A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Modifications outside CDR1, 2 and 3 of the TCR variable ß domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable ß domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner.

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1.

Vγ9Vδ2 T cells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor," and BTN2A1, a direct ligand for T cell receptor (TCR) Vγ9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vγ9Vδ2 TCR and BTN3A1. Nuclear magnetic resonance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV structural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis indicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the γδ-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vγ9Vδ2 TCR triggering.

Division of labor and cooperation between different butyrophilin proteins controls phosphoantigen-mediated activation of human γδ T cells.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Transcriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

Missense Mutations in Desmoplakin Plakin Repeat Domains Have Dramatic Effects on Domain Structure and Function.

Plakin repeat domains (PRDs) are globular modules that mediate the interaction of plakin proteins with the intermediate filament (IF) cytoskeleton. These associations are vital for maintaining tissue integrity in cardiac muscle and epithelial tissues. PRDs are subject to mutations that give rise to cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy, characterised by ventricular arrhythmias and associated with an increased risk of sudden heart failure, and skin blistering diseases. Herein, we have examined the functional and structural effects of 12 disease-linked missense mutations, identified from the human gene mutation database, on the PRDs of the desmosomal protein desmoplakin. Five mutations (G2056R and E2193K in PRD-A, G2338R and G2375R in PRD-B and G2647D in PRD-C) rendered their respective PRD proteins either fully or partially insoluble following expression in bacterial cells. Each of the residues affected are conserved across plakin family members, inferring a crucial role in maintaining the structural integrity of the PRD. In transfected HeLa cells, the mutation G2375R adversely affected the targeting of a desmoplakin C-terminal construct containing all three PRDs to vimentin IFs. The deletion of PRD-B and PRD-C from the construct compromised its targeting to vimentin. Bioinformatic and structural modelling approaches provided multiple mechanisms by which the disease-causing mutations could potentially destabilise PRD structure and compromise cytoskeletal linkages. Overall, our data highlight potential molecular mechanisms underlying pathogenic missense mutations and could pave the way for informing novel curative interventions targeting cardiomyopathies and skin blistering disorders.

The chicken or the egg? A case of sympathetic staphylococcal empyema thoracis and splenic abscess.

Multicavitary abscesses are uncommon in immunocompetent individuals. Splenic abscesses being relatively uncommon with an incidence of 0.1-0.7% in quoted series [ 1]. Commonly reported cases are secondary infective endocarditis. To have both a splenic abscess and empyema concurrently is rare. We describe a case of a patient with a large left-sided empyema thoracis and concurrent splenic abscess.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2.

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Desmosomal protein structure and function and the impact of disease-causing mutations.

In this graphical review we focus on the structural characteristics of desmosomal proteins, their interactions with each other and with the intermediate filament cytoskeleton. The wealth of structural information that is now available allows predictions to be made about the pathogenic effect of disease-causing mutations. We have selected representative examples of missense mutations that are buried, semi-buried or surface exposed, and demonstrate how such variants could affect the structural fold of desmosomal proteins that are expressed in the heart. We explain how such alterations could compromise desmosomal adhesion, resulting in life threatening diseases including arrhythmogenic right ventricular cardiomyopathy.

The distinct MHC-unrestricted immunobiology of innate-like and adaptive-like human γδ T cell subsets-Nature's CAR-T cells.

Distinct innate-like and adaptive-like immunobiological paradigms are emerging for human γδ T cells, supported by a combination of immunophenotypic, T cell receptor (TCR) repertoire, functional, and transcriptomic data. Evidence of the γδ TCR/ligand recognition modalities that respective human subsets utilize is accumulating. Although many questions remain unanswered, one superantigen-like modality features interactions of germline-encoded regions of particular TCR Vγ regions with specific BTN/BTNL family members and apparently aligns with an innate-like biology, albeit with some scope for clonal amplification. A second involves CDR3-mediated γδ TCR interaction with diverse ligands and aligns with an adaptive-like biology. Importantly, these unconventional modalities provide γδ T cells with unique recognition capabilities relative to αß T cells, B cells, and NK cells, allowing immunosurveillance for signatures of "altered self" on target cells, via a membrane-linked γδ TCR recognizing intact non-MHC proteins on the opposing cell surface. In doing so, they permit cellular responses in diverse situations including where MHC expression is compromised, or where conventional adaptive and/or NK cell-mediated immunity is suppressed. γδ T cells may therefore utilize their TCR like a cell-surface Fab repertoire, somewhat analogous to engineered chimeric antigen receptor T cells, but additionally integrating TCR signaling with parallel signals from other surface immunoreceptors, making them multimolecular sensors of cellular stress.

Molecular mechanism of intermediate filament recognition by plakin proteins.

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.

γδ TCR Recognition of MR1: Adapting to Life on the Flip Side.

Nonclassical class I MHC-like molecules are ligands for several unconventional T cell populations. Recently, Le Nours et al. identified human γδ T cells recognising MHC-related protein-1 (MR1) via their T cell receptor (TCR). Also recognised by the αß-TCR of mucosal associated invariant T cells, MR1 interacts with specific γδ-TCRs using strikingly diverse binding modes, suggesting fundamental differences in γδ T cell recognition.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Binding of the periplakin linker requires vimentin acidic residues D176 and E187.

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.

HLA DR2b-binding peptides from human endogenous retrovirus envelope, Epstein-Barr virus and brain proteins in the context of molecular mimicry in multiple sclerosis.

The aetiology of multiple sclerosis (MS) is as yet poorly understood. Multiple mechanisms in different disease stages are responsible for immunopathology in MS. HLA Class II DR2b (DRB1*1501 ß, DRA1*0101 α) is the strongest genetic risk factor for MS. Remnants of ancient retroviruses in the human genome, termed human endogenous retroviruses (HERV), and Epstein-Barr virus (EBV) infection are also associated with MS. In silico analyses of human endogenous retroviral envelope (HERV env) proteins and three myelin proteins that are principal targets of an autoimmune response in MS showed sequence similarities between potential TH epitopes within pairs of viral and myelin peptides predicted to bind HLA DR2b. This led to the proposal that such molecular mimicry may potentially trigger MS. HLA DR2b binding characteristics of previously identified peptides from the three myelin proteins and HERV env proteins as well as additional in silico predicted peptides from other encephalitogenic brain proteins and EBV proteins were studied to further investigate molecular mimicry. Peptides containing potential TH epitopes from the myelin oligodendrocyte glycoprotein and HERV env previously predicted to bind HLA DR2b as well as other pertinent potential HLA DR2b-restricted TH epitopes were confirmed to bind HLA DR2b molecules. Molecular modelling of HLA DR2b in complex with high affinity peptides derived from MOG and HERV env proteins showed that their binding could occur in a similar manner to a HLA DR2b-binding peptide containing a known TH epitope. A structurally related pair of peptides predicted to bind HLA DR2b from the EBV protein EBNA1 and ß synuclein, a brain protein implicated in MS, were also shown to similarly bind HLA DR2b. The findings justify investigating CD4+ T cell responses to the identified peptides.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function.

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.

C-type lectin domain group 14 proteins in vascular biology, cancer and inflammation.

The C-type lectin domain (CTLD) group 14 family of transmembrane glycoproteins consist of thrombomodulin, CD93, CLEC14A and CD248 (endosialin or tumour endothelial marker-1). These cell surface proteins exhibit similar ectodomain architecture and yet mediate a diverse range of cellular functions, including but not restricted to angiogenesis, inflammation and cell adhesion. Thrombomodulin, CD93 and CLEC14A can be expressed by endothelial cells, whereas CD248 is expressed by vasculature associated pericytes, activated fibroblasts and tumour cells among other cell types. In this article, we review the current literature of these family members including their expression profiles, interacting partners, as well as established and speculated functions. We focus primarily on their roles in the vasculature and inflammation as well as their contributions to tumour immunology. The CTLD group 14 family shares several characteristic features including their ability to be proteolytically cleaved and engagement of some shared extracellular matrix ligands. Each family member has strong links to tumour development and in particular CD93, CLEC14A and CD248 have been proposed as attractive candidate targets for cancer therapy.