Faculty Bibliography
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396
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.
PMCID:7596257
PMID: 33178706
ISSN: 2296-634x
CID: 4665362
Maturation of Monocyte-Derived DCs Leads to Increased Cellular Stiffness, Higher Membrane Fluidity, and Changed Lipid Composition
Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.
PMCID:7728921
PMID: 33329576
ISSN: 1664-3224
CID: 4717962
Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.
PMCID:7848080
PMID: 33537301
ISSN: 2296-634x
CID: 4776522
Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
PMID: 31633216
ISSN: 1521-4141
CID: 4146932
A tissue-like platform for studying engineered quiescent human T-cells' interactions with dendritic cells
Research in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situ activation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 help to CD8 T-cells through enhanced maturation of DCs and effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.
PMID: 31552826
ISSN: 2050-084x
CID: 4107672
Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T-cells
Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signalling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.
PMID: 31469364
ISSN: 2050-084x
CID: 4066572
The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis
The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived BÂ cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing BÂ cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into TÂ cells through the phosphatase SHP1 reduced TÂ cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, TÂ cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC BÂ cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.
PMID: 31204070
ISSN: 1097-4180
CID: 3955922
Immuno-phenotypes of Pancreatic Ductal Adenocarcinoma: Meta-analysis of transcriptional subtypes
Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas and has one of the highest mortality rates of any cancer type with a 5-year survival rate of <5%. Recent studies of PDAC have provided several transcriptomic classifications based on separate analyses of individual patient cohorts. There is a need to provide a unified transcriptomic PDAC classification driven by therapeutically relevant biologic rationale to inform future treatment strategies. Here, we used an integrative meta-analysis of 353 patients from four different studies to derive a PDAC classification based on immunologic parameters. This consensus clustering approach indicated transcriptomic signatures based on immune infiltrate classified as adaptive, innate and immune-exclusion subtypes. This reveals the existence of microenvironmental interpatient heterogeneity within PDAC and could serve to drive novel therapeutic strategies in PDAC including immune modulation approaches to treating this disease.
PMID: 30720864
ISSN: 1097-0215
CID: 3632072
Cutting Edge: Synapse Propensity of Human Memory CD8 T Cells Confers Competitive Advantage over Naive Counterparts
Memory T cells are endowed with multiple functional features that enable them to be more protective than naive T cells against infectious threats. It is not known if memory cells have a higher synapse propensity (SP; i.e., increased probability to form immature immunological synapses that then provide an entry into different modes of durable interaction with APCs). In this study, we show that only human memory CD8 T cells have remarkably high SP compared with naive counterparts. Such a dichotomy between naive and memory cells is not observed within the human CD4 or murine CD8 T cell population. Higher SP in human memory CD8 T cells allows them to outcompete and prevent naive CD8 T cells from getting recruited to the response. This observation has implications for original antigenic sin and aging of the immune system in humans.
PMID: 31201237
ISSN: 1550-6606
CID: 3930302
Integrins and Their Role in Immune Cell Adhesion
One of the 2019 Canada Gairdner International Awards recognizes Timothy Springer's discovery of the first immune system adhesion molecules involved in lymphocyte homing and the translation of those discoveries into therapeutics for autoimmune disease and cancer.
PMID: 30952447
ISSN: 1097-4172
CID: 4095272
