Immunotherapy, harnessing the immune system to combat cancers, has yielded promising results in patient care. Monoclonal antibodies that block the “immune checkpoint” through CTLA-4, PD-1, or its ligand PD-L1 have demonstrated impressive clinical activities against a variety of tumors. Increasing number of immune checkpoints have been identified and clinically targeted. However, clinical benefit of immune checkpoint blockade therapy is limited in a small fraction of patients. Identifying patients that will likely respond and extending the therapies to a larger population both require a better understanding of immune checkpoints at the cell biological level.
The goal of Hui lab is to provide an in-depth mechanistic understanding of immune checkpoints. We are interested in both the intracellular signaling pathways and cell surface ligand-receptor interactions.
1. Regulation of Immune Checkpoints by Cis-interactions.
Receptors on T cells are triggered when they interact, in trans, with their respective ligands on the target cells. Using membrane reconstitution assays, we recently showed that in addition to trans-interactions, PD-1 and PD-L1 can interact in cis on the same cells, and the cis-PD-1:PD-L1 interaction on the antigen presenting cells (APCs) can block the their trans-interaction, thereby inhibits PD-1 signaling in T cells (Zhao et al. Cell Reports, 2018). This study indicates that APC-intrinsic PD-1 acts as a "decoy" to neutralize PD-L1 in cis. More recently, we found that PD-L1 also interacts in cis with CD80, a shared ligand of CTLA-4 and the costimulatory receptor CD28. Moreover, we showed that 1) cis-CD80 blocks PD-L1:PD-1 interaction, 2) cis-PD-L1 weakens CD80:CTLA-4 interaction to restrict CTLA-4 and Treg functions, 3) cis-PD-L1 does not affect CD80:CD28 interaction. We further showed that anti-PD-L1, but not anti-PD-1, depletes CD80 from tumor infiltrating APCs, and this effect can be negated by anti-CTLA-4 (Zhao et al. Immunity, 2019). This finding revealed a novel layer of crosstalk between PD-L1/PD-1 and CTLA-4 axes, with implications to the synergy observed in combination therapies. Besides the clinical implications, these two studies suggest another dimension of regulatory mechanism of immune checkpoints. Moving forward, we are identifying and characterizing other cis-interacting pairs at the immunological synapse.
2. Intracellular pathways of Immune Checkpoints.
Despite the clinical success of immune checkpoint inhibitors, very little is known regarding the intracellular mechanism by which each immune checkpoint receptors inhibit T cell function. Which molecules do they recruit? Which molecular targets do they inhibit? Recently, we investigated the intracellular mechanism of PD-1 and showed that PD-1 preferentially inhibits the costimulatory receptor CD28 over the T cell receptor (TCR). We demonstrated the CD28 preference in both a biochemical reconstitution system and a cell culture assay (Hui et al. Science, 2017). More recently, we found that while PD-1 recruits SHP2 to exert its inhibitory effect, a structurally related immune checkpoint receptor BTLA primarily employs SHP1 to inhibit T cell signaling (Xu et al., BioRxiv, 2019). We are currently studying the intracellular mechanisms of additional immune checkpoints as well as their biochemical and functional crosstalk using a multidisciplinary approach.
We combine in vivo models, cell cultures, proteomics and cell-free reconstitution assays. We probe the spatiotemporal dynamics of immune checkpoint signaling using fluorescence and microscopy assays.
1. Reconstitution of intracellular signaling: We directly and quantitatively study T cell signaling reactions using membrane reconstitution assays, in which purified proteins are reconstituted to synthetic lipid bilayers. In membrane reaction kinetics and net output of signaling modules, can be probed by fluorescence and microscopy readouts.
2. Reconstitution of cis versus trans-interactions: We developed membrane reconstitution assays to detect and characterize either cis or trans-interactions among T cell surface proteins.
3. Live Cell : bilayer hybrid: We visualize the spatiotemporal dynamics of immune checkpoints using live T-cell : bilayer hybrids, in which the APCs are replaced with a ligands-containing supported lipid bilayer.
4. Proteomic analysis of immune checkpoint interactomes. We discover new interactors of immune checkpoints using quantitative proteomics.
5. T : APC co-cultures & animal models: We cross-check our findings in conventional T cell signaling assays in both ex vivo cultures and in vivo models.