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.

Large 22q13.3 deletions perturb peripheral transcriptomic and metabolomic profiles in Phelan-McDermid syndrome.

Phelan-McDermid syndrome (PMS) is a rare neurodevelopmental disorder caused at least in part by haploinsufficiency of the SHANK3 gene, due to sequence variants in SHANK3 or subtelomeric 22q13.3 deletions. Phenotypic differences have been reported between PMS participants carrying small "class I" mutations and large "class II" mutations; however, the molecular perturbations underlying these divergent phenotypes remain obscure. Using peripheral blood transcriptome and serum metabolome profiling, we examined the molecular perturbations in the peripheral circulation associated with a full spectrum of PMS genotypes spanning class I (n = 37) and class II mutations (n = 39). Transcriptomic data revealed 52 genes with blood expression profiles that tightly scale with 22q.13.3 deletion size. Furthermore, we uncover 208 underexpressed genes in PMS participants with class II mutations, which were unchanged in class I mutations. These genes were not linked to 22q13.3 and were strongly enriched for glycosphingolipid metabolism, NCAM1 interactions, and cytotoxic natural killer (NK) immune cell signatures. In silico predictions estimated a reduction in CD56+ CD16- NK cell proportions in class II mutations, which was validated by mass cytometry time of flight. Global metabolomics profiling identified 24 metabolites that were significantly altered in PMS participants with class II mutations and confirmed a general reduction in sphingolipid metabolism. Collectively, these results provide new evidence linking PMS participants carrying class II mutations with decreased expression of cytotoxic cell signatures, reduced relative proportions of NK cells, and lower sphingolipid metabolism. These findings highlight alternative avenues for therapeutic development and offer new mechanistic insights supporting genotype-to-phenotype associations in PMS.

PD-1-stimulated T cell subsets are transcriptionally and functionally distinct.

Despite the obvious inhibitory outcome of PD-1 signaling, an additional series of functions are activated. We have observed that T cells stimulated through the T cell receptor (TCR) and PD-1 primarily do not proliferate; however, there is a population of cells that proliferates more than through TCR stimulation alone. In this study, we performed flow cytometry and RNA sequencing on individual populations of T cells and discovered that unlike naive T cells, which were inhibited following PD-1 ligation, T cells that proliferated more following PD-1 ligation were associated with effector and central memory phenotypes. We showed that these populations had different gene expression profiles following PD-1 ligation with PD-L1 compared to PD-L2. The presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation provides new insights into the biology of PD-1 and suggest the use of T cell subset-specific approaches to improve the clinical outcome of PD-1 blockade.

PD-1-induced proliferating T cells exhibit a distinct transcriptional signature.

Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signalling, there are additionally a collection of functions that are activated. We have observed that CD4+ T cells stimulated through the T-cell receptor and PD-1 primarily do not proliferate; however, there is a population of cells that proliferates more than T-cell receptor stimulation alone. These highly proliferating cells could potentially be associated with PD-1-blockade unresponsiveness in patients. In this study, we have performed RNA sequencing and found that following PD-1 ligation proliferating and non-proliferating T cells have distinct transcriptional signatures. Remarkably, the proliferating cells showed an enrichment of genes associated with an activated state despite PD-1 signalling. Additionally, circulating follicular helper T cells were significantly more prevalent in the non-proliferating population, demonstrated by enrichment of the associated genes CXCR5, CCR7, TCF7, BCL6 and PRDM1 and validated at the protein level. Translationally, we also show that there are more follicular helper T cells in patients that respond favourably to PD-1 blockade. Overall, the presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation may provide insights into the clinical differences observed following therapeutic PD-1 blockade.

VRK2 inhibition synergizes with PD-1 blockade to improve T cell responses.

Therapeutic programmed cell death protein 1 (PD-1) blockade enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To develop better therapeutics and to understand the signaling pathways downstream of PD-1 we performed phosphoproteomic analysis of PD-1 and identified vaccinia related kinase 2 (VRK2) as a key mediator of PD-1 signaling. Using genetic and pharmacological approaches, we discovered that VRK2 is required for PD-1-induced phosphorylation of the protein p21 activated kinase 2 (PAK2), and for the inhibition of IL-2, IL-8, and IFN-γ secretion. Moving into in vivo syngeneic tumor models, pharmacologic inhibition of VRK2 in combination with PD-1 blockade enhanced tumor clearance through T cell activation. This study suggests that VRK2 is a unique therapeutic target and that combination of VRK2 inhibitors with PD-1 blockade may improve cancer immunotherapy.

A novel mouse model for checkpoint inhibitor-induced adverse events.

Immune checkpoint inhibitors have demonstrated significant efficacy in the treatment of a variety of cancers, however their therapeutic potential is limited by abstruse immune related adverse events. Currently, no robust animal model exists of checkpoint inhibitor-induced adverse events. Establishing such a model will improve our mechanistic understanding of this process, which in turn will inform design of improved therapies. We developed a mouse model to determine inflammatory toxicities in response to dual checkpoint blockade in the presence of syngeneic tumors. Mice from susceptible genetic backgrounds received intraperitoneal injections of anti-mouse PD-1 and CTLA-4 antibodies. The mice were monitored for weight loss and histologic evidence of inflammation. Blood was collected for basic metabolic panels and titers of anti-nuclear antibodies. In parallel, mice were also treated with prednisolone, which is commonly used to treat immune related adverse events among cancer patients. Among all the genetic backgrounds, B6/lpr mice treated with anti-CTLA-4 and anti-PD-1 antibodies developed more substantial hepatitis, pancreatitis, colitis, and pneumonitis characterized by organ infiltration of immune cells. Mice that developed tissue infiltration demonstrated high serum levels of glucose and high titers of anti-nuclear antibodies. Finally, while administration of prednisolone prevented the development of the inflammatory adverse events, it also abrogated the protective anti-tumor effect of the checkout inhibitors. Genetic background and treatment modalities jointly modified the inflammatory adverse events in tumor bearing mice, suggesting a complex mechanism for checkpoint inhibitor-related inflammation. Future studies will assess additional genetic susceptibility factors and will examine possible contributions from the administration of other anti-inflammatory drugs.

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

In Vitro Assays to Study PD-1 Biology in Human T Cells.

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

Correction: Differential transcriptional response following glucocorticoid activation in cultured blood immune cells: a novel approach to PTSD biomarker development.

This Article was originally published without the correct Supplemental Table file (Table S1 was missing). In total, there are seven Supplemental Tables, and six were in the original submission. Furthermore, Fig. 1 was misplaced in the main text; it was embedded in the manuscript file even before the results section. Both issues have now been fixed in the HTML and PDF versions of this Article.

The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals.

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D ß-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.

Differential transcriptional response following glucocorticoid activation in cultured blood immune cells: a novel approach to PTSD biomarker development.

Post-traumatic stress disorder (PTSD) is a condition of stress reactivity, whose clinical manifestations are evident when patients are triggered following exposure to a traumatic event. While baseline differences in gene expression of glucocorticoid signaling and inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) have been associated with PTSD, these alterations do not fully recapitulate the molecular response to physiological triggers, such as stress hormones. Therefore, it is critical to develop new techniques that will capture the dynamic transcriptional response associated with stress-activated conditions relative to baseline conditions. To achieve this goal, cultured PBMCs from combat-exposed veterans with PTSD(+) (n = 10) and without PTSD(-) (n = 10) were incubated with increasing concentrations (vehicle, 2.5 nM, 5 nM, 50 nM) of dexamethasone (DEX). Across diagnosis and dosage, several genes and gene networks were reliable markers of glucocorticoid stimulation (FDR < 5%), including enhanced expression of FKPB5, VIPR1, NR1I3, and apoptosis-related pathways, and reduced expression of NR3C1, STAT1, IRF1, and related inflammatory and cellular stress-responsive pathways. Dose-dependent differential transcriptional changes in several genes were also identified between PTSD+ and PTSD-. Robust changes in expression were observed at 2.5 nM DEX in PTSD- but not PTSD+ participants; whereas, with increasing concentrations (5 nM and 50 nM), several genes were identified to be uniquely up-regulated in PTSD+ but not PTSD- participants. Collectively, these preliminary findings suggest that genome-wide gene expression profiling of DEX-stimulated PBMCs is a promising method for the exploration of the dynamic differential molecular responses to stress hormones in PTSD, and may identify novel markers of altered glucocorticoid signaling and responsivity in PTSD.

SLAMF6 clustering is required to augment T cell activation.

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors that are expressed exclusively on hematopoietic cells. Most of these transmembrane receptors are homotypic by nature and downstream signaling occurs when cells that express the same SLAM receptor interact. Previous studies have determined that anti-SLAMF6 antibodies can have a therapeutic effect in autoimmunity and cancer. However, little is known about the role of SLAMF6 in the adaptive immune responses and in order to utilize SLAMF6 interventional approaches, a better understanding of the biology of this receptor in T cell is warranted. Accordingly, the objective of our study was to investigate both functionally and structurally the role of SLAMF6 in T cell receptor (TCR) mediated responses. Biochemical and genetic experiments revealed that SLAMF6 was required for productive TCR downstream signaling. Interestingly, SLAMF6 ectodomain was required for its function, but not for its recruitment to the immunological synapse. Flow-cytometry analysis demonstrated that tyrosine 308 of the tail of SLAMF6 was crucial for its ability to enhance T cell function. Imaging studies revealed that SLAMF6 clustering, specifically with the TCR, resulted in dramatic increase in downstream signaling. Mechanistically, we showed that SLAMF6 enhanced T cell function by increasing T cell adhesiveness through activation of the small GTPase Rap1. Taken together SLAMF6 is an important regulator of T cell activation where both its ectodomain and its endodomain contribute differentially to T cell functions. Additional studies are underway to better evaluate the role of anti-SLAMF6 approaches in specific human diseases.

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity.

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

CD1b-restricted GEM T cell responses are modulated by Mycobacterium tuberculosis mycolic acid meromycolate chains.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.

Checkpoint Inhibitors: Applications for Autoimmunity.

To limit excessive T cell-mediated inflammatory responses, the immune system has a milieu of inhibitory receptors, called immune checkpoints. Cancer cells have evolved to seize those inhibitory pathways and to prevent T cell-mediated killing of tumor cells. Therefore, immune checkpoint inhibitors (ICI) consisting of blocking antibodies against these receptors present an exciting avenue in the fight against cancer. The last decade has seen the implementation of ICI against a variety of cancer indications that have improved the overall anti-tumor responses and patient survival. However, inflammatory toxicities and autoimmunity are a significant adverse event of ICI therapies. In this review, we will discuss the biology of immune checkpoints, highlight research strategies that may help reduce the incidence of immune-related adverse events associated with ICI therapies, and also suggest investigational approaches to manipulate immune checkpoints to treat primary autoimmune disorders.

Quantitative and qualitative iNKT repertoire associations with disease susceptibility and outcome in macaque tuberculosis infection.

Correlates of immune protection that reliably predict vaccine efficacy against Mycobacterium tuberculosis (Mtb) infection are urgently needed. Invariant NKT cells (iNKTs) are CD1d-dependent innate T cells that augment host antimicrobial immunity through production of cytokines, including interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We determined peripheral blood iNKT numbers, their proliferative responses and iNKT subset proportions after in vitro antigen expansion by α-galactosylceramide (αGC) in a large cohort of mycobacteria-naïve non-human primates, and macaques from Bacillus Calmette-Guerin (BCG) vaccine and Mtb challenge studies. Animals studied included four genetically distinct groups of macaques within cynomolgus and rhesus species that differ in their susceptibility to Mtb infection. We demonstrate significant differences in ex vivo iNKT frequency between groups, which trends towards an association with susceptibility to Mtb, but no significant difference in overall iNKT proliferative responses. Susceptible animals exhibited a skewed CD4+/CD8+ iNKT subset ratio in comparison to more Mtb-resistant groups. Correlation of iNKT subsets post BCG vaccination with clinical disease manifestations following Mtb challenge in the Chinese cynomolgus and Indian rhesus macaques identified a consistent trend linking increased CD8+ iNKTs with favourable disease outcome. Finally, a similar iNKT profile was conferred by BCG vaccination in rhesus macaques. Our study provides the first detailed characterisation of iNKT cells in macaque tuberculosis infection, suggesting that iNKT repertoire differences may impact on disease outcome, which warrants further investigation.

The Clonal Invariant NKT Cell Repertoire in People with Type 1 Diabetes Is Characterized by a Loss of Clones Expressing High-Affinity TCRs.

Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.

Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36.

Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM) were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV) or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNß gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031) and CD36 gene expression (p = 0.031) by MDM cultured for 24 h in 50IU/ml IFNß. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNß production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells.

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.