

Appropriate immune responses require integration of a variety of activating, coinhibitory, and co-stimulatory signals from cell surface receptors. One important class of immune receptors are the non-catalytic tyrosine-phosphorylated receptors (NTRs), a group of over 100 receptors, such as PD-1, CTLA-4, CD28, and the T and B cell antigen receptors. These receptors have a number of shared features that we propose have a functional significance, such as relatively small receptor-ligand dimensions and binding of surface-associated ligands. The mechanism through which NTR-ligand engagement leads to signal initiation and integration is not well understood and may offer novel targets for emerging immunotherapies, including checkpoint blockade therapies and adoptive cell transfer approaches such as the use of chimeric antigen receptors.
We have developed a generic cellular two-ligand system to investigate signal integration between NTRs by manipulating their extra- and intra-cellular dimensions. We hypothesise that close co-localisation of receptors is important for optimal signal integration and that this requires compatible receptor-ligand dimensions. We also hypothesise that intracellular tail length influences the effectiveness of inhibitory NTRs by determining the “reach” of the tail and any effector molecules recruited to it.
To manipulate the dimensions of receptor-ligand interactions, CHO.K1 cells ("ligand cells") were transfected with SpyTag constructs of different lengths and either CD48- or HLA-A2 single chain dimer-based constructs. SpyTag can be covalently coupled via SpyCatcher to StrepTactin, which binds to NTRs tagged with the short peptide tag Strep-tag II, such as the co-inhibitory receptor PD-1 studied here. Ligand cells expressing HLA-A2 SCD presenting cognate peptide or expressing CD48 can be used to provide activating signals through binding of the 1G4 TCR and a 2B4-CD64 fusion protein respectively, in THP-1 and Jurkat "receptor cells".
CD48-based and SCD-based ligand cells were able to activate IL-8 production orupregulation of NF-{deletd} kB-eGFP gMFI by THP-1 receptor cells expressing 2B4-CD64 and Jurkat receptor cells expressing the 1G4 TCR, respectively. This activation of THP-1 and Jurkat receptor cells was partially inhibited by engagement of Strep-tag II-tagged PD-1 with the StrepTactin-based ligand coupled to CHO.K1 ligand cells, demonstrating PD-1-mediated inhibition in the two-ligand system.
Experiments testing the study hypotheses have provided promising preliminary results, including that mismatching of receptor-ligand dimensions partially abrogates PD-1-mediated signalling and that the inhibitory function of PD-1 variants of different lengths is dependent on the presence of an immunoreceptor tyrosine-based switch motif (ITSM). Truncation of the PD-1 intracellular tail abolished PD-1-mediated inhibition, even in the presence of an ITSM. There was no evidence that elongation of the intracellular tail affected PD-1 inhibitory function using preliminary experimental conditions, although this result needs to be confirmed using alternative conditions such as lower ligand density.
These experiments have provided several insights into areas for further development of the two-ligand system. Optimisation of some aspects of the system, such as improving coupling of the soluble StrepTactin-based ligand, increasing the StrepTactin-Strep-tag II affinity, and incorporating co-stimulatory signalling, should provide a suitable platform for studying signal integration between a wide range of NTRs of interest.

Advisor: van der Merwe, P
Goyette, J