ABSTRACT
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Subject(s)
Dendritic Cells, Follicular/immunology , Fibroblasts/immunology , Lymph Nodes/immunology , Stromal Cells/immunology , Aged , Animals , Apoptosis/genetics , Apoptosis/immunology , Cell Proliferation/genetics , Cell Survival/genetics , Cell Survival/immunology , Dendritic Cells, Follicular/metabolism , Female , Fibroblasts/metabolism , Gene Expression Regulation/immunology , Gene Knock-In Techniques , Humans , Immunity, Cellular/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Lymph Nodes/cytology , Male , Mice , Mice, Transgenic , RNA-Seq , Single-Cell Analysis , Stromal Cells/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.
Subject(s)
Histocompatibility Antigens Class I/physiology , Lymphocyte Activation/physiology , Adult , Female , Humans , Kinetics , Ligands , Major Histocompatibility Complex/physiology , Male , Middle Aged , Molecular Dynamics Simulation , Oligopeptides , Peptides , Protein Binding/physiology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/physiology , Signal Transduction , Single Molecule Imaging , T-Lymphocytes/physiologyABSTRACT
The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.
Subject(s)
Neoplasms , T-Lymphocytes , Tumor Microenvironment , Humans , Immunotherapy , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy , T-Lymphocytes/immunology , Phenotype , Fibroblasts , Monocytes , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic useABSTRACT
Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.
Subject(s)
Membrane Lipids , Membrane Proteins , GRB2 Adaptor Protein/metabolism , Membrane Proteins/metabolism , Phosphorylation , Phosphotyrosine , Son of Sevenless Proteins/metabolismABSTRACT
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 (â¼50%) than the smaller R0 isoform (â¼20%), 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.