Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.

Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

Curving out a new path: CD28/B7 cis interactions.

Important signaling events at the immunological synapse have increasingly been linked to cis interactions between receptors on T cells. In this issue of Immunity, Zhao et al.1 implicate cis CD28/B7 interactions facilitated by curved membrane invaginations in boosting tumor immunity.

Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).

Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.

The kinase occupancy of T cell coreceptors reconsidered.

The sensitivity of the αß T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αß, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αß/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to also show that stoichiometric coupling to Lck required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 outcompeted CD8αß for Lck. In T cells, TCR signaling induced CD4/Lck oligomerization but did not affect the high levels of CD4/Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.

Coreceptors and TCR Signaling - the Strong and the Weak of It.

The T-cell coreceptors CD4 and CD8 have well-characterized and essential roles in thymic development, but how they contribute to immune responses in the periphery is unclear. Coreceptors strengthen T-cell responses by many orders of magnitude - beyond a million-fold according to some estimates - but the mechanisms underlying these effects are still debated. T-cell receptor (TCR) triggering is initiated by the binding of the TCR to peptide-loaded major histocompatibility complex (pMHC) molecules on the surfaces of other cells. CD4 and CD8 are the only T-cell proteins that bind to the same pMHC ligand as the TCR, and can directly associate with the TCR-phosphorylating kinase Lck. At least three mechanisms have been proposed to explain how coreceptors so profoundly amplify TCR signaling: (1) the Lck recruitment model and (2) the pseudodimer model, both invoked to explain receptor triggering per se, and (3) two-step coreceptor recruitment to partially triggered TCRs leading to signal amplification. More recently it has been suggested that, in addition to initiating or augmenting TCR signaling, coreceptors effect antigen discrimination. But how can any of this be reconciled with TCR signaling occurring in the absence of CD4 or CD8, and with their interactions with pMHC being among the weakest specific protein-protein interactions ever described? Here, we review each theory of coreceptor function in light of the latest structural, biochemical, and functional data. We conclude that the oldest ideas are probably still the best, i.e., that their weak binding to MHC proteins and efficient association with Lck allow coreceptors to amplify weak incipient triggering of the TCR, without comprising TCR specificity.

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur when these parasites replicate in human blood. Despite the risk of immune detection, the parasite delivers proteins that bind to host receptors on the cell surfaces of infected erythrocytes. In the causative parasite of the most deadly form of malaria in humans, Plasmodium falciparum, RIFINs form the largest family of surface proteins displayed by erythrocytes1. Some RIFINs can bind to inhibitory immune receptors, and these RIFINs act as targets for unusual antibodies that contain a LAIR1 ectodomain2-4 or as ligands for LILRB15. RIFINs stimulate the activation of and signalling by LILRB15, which could potentially lead to the dampening of human immune responses. Here, to understand how RIFINs activate LILRB1-mediated signalling, we determine the structure of a RIFIN bound to LILRB1. We show that this RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single mutation in the RIFIN disrupts the complex, blocks LILRB1 binding of all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics the activation of natural killer (NK) cells by antibody-dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the immunological synapse of NK cells and reduce the activation of NK cells, as measured by the mobilization of perforin. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress the function of NK cells.