Biology subjects

Dushek, O.

Publications and source records attributed to Dushek, O..

At least 19 recordsLinked to original sources

bioRxiv2026

Membrane-mediated cooperativity of antigen receptors and CD2 adhesion receptors in CAR-T cell activation

Chimeric antigen receptor (CAR)-T cells exhibit a major defect in antigen recognition, often requiring 100-1000-fold higher antigen densities for activation than the native T cell receptor (TCR). Recent work has traced this defect to the inefficient use of CD2/CD58 adhesion by CAR-T cells and demonstrated that matching the extracellular dimensions of CD2/CD58 and CAR/antigen complexes can dramatically enhance antigen sensitivity. Remarkably, modest nanometre-scale changes in CD2/CD58 dimensions produce orders-of-magnitude changes in antigen sensitivity, while having little effect on T cell activation at high antigen densities. Here, we present a general model for the length-dependent cooperativity of antigen receptors and CD2 adhesion receptors in CAR-T cell activation, combining statistical-physical theory with coarse-grained simulations. The model predicts that the antigen concentration required for activation increases sharply with the length mismatch between antigen-receptor and adhesion-receptor complexes until a critical mismatch is reached, at which the complexes segregate. Beyond this critical mismatch when complexes segregate, antigen sensitivity becomes largely independent of complex length, consistent with experimental observations. For small to moderate mismatches, the antigen concentration required for activation exhibits a Gaussian dependence on length mismatch with a characteristic width of only a few nanometres, explaining how nanometre-scale changes in CD2 length can alter antigen sensitivity by orders of magnitude. Fitting the model to measurements of CD2-length-dependent CAR-T cell activation further enables estimation of characteristic molecular length scales and concentrations of antigen-receptor and adhesion-receptor complexes. These results provide a quantitative physical framework connecting nanometre-scale organization of the cell-cell interface to CAR-T cell antigen sensitivity and highlight molecular dimensions as a design parameter for T cell-based immunotherapies.

immunology↗

bioRxiv2026

Using peptide-exchange systems to interrogate peptide-specific KIR binding to HLA Class -I

The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory HLA class I (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide-specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I. We tested two methods; dipeptide-mediated peptide exchange and open-HLA-I, HLA-I molecules consisting of heavy chain-{beta}2m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells. We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells. Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide-sequence dependent recognition of HLA-I by KIR.

immunology↗

bioRxiv2025

Mixed-mobility supported lipid bilayers reveal the modulatory role ofimmobilized ICAM1 on T cell activation, effector functions and immune synapse organization

The immunological synapse (IS) integrates antigen recognition and adhesion to control T cell activation and effector functions. Reductionist systems have been instrumental in dissecting IS organization, but conventional systems constrain all ligands to be either mobile or immobile, unlike antigen-presenting cells where intercellular adhesion molecule 1 (ICAM1) is cytoskeletally anchored while T cell receptor (TCR) ligands remain mobile. Here, we establish mixed-mobility supported lipid bilayers (SLBs) that simultaneously present mobile TCR agonists and immobile ICAM1. Selective immobilization of ICAM1 disrupts centripetal F-actin flow, prevents centralization of TCR microclusters and shifts signaling to peripheral microclusters. This attenuates TCR downregulation through ectocytosis while maintaining recycling, and enhances integrin mechanotransduction, reflected in increased phosphorylation of Focal Adhesion Kinase, Paxillin and the stretch-sensitive adaptor CasL. Functionally, immobilized ICAM1 augments T cell activation, degranulation, Perforin release and cytotoxicity. Importantly, these findings were recapitulated in a cell-cell system engineered to express either full-length, cytoskeleton-anchored ICAM1 or a truncated form lacking cytoskeletal association, with full-length ICAM1 consistently promoting stronger effector responses. These findings identify ligand mobility as a key biophysical parameter that shapes IS organization and T cell effector responses and establish mixed-mobility SLBs as a powerful tool for probing receptor mechanics in immunity.

immunology↗

bioRxiv2025

NanoBondy reacting through NeissLock anhydride allows covalent immune cell decoration

Cell-surface conjugation has enormous therapeutic and research potential. Existing technologies for cell-surface modification are usually reversible, non-specific, or rely on genetic editing of target cells. Here we present the NanoBondy, a nanobody modified for covalent ligation to an endogenous protein target at the cell-surface. The NanoBondy utilizes the 20 naturally occurring amino acids, harnessing NeissLock chemistry engineered from Neisseria meningitidis. We evaluated binding and specificity of a panel of nanobodies to CD45, a long-lived surface marker of nucleated hematopoietic cells. We demonstrated conversion of existing nanobodies to covalently reacting NanoBondies using a disulfide clamp to position the self-processing module of FrpA close to the nanobody antigen-binding site. Addition of calcium induces anhydride formation at the NanoBondy C-terminus and proximity-directed ligation to surface amines on CD45. We optimized NanoBondy reaction by fine-tuning linkers and disulfide clamp sites to modulate anhydride positioning. Tandem mass spectrometry mapped reaction sites between the NanoBondy and CD45. NanoBondy ligation was robust to buffer, pH and temperature and was detected within 2 minutes. We established reaction specificity of NanoBondies to endogenous CD45 at the surface of NK cells and T cells. NanoBondy technology provides a modular approach for targeted, inducible and covalent cell-surface modification of immune cells.

synthetic biology↗

bioRxiv2025

Precise engineering of chimeric antigen receptor expression levels defines T cell identity and function

Chimeric Antigen Receptor (CAR) T therapy is a potent treatment for haematological malignancies, but T cell exhaustion reduces its efficacy in many patients. Although high CAR transgene levels appear to drive T cell exhaustion, the relationship between CAR expression levels, T cell function, and transcriptional identity is yet to be mapped at high resolution. Here, we harness a high-resolution microRNA-based control system to precisely modulate CAR transgene expression levels and assess the impact on T cell activation, gene expression and function. By post-transcriptionally modulating CAR abundance, we show that differential CAR levels significantly impact T cell proliferation, cytokine production and tonic signalling. T cells with high CAR expression become strongly activated even at low target antigen densities, while those with low CAR expression are triggered only by high concentrations of their target. Single-cell RNA sequencing of primary T cells expressing a broad range of CAR transcript levels revealed global transcriptional programmes that become dysfunctional with increased CAR abundance, expanding our understanding of T cell exhaustion. Notably, we identified a narrow CAR expression range where the exhaustion transcriptional state is not triggered, demonstrating that T cell exhaustion can be controlled by fine-tuning CAR levels. This work demonstrates that CAR expression levels are key determinants of T cell transcriptional identity and function and introduces a tractable method to precisely tune CAR expression and T cell activity.

synthetic biology↗

bioRxiv2025

The 3D affinities of the OT-I TCR to foreign and self-antigens predict their 2D affinities and reveal imperfect antigen discrimination

T cells use the T cell antigen receptor (TCR) to discriminate between higher-affinity foreign and lower-affinity self peptide-MHC (pMHC) antigens. The OT-I mouse TCR is widely used to study anti-gen discrimination utilising many pMHCs, including foreign and self antigens. Previous studies sug-gested that OT-I T cells achieve near-perfect discrimination between higher and lower affinity antigens. Moreover, these 3D affinities measured in solution did not correlate with the 2D membrane affinities, suggesting a complex relationship between 3D and 2D affinities. In contrast, other TCRs have shown imperfect antigen discrimination and strong correlations between 3D and 2D affinities. To resolve these discrepancies, we extended a protocol for measuring ultra-low TCR/pMHC affinities to accurately de-termine the 3D affinities of the OT-I TCR binding 19 pMHC complexes. These revised 3D affinities now strongly correlate with the 2D affinities, and accurately predict functional responses. Importantly, we now find that the OT-I TCR exhibits enhanced yet imperfect antigen discrimination, similar to other TCRs, allowing it to detect abnormally high levels of low-affinity self-antigens. Finally, we show that discrimination is highest with low-affinity pMHC ligands, a finding predicted by the kinetic-proofreading model of antigen discrimination. This work underscores the ability of T cells to effectively gauge prox-ies for 3D affinity within the 2D cell-cell interface, with significant implications for the mechanisms underlying antigen discrimination. Lay abstractT cells protect the body from infection by distinguishing between foreign and self molecules. They do this using a specialized receptor called the T cell receptor (TCR), which senses differences in how strongly it binds to foreign versus self molecules. Scientists have used a mouse TCR called OT-I to understand this process, concluding that OT-I T cells exhibit near perfect discrimination between foreign and self antigens based on a sharp affinity threshold. However, conflicting results from experiments on other TCRs hinted at a more complicated picture. In this study, we used an improved method to accurately measure OT-I TCR binding affinity to 19 peptides, including foreign and self antigens. In contrast to previous results and like other TCRs, we found that OT-I T cells display enhanced but im-perfect discrimination, enabling them to be activated by high levels of self antigen. The new data also revealed that the ability of T cells to discriminate antigens is particularly high at low affinities. These findings reconcile apparent discrepancies between the OT-I TCR and other TCRs, and have implications for understanding diseases where T cells often respond to self antigen, such as autoimmunity and cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/632665v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@977df5org.highwire.dtl.DTLVardef@1ce7bd3org.highwire.dtl.DTLVardef@12363cdorg.highwire.dtl.DTLVardef@6d5573_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Optimising CAR-T cell sensitivity by engineering matched extracellular sizes between CAR/antigen and CD2/CD58 adhesion complexes

Chimeric antigen receptor (CAR)-T cells exhibit low antigen sensitivity, which restricts their therapeutic efficacy and leads to patient relapses when cancer cells downregulate antigen expression. Despite the pressing need to overcome this limitation, the underlying mechanisms remain poorly understood. Here, we demonstrate that enhancing CAR sensitivity to match the sensitivity of the T-cell receptor (TCR) can be achieved by engineering matched extracellular sizes of CAR/antigen and CD2/CD58 complexes. We find that different CAR/antigen sizes, which are generated by different CAR architectures and different target antigens, require a different CD2/CD58 size to optimise sensitivity. This extracellular size-matching improves antigen engagement and co-localisation of CAR/antigen and CD2/CD58 complexes. We also find that size-matching controls co-inhibition of CARs by PD-1/PD-L1. These findngs highlight the importance of size-matching for signal integration by surface receptors and offers a new approach to tune CAR-T cell sensitivity by matching or mismatching extracellular sizes. One sentence summaryThe antigen sensitivity of CAR-T cells can be tuned to match the sensitivity of TCR-T cells by varying the relative extracellular size of CAR/antigen and CD2/CD58 complexes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/631424v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17e6a55org.highwire.dtl.DTLVardef@771b38org.highwire.dtl.DTLVardef@19c3544org.highwire.dtl.DTLVardef@6d1fe1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Generation of T cells with reduced off-target cross-reactivities by engineering co-signalling receptors

Adoptive T cell therapy using T cells engineered with novel T cell receptors (TCRs) targeting tumor-specific peptides is a promising immunotherapy. However, these TCR-T cells can cross-react with off-target peptides, leading to severe autoimmune toxicities. Current efforts focus on identifying TCRs with reduced cross-reactivity. Here, we show that T cell cross-reactivity can be controlled by the co-signalling molecules CD5, CD8, and CD4, without modifying the TCR. We find the largest reduction in cytotoxic T cell cross-reactivity by knocking out CD8 and expressing CD4. Cytotoxic T cells engineered with a CD8-to-CD4 co-receptor switch show reduced cross-reactivity to random and positional scanning peptide libraries, as well as to self-peptides, while maintaining their on-target potency. Therefore, co-receptor switching generates super selective T cells that reduce the risk of lethal off-target cross-reactivity, and offers a universal method to enhance the safety of T cell immunotherapies for any TCR. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/620274v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@9b65dborg.highwire.dtl.DTLVardef@3c1354org.highwire.dtl.DTLVardef@190782org.highwire.dtl.DTLVardef@13c1e39_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summarySwitching the CD8 for the CD4 co-receptor in cytotoxic T cells reduces the functional cross-reactivity of T cells without modifying the TCR.

immunology↗

bioRxiv2024

G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

Key PointsO_LIBispecific cis-acting platelet receptor inhibitors (CAPRIs) mediate hetero-clustering of the ITIM receptor G6B with ITAM receptors. C_LIO_LIG6B-GPVI and G6B-CD32A CAPRIs specifically inhibit collagen- and immune complex-induced thrombus formation, respectively. C_LI Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR {gamma}-chain complex or Fc{gamma}RIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.

cell biology↗

bioRxiv2024

Regulation of temporal cytokine production by co-stimulation receptors in TCR-T cells is lost in CAR-T cells

CD8+ T cells contribute to immune responses by producing cytokines when their T cell receptors (TCRs) recognise peptide antigens on major-histocompability-complex (pMHC) class I. However, excessive cytokine production can be harmful. For example, cytokine release syndrome (CRS) is a common toxicity observed in treatments that activate T cells, including chimeric antigen receptor (CAR)-T cell therapy. While engagement of costimulatory receptors is well known to enhance cytokine production, we have limited knowledge of their ability to regulate the kinetics of cytokine production by CAR-T cells. Here we compare early (0-12 hours) and late (12-20 hours) production of IFN-{gamma}, IL-2, and TNF- production by T cells stimulated via TCR or CARs in the presence or absence ligands for CD2, LFA-1, CD28, CD27, and 4-1BB. For T cells expressing TCRs and 1st-generation CARs, activation by antigen alone was sufficient to stimulate early cytokine production, while co-stimulation by CD2 and 4-1BB was required to maintain late cytokine production. In contrast, T cells expressing 2nd-generation CARs, which have intrinsic costimulatory signalling motifs, produce high levels of cytokines in both early and late periods in the absence of costimulatory receptor ligands. Losing the requirement for costimulation for sustained cytokine production may contribute to the effectiveness and/or toxicity of 2nd-generation CAR-T cell therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/581341v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1d59d68org.highwire.dtl.DTLVardef@1512832org.highwire.dtl.DTLVardef@126134corg.highwire.dtl.DTLVardef@bd104d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

The molecular reach of antibodies determines their SARS-CoV-2 neutralisation potency

Key functions of antibodies, such as viral neutralisation, depend on bivalent binding but the factors that influence it remain poorly characterised. Here, we develop and employ a new bivalent model to mechanistically analyse binding between >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. Our method reproduces the monovalent on/off-rates and enables measurements of the bivalent on-rate and the molecular reach: the maximum antigen separation that supports bivalent binding. We find large variations in these parameters across antibodies, including variations in reach (22-46 nm) that exceed the physical antibody size ([~]15 nm) due to the antigen size. The bivalent model integrates all parameters, including reach and antigen density, to predict an emergent binding potency for each antibody that matches their neutralisation potency. Indeed, antibodies with similar monovalent affinities to the same RBD-epitope but with different reaches display differences in emergent bivalent binding that match differences in their neutralisation potency. Together, our work highlights that antibodies within an isotype class binding the same antigen can display differences in molecular reach that can substantially modulate their emergent binding and functional properties. Lay SummaryAntibodies are soluble proteins that can neutralise pathogens by sticking to them. They contain two identical arms that allow them to simultaneously bind two identical antigen molecules on pathogen surfaces. Although we know that bivalent binding is important for neutralisation, we dont know how different antibodies achieve it. We developed a new model to analyse the mechanism of bivalent binding and used it to study over 45 antibodies from COVID-19 patients that bind the RBD antigen of SARS-CoV-2. Unexpectedly, we found that the molecular reach of an antibody, which is the maximum antigen separation that supports bivalent binding, varied widely between antibodies and exceeded their physical size. We show how antibody binding emerges from the interplay of multiple factors, including reach, and that this emergent binding predicts their neutralisation function. The ability to analyse and predict bivalent binding should improve our understanding and exploitation of antibodies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/556503v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1674563org.highwire.dtl.DTLVardef@fc7feborg.highwire.dtl.DTLVardef@1325847org.highwire.dtl.DTLVardef@41ad51_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that act as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings reveal a novel glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

cell biology↗

bioRxiv2023

Using CombiCells, a platform enabling titration and combinatorial display of cell surface ligands, to investigate the sensitivity and costimulatory requirements of TCRs and CARs

Understanding how cellular decisions by receptor/ligand interactions at cell/cell interface has been challenging because it is difficult to independently vary the surface density of multiple ligands. Here, we exploit the SpyCatcher/SpyTag split-protein system for rapid combinatorial display of native ligands on cells (Combicells). We use this platform to assess T cell antigen sensitivity and the impact of T cell co-stimulation/co-inhibition receptors. The TCR displayed much greater sensitivity to pMHC than CARs and BiTES did to CD19. While TCR sensitivity was greatly enhanced by CD2 ligand, CAR sensitivity to CD19 was primarily but more modestly enhanced by LFA-1 ligand. Lastly, we show that the PD-1/ligand engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor/ligand interactions at cell/cell interfaces. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/545075v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@d1ec95org.highwire.dtl.DTLVardef@33c0forg.highwire.dtl.DTLVardef@5bfed7org.highwire.dtl.DTLVardef@1b80ee2_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryUsing CombiCells, a platform for the combinatorial display of cell surface ligands, to compare T cell antigen sensitivity mediated by TCRs, CARs, and BiTEs and its dependence on co-stimulation/co-inhibition receptor ligands

immunology↗

bioRxiv2023

TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)-modified T-cells and call for alternative antigen receptor designs for effective T-cell-based cancer immunotherapy. Here we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousand-fold. TCC-based antigen recognition occurred without adverse non-specific signaling, which is typically observed in CAR-T-cells, and did not depend - unlike sensitized peptide/MHC detection by conventional T-cells - on CD4- or CD8- coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anti-cancer response.

immunology↗

bioRxiv2022

Tuning the potency and therapeutic window of ImmTAC molecules by affinity modulation

T cell engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the re-directed T cell response. This class of bispecifics, exemplified by tebentafusp which has recently shown survival benefit in a randomized phase 3 clinical trial1, bind specific target peptides presented by human leukocyte antigen (HLA) on the cell surface via an affinity-enhanced T cell receptor and can redirect T cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T cell activation, while strong affinity of both targeting and effector domains significantly reduces efficacy. Moreover, by optimising the affinity of both parts of the molecule, it is possible to improve the therapeutic window. These results could be effectively modelled based on kinetic proof-reading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark-state. These findings have important implications for the design of anti-CD3 based bispecifics with optimal biophysical parameters for both activity and specificity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/511676v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@19ac262org.highwire.dtl.DTLVardef@14fb9fcorg.highwire.dtl.DTLVardef@13b5a52org.highwire.dtl.DTLVardef@1a6d096_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Mechanical forces impair antigen discrimination by reducing differences in T cell receptor off-rates

T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic simulations. Consistent with these findings, we show that the best characterized catch-bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination and we suggest this new force-shielding role for the adhesion receptors CD2 and LFA-1. One sentence summaryMechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination

immunology↗

bioRxiv2021

T-cell microvilli simulations show operation near packing limit and impact on antigen recognition.

T-cells are immune cells that continuously scan for foreign-derived antigens on the surfaces of nearly all cells, termed antigen presenting cells (APCs). They do this by dynamically extending numerous protrusions called microvilli (MV) that contain T-cell receptors (TCRs) towards the APC surface in order to scan for antigens. The number, size, and dynamics of these MV, and the complex multi-scale topography that results, play a yet unknown role in antigen recognition. We develop an anatomically informed model of the T-cell/APC interface to elucidate the role of MV dynamics in antigen sensitivity and discrimination. We find that MV surveillance reduces antigen sensitivity compared to a completely flat interface unless MV are stabilized in an antigen-dependent manner and find that MV have only a modest impact on antigen discrimination. The model highlights that MV contacts optimise the competing demands of fast scanning speeds of the APC surface with antigen sensitivity and that T-cells operate their MV near the interface packing limit. Finally, we find that observed MV contact lifetimes can be largely influenced by conditions in the T-cell/APC interface with these lifetimes often being longer than the simulation or experimental observation period. The work highlights the role of MV in antigen recognition. Significance StatementT-cells search for foreign-derived antigens on the surface of antigen presenting cells (APC) by dynamically extending tubular protrusions called microvilli (MV) which form membrane close-contacts. Although known for decades, their role in antigen recognition remains unclear. Guided by recent experiments, we built an anatomically informed stochastic model of MV scanning and compared with a topologically flat interface. We find that MV scanning modestly impacts antigen discrimination, yet it enables T-cells to balance the competing effects of maintaining sensitivity while conducting rapid APC surveillance. The model can reconcile discrepancies in observed MV lifetimes and demonstrates that observed area coverage fractions correspond to geometric packing limits. Our work suggests that MVs promote positive signaling outcomes despite anatomical constraints to close contact formation.

biophysics↗

bioRxiv2021

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can re-direct T cells to target abnormal cells but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here we show that, while CARs have a >100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting-cells (APCs), they have nearly identical sensitivity when antigen is presented as purified protein on artificial surfaces. We next measured the impact of engaging accessory receptors (CD2, LFA-1, CD28, CD27, 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved TCR antigen sensitivity by 125 and 22-fold, respectively, but only improved CAR sensitivity by <5-fold. This differential effect of CD2 and LFA-1 engagement on TCR versus CAR sensitivity was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3{varepsilon} subunit (also known as a TRuC), and fully restored by exchanging the CAR variable domains with the TCR{beta} variable domains (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors, and suggest approaches to increase sensitivity.

immunology↗