Innate extracellular Hsp70 inflammatory properties are mediated by the interaction of Siglec-E and LOX-1 receptors.

Innate immune responses to cell damage-associated molecular patterns induce a controlled degree of inflammation, ideally avoiding the promotion of intense unwanted inflammatory adverse events. When released by damaged cells, Hsp70 can stimulate different responses that range from immune activation to immune suppression. The effects of Hsp70 are mediated through innate receptors expressed primarily by myeloid cells, such as dendritic cells (DCs). The regulatory innate receptors that bind to extracellular mouse Hsp70 (mHsp70) are not fully characterized, and neither are their potential interactions with activating innate receptors. Here, we describe that extracellular mHsp70 interacts with a receptor complex formed by inhibitory Siglec-E and activating LOX-1 on DCs. We also find that this interaction takes place within lipid microdomains, and Siglec-E acts as a negative regulator of LOX-1-mediated innate activation upon mHsp70 or oxidized LDL binding. Thus, HSP70 can both bind to and modulate the interaction of inhibitory and activating innate receptors on the cell surface. These findings add another dimension of regulatory mechanism to how self-molecules contribute to dampening of exacerbated inflammatory responses.

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity.

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47-SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

Cis Interactions of Membrane Receptors and Ligands.

Cell-cell communication is critical for the development and function of multicellular organisms. A crucial means by which cells communicate with one another is physical interactions between receptors on one cell and their ligands on a neighboring cell. Trans ligand:receptor interactions activate the receptor, ultimately leading to changes in the fate of the receptor-expressing cells. Such trans signaling is known to be critical for the functions of cells in the nervous and immune systems, among others. Historically, trans interactions are the primary conceptual framework for understanding cell-cell communication. However, cells often coexpress many receptors and ligands, and a subset of these has been reported to interact in cis and profoundly impact cell functions. Cis interactions likely constitute a fundamental, understudied regulatory mechanism in cell biology. Here, I discuss how cis interactions between membrane receptors and ligands regulate immune cell functions, and I also highlight outstanding questions in the field.

cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

CTLA4 depletes T cell endogenous and trogocytosed B7 ligands via cis-endocytosis.

CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Enhancing the therapeutic efficacy of programmed death ligand 1 antibody for metastasized liver cancer by overcoming hepatic immunotolerance in mice.

BACKGROUND AND AIMS: Immunotherapy with programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade has shown low response rates in liver cancer patients, with the underlying mechanisms unclear. To decipher a specific impact of the liver microenvironment, we compared the effects of anti-PD-L1 antibody (αPD-L1) blockade on the same tumor grown s.c. or in the liver. APPROACH AND RESULTS: We generated s.c. tumors in mice by inoculating MC38 colorectal cancer (CRC) cells under the skin and metastatic liver tumors by portal vein or splenic injection of CRC cells. Tumor-bearing mice were treated by i.p. injection of αPD-L1, polyinosinic:polycytidylic acid (poly[I:C]), or both. αPD-L1 monotherapy significantly suppressed s.c. tumor growth, but showed no effect on metastatic liver tumors. However, the combination of αPD-L1 with poly(I:C), an innate immunity-stimulating reagent, robustly inhibited tumor progression in liver. The combination therapy effectively down-regulated myeloid-derived suppressor cells (MDSCs), but up-regulated ratios of M1/M2 macrophages, CD8/CD4, and CD8/regulatory T (Treg) cells infiltrated into liver tumors and whole liver. A group of long-lasting T-bet+ Eomes- PD-1- cytotoxic T cells was maintained in the combo-treated liver, leading to resistance to tumor recurrence. Depleting macrophages or blocking type Ⅰ interferon signaling abrogated the synergistic antitumor effect of αPD-L1 and poly(I:C), indicating a requirement of boosting innate immunity for optimized activation of cytotoxic T cells by PD-1/PD-L1 blockade. CONCLUSIONS: The poor response of liver cancers to αPD-L1 therapy is largely attributable to a unique hepatic immunotolerant microenvironment, independent of tumor origins or types. The success of a combinatorial immunotherapy relies on coordinated inhibition or activation of various innate and adaptive immune cell activities.

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

PD-1 and BTLA regulate T cell signaling differentially and only partially through SHP1 and SHP2.

Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.

TCR-pMHC bond conformation controls TCR ligand discrimination.

A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR-pMHC bonds and the conformations of individual TCR-CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR-pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR-pMHC bond conformation.

PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways.

Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.

Understanding T cell signaling using membrane reconstitution.

T cells are central players of our immune system, as their functions range from killing tumorous and virus-infected cells to orchestrating the entire immune response. In order for T cells to divide and execute their functions, they must be activated by antigen-presenting cells (APCs) through a cell-cell junction. Extracellular interactions between receptors on T cells and their ligands on APCs trigger signaling cascades comprised of protein-protein interactions, enzymatic reactions, and spatial reorganization events, to either stimulate or repress T cell activation. Plasma membrane is the major platform for T cell signaling. Recruitment of cytosolic proteins to membrane-bound receptors is a common critical step in many signaling pathways. Membranes decrease the dimensionality of protein-protein interactions to enable weak yet biologically important interactions. Membrane resident proteins can phase separate into micro-islands that promote signaling by enriching or excluding signal regulators. Moreover, some membrane lipids can either mediate or regulate cell signaling by interacting with signaling proteins. While it is critical to investigate T cell signaling in a cellular environment, the large number of signaling pathways involved and potential crosstalk have made it difficult to obtain precise, quantitative information on T cell signaling. Reconstitution of purified proteins to model membranes provides a complementary avenue for T cell signaling research. Here, I review recent progress in studying T cell signaling using membrane reconstitution approaches.

Antigen-Presenting Cell-Intrinsic PD-1 Neutralizes PD-L1 in cis to Attenuate PD-1 Signaling in T Cells.

The PD-1 pathway, consisting of the co-inhibitory receptor PD-1 on T cells and its ligand (PD-L1) on antigen-presenting cells (APCs), is a major mechanism of tumor immune evasion. PD-1 and PD-L1 blockade antibodies have produced remarkable clinical activities against a subset of cancers. Binding between T cell-intrinsic PD-1 and APC-intrinsic PD-L1 triggers inhibitory signaling to attenuate the T cell response. Here, we report that PD-1 is co-expressed with PD-L1 on tumor cells and tumor-infiltrating APCs. Using reconstitution and cell culture assays, we demonstrate that the co-expressed PD-1 binds to PD-L1 in cis. Such interaction inhibits the ability of PD-L1 to bind T cell-intrinsic PD-1 in trans and, in turn, represses canonical PD-L1/PD-1 inhibitory signaling. Selective blockade of tumor-intrinsic PD-1 frees up tumor-intrinsic PD-L1 to inhibit T cell signaling and cytotoxicity. Our study uncovers another dimension of PD-1 regulation, with important therapeutic implications.

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase.

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.

T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.

Programmed cell death-1 (PD-1) is a coinhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). By titrating PD-1 signaling in a biochemical reconstitution system, we demonstrate that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1-recruited Shp2 phosphatase. We also show that CD28, but not the TCR, is preferentially dephosphorylated in response to PD-1 activation by PD-L1 in an intact cell system. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways play key roles in regulating effector T cell function and responses to anti-PD-L1/PD-1 therapy.

Phase separation of signaling molecules promotes T cell receptor signal transduction.

Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micrometer- or submicrometer-sized clusters. However, the functional consequences of such clustering have been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling.

A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores.

Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two α-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD.

Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment.

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca(2+)-dependent and Ca(2+)-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca(2+)-dependent pathway. Mutations that disrupt Ca(2+)-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca(2+)-sensor for replenishment. The Ca(2+)-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca(2+)-dependent manner; this interaction requires intact Ca(2+)-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca(2+)-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001.