2D Affinity Research Articles

Strategic approaches to enhance efficiency and commercial feasibility of copper-based surface plasmon resonance sensing

Despite enormous progress in the field of surface plasmon resonance (SPR) imaging sensor technology in the past three decades, noble metals like Au and Ag, despite their drawbacks, continue to be the metals of choice for plasmonic sensing layers. The conventional architecture of the SPR sensor based on gold/silver and glass prism hinders scaling, portability, and industrial manufacturing. In this contribution, we present a novel architecture of SPR sensor using copper on polycarbonate prism as a cost-effective, scalable, and mass-producible alternative. Various optimization techniques, such as interface layer, protective encapsulation, and 2D affinity layer are explored to address the challenges related to the practical application of copper in a plasmonic sensor. Our results show that optimized architectures of SPR sensor based on copper has a sensitivity of 235.01°/RIU. From a quantitative analysis of the interrelationships among various performance parameters, we have derived a comprehensive sensing parameter that integrates signal quality and sensitivity. The proposed architecture of the copper based SPR sensor can lead to inexpensive, compact, and handy probes that do not sacrifice accuracy or reliability.

Human versus mouse Beta 2 microglobulin alters TCR antigen recognition kinetics with mouse MHCI

Abstract There are three isoforms of Beta2 microglobulin (B2m) identified in mice and they differ by a single amino acid residue. To study how B2m can modify the peptide binding pocket of MHCI we turned to the comparison of mouse (mB2m) and human (hB2m). It is known that hB2m forms more stable with mouse MHCI heavy chain as compared to mb2m and hB2m is often the choice for most pMHCI tetramer production. Analysis of mB2m and hB2m tetramer (GP33 41M) following LCMV infection revealed no difference in percentage of tetramer positive cells but an increase was observed in staining intensity (MFI) of hB2m. Using steered molecular dynamics we found that there were allosteric effects with mB2m displaying more movement within the binding pocket than hB2m. Consistent with this we saw increased 2D affinity for antigen MHC D band K bhB2m complexes for OT1 and P14 TCRs, respectively. We also measured the force effects on bond lifetime between TCR and pMHC using biomembrane force probe (BFP) and DNA tension sensor. For OTI, we found that mB2m complexes had a longer bond lifetime measured by both methods. We observed signaling and functional effects consistent with the 2D affinity and bond lifetime measurements in mouse verses human B2m. For example, mB2m leads to increased levels of activated LCK (pY394/505) in OT1 T cells. Therefore, alterations in B2m effect the distal peptide binding domain of MHCI and suggests dynamic allostery may influence antigen recognition by TCR. 5T32NS115664-03 5R01AI147641-03

Single-cell measurements of two-dimensional binding affinity across cell contacts

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/μm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/μm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

Dietary fish oil directly impact viral immunity by modifying CD8 T cell membrane flexibility and bond lifetime under force

Abstract Fish oil supplements enriched in omega-3s are increasingly consumed for their health benefits and exhibit immunosuppressive effects on both innate and adaptive immunity. While omega-3s have been shown to reduced inflammatory metabolites, we focused on the direct effects of a high omega-3 diet on T cell activation during viral infection. Using lymphocytic choriomeningitis (LCMV) infection we analyzed CD8 T cells response to antigen. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to stiffer T cells which subsequently resulted in reduced frequency and activation of the prevalence immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime under force of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicates that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.

Cooperative ectodomain interaction among TCRαβ, CD3δɛ and CD3γɛ

Abstract The T-cell receptor (TCR) complex comprises of the ligand-binding subunit TCRαβ, and the signaling subunits CD3δɛ, CD3γɛ and CD3ζζ, with the Cα/Cβ in proximity to both CD3δɛ and CD3γɛ extracellularly. Direct measurements of the ectodomain interactions had not been successful, although they are believed to be important for TCR stability and functionality. Mechanical force has been shown to modulate TCR–ligand interactions. The TCR mechanosensor hypothesis predicts that force-encoded information may transmit from pMHC to CD3 via TCR-CD3 interaction. Evaluating ectodomain interactions among TCRαβ, CD3δɛ and CD3γɛ can help elucidate the TCR triggering mechanism and further guide the design of TCR-based immunotherapy. Using two mechanical based assays, we were able to measure the weak two-dimensional (2D) affinities among ectodomains of human TCRαβ (2B4-LC13), and human CD3δɛ or CD3γɛ and showed catch bond formation where lifetimes of TCRαβ–CD3δɛ and TCRαβ–CD3γɛ bonds are prolonged by forces <15 pN. Remarkably, CD3δɛ and CD3γɛ bind TCRαβ cooperatively, forming more bonds that last longer when both CD3s interact with TCRαβ as a whole than the sum of either CD3 interacting with TCRαβ individually. Interestingly, these measurements are comparable to 2D affinity and force-dependent bond lifetime of the 2B4 TCR interaction with its cognate ligand K5:I-Ek, supporting their relevance to TCR function. Using molecular dynamics simulations based on a published Cryo-EM structure, we identified the formation of long-lasting CD3δɛ–TCRαβ–CD3γɛ trimolecular bonds as the structural mechanisms of the cooperativity. Our work helps explain TCR function under force and suggests strategies for engineering of TCR for immunotherapy applications.

Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling.

SUMMARYThe interaction of the human FcγRIIA with immune complexes (ICs) promotes neutrophil activation and thus must be tightly controlled to avoid damage to healthy tissue. Here, we demonstrate that a fungal-derived soluble β-1,3/1,6-glucan binds to the glycosphingolipid long-chain lactosylceramide (LacCer) to reduce FcγRIIA-mediated recruitment to immobilized ICs under flow, a process requiring high-affinity FcγRIIA-immunoglobulin G (IgG) interactions. The inhibition requires Lyn phosphorylation of SHP-1 phosphatase and the FcγRIIA immunotyrosine-activating motif. β-glucan reduces the effective 2D affinity of FcγRIIA for IgG via Lyn and SHP-1 and, in vivo, inhibits FcγRIIA-mediated neutrophil recruitment to intravascular IgG deposited in the kidney glomeruli in a glycosphingolipid- and Lyn-dependent manner. In contrast, β-glucan did not affect FcγR functions that bypass FcγR affinity for IgG. In summary, we have identified a pathway for modulating the 2D affinity of FcγRIIA for ligand that relies on LacCer-Lyn-SHP-1-mediated inhibitory signaling triggered by β-glucan, a previously described activator of innate immunity.

Relationship of 2D Affinity to T Cell Functional Outcomes.

T cells are critical for a functioning adaptive immune response and a strong correlation exists between T cell responses and T cell receptor (TCR): peptide-loaded MHC (pMHC) binding. Studies that utilize pMHC tetramer, multimers, and assays of three-dimensional (3D) affinity have provided advancements in our understanding of T cell responses across different diseases. However, these technologies focus on higher affinity and avidity T cells while missing the lower affinity responders. Lower affinity TCRs in expanded polyclonal populations almost always constitute a significant proportion of the response with cells mediating different effector functions associated with variation in the proportion of high and low affinity T cells. Since lower affinity T cells expand and are functional, a fully inclusive view of T cell responses is required to accurately interpret the role of affinity for adaptive T cell immunity. For example, low affinity T cells are capable of inducing autoimmune disease and T cells with an intermediate affinity have been shown to exhibit an optimal anti-tumor response. Here, we focus on how affinity of the TCR may relate to T cell phenotype and provide examples where 2D affinity influences functional outcomes.

Modulating extracellular TCR-CD3 interaction to identify new immunotherapy targets against cancer

Abstract T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCR-CD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for efficacy and toxicity to develop more effective T cell therapies for human targets.

Dietary fats directly impact viral immunity by modifying membrane flexibility and how T cells see antigen

Abstract Dietary supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) have are increasingly consumed for their health benefits and exhibit immunosuppressive effects on both innate and adaptive immunity. Although omega-3s can lead to the production of less inflammatory metabolites, we focused on the direct effects of a high omega 3 diet on T cell activation during viral infection. Specifically, we analyzed T cell recognition of antigen and the resulting functional responses during lymphocytic choriomeningitis (LCMV) infection. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to stiffer T cells which subsequently resulted in reduced frequency and activation of the prevalence immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicates that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.

A Hybrid Insulin Epitope Maintains High 2D Affinity for Diabetogenic T Cells in the Periphery.

β-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29 -42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

TCR-pMHC bond length controls TCR ligand discrimination

Abstract T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-pMHCs to trigger antigen-specific adaptive immune responses. Despite intense efforts, the mechanism underlying TCR ligand discrimination remains a major unanswered question in immunology. Here we show that a TCR discriminates between closely related peptides by forming TCR-pMHC bonds with different lengths, which precisely control the accessibility of CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. Using in situ fluorescence resonance energy transfer (FRET), we measured the intermolecular length of single TCR-pMHC bonds and the intramolecular distance of individual TCR-CD3ζ complexes at the membrane of live primary T cells. We found that an agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3ζ off the inner leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond with the TCR, resulting in partial dissociation of CD3ζ from the membrane and weak phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the entire process of signal reception, transduction and regulation. Thus, our data reveal the fundamental mechanism by which a TCR deciphers the structural differences between foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR signaling for ligand discrimination.

Diets rich in omega-3s decrease CD8 T cell 2-dimensional affinity and short lived effector cell development and function during viral infection

Abstract Omega-3 polyunsaturated fatty acids (n-3 PUFAs), which are key components of fish oil, are increasingly consumed for their health benefits and have been shown to dramatically influence chronic disease. Although omega-3s can lead to the production of less inflammatory metabolites, we focused on the direct effects of a high omega-3 diet on T cell activation during viral infection. Specifically, we analyzed T cell recognition of antigen and the resulting functional responses during lymphocytic choriomeningitis (LCMV) infection. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to reduced T cell frequency and changes in the prevalence of immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicate that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.

2D Kinetic Analysis of TCR and CD8 Coreceptor for LCMV GP33 Epitopes.

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.

Activation induced decrease and recovery of TCR 2D affinity and bond lifetime

Abstract Shortly after encountering antigen, T cells are considered transiently unresponsive or refractory to subsequent simulation. To determine whether antigen stimulation also changes the cell surface biophysical interaction between TCR and pMHC, we assessed the 2D TCR affinity and bond lifetime under force between this receptor ligand pair. Virus (LCMV) specific SMARTA transgenic CD4+ T cells were stimulated with peptide in vitro and 2D TCR affinity and bond lifetime determined at various time points after stimulation using the 2D micropipette adhesion frequency assay and the biomembrane force probe (BFP) respectively. We found activated T cells downregulated 2D affinity at twelve and twenty-four hours after seeing antigen with affinity recovering to naïve levels by forty-eight hours. Unlike pMHC tetramer staining which was dependent on TCR expression levels, the decrease in affinity was independent of antigen dose and degree of TCR downregulation. TCR:pMHC bond lifetime under force was also reduced early after antigen encounter with a steady recovery observed with time. Our data demonstrate a decrease in bond lifetime and relative 2D affinity of TCR and pMHC result from early T cell activation events followed by a recovery phase with both changes occurring independent of activation induced modulation of TCR expression. Thus, TCR affinity for pMHC is dynamic in the 2D T cell membrane context with cellular events allowing the T cell to fine-tune the ability of TCR to interact with antigen.

Profiling TCR affinity distribution in human CD8+ T cell subsets responding to a cytomegalovirus epitope

Abstract CD8 T cell subsets have long been distinguished using phenotypic markers like CCR7, CD45RA, and IL-7Rα. These markers have allowed for the isolation of CD8 T cell populations with distinct functional and proliferative capacities as well as those that maintain better proliferative responses in vivo without antigenic stimulation thus contributing more to immunological memory. Although phenotypic analysis has allowed for the discerning of functional, proliferative, and homeostatic proliferative potential, there is still little known about the role TCR affinity plays in recruitment of various affinity TCRs into memory and effector phenotypes that generate long term immunological memory in human infection. Using the newly developed iTAST technique, CMV specific CD8 T cells were isolated from the blood and spleen of human patients seropositive for CMV. CMV specific T cells were split into memory and effector subsets using phenotypic markers CCR7, CD45RA, and IL-7Rα before isolation and quantification of 2D TCR affinity and sequence. A roughly 100-fold difference of 2D affinity was present in CMV specific cells within the immunological memory response; however, with an enrichment for high affinity TCRs across all phenotypes assessed. This is consistent with previous studies demonstrating high avidity TCR selection in CD8 T cell responses to CMV infection, coupled with a greater clonality observed in CMV specific TCRs within healthy older individuals infected with CMV. Overall the iTAST technique allows for quick and easy isolation of antigen specific populations, quantification of TCR affinity, and TCR sequence thus giving unique insight into immunological memory responses in individuals infected with human persistent viruses.

Autoimmune T cells binds to self antigen weakly in the thymus but greatly upregulate the binding strength in the periphery

Abstract How autoimmune TCRs progress through thymocyte selection to egress in the periphery and induce disease is unclear. Autoimmune TCRs are believed to be of low affinity for self antigens due to the process of thymocyte negative selection. Using an experimental autoimmune encephalomyelitis (EAE) mouse model, we have cloned two tetramer positive myelin oligodendrocyte glycoprotein (MOG)35-55-specific TCRs (named B8 and 1C9) from polyclonal CD4+ T cells; both TCRs have a high two dimensional (2D) affinity for MOG (similar to a pathogen-specific TCR SMARTA for an LCMV epitope gp66-77) by the micropipette adhesion frequency assay. A second critical feature of antigen recognition is the bond lifetime between the TCR and pMHC that occurs under applied force. To this end, we have produced retrogenic mice for the two TCRs and used their thymocytes on biomembrane force probe (BFP) to measure single TCR-pMHC bond lifetimes. Despite their high affinity, both TCRs had short bond lifetimes with the MOG antigen displaying a slip bond where bond lifetimes decrease with increasing force. In contrast, the same TCRs on naïve T cells in the periphery displayed much longer bond lifetimes (up to 100 fold) and strong catch bonds (a counterintuitive type of molecular bond that strengthens under force), suggesting physiological regulation of TCR kinetics within single T cell clones. In comparison, the SMARTA TCR formed indistinguishable catch bonds with gp66-77 on thymocytes and naïve T cells. Therefore, these data identify bond lifetime under force as a unique kinetic property of autoimmune TCRs where weak interactions in the thymus potentially avoid negative selection yet in the periphery the TCRs bind with sufficiently long lifetime to promote autoimmunity.

Force-Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells.

We have recently shown that two-dimensional (2D) and force-regulated kinetics of TCR-peptide-bound MHC class I (pMHC-I) interactions predict responses of CD8(+) T cells. To test whether these findings are applicable to CD4(+) T cells, we analyzed the in situ 3.L2 TCR-pMHC-II interactions for a well-characterized panel of altered peptide ligands on the T cell surface using the adhesion frequency assay with a micropipette and the thermal fluctuation and force-clamp assays with a biomembrane force probe. We found that the 2D effective TCR-pMHC-II affinity and off-rate correlate with, but better predict the T cell response than, the corresponding measurements with the surface plasmon resonance in three dimensions. The 2D affinity of the CD4 for MHC-II was very low, approaching the detection limit, making it one to two orders of magnitude lower than the affinity of CD8 for MHC-I. In addition, the signal-dependent cooperation between TCR and coreceptor for pMHC binding previously observed for CD8 was not observed for CD4. Interestingly, force elicited TCR-pMHC-II catch-slip bonds for agonists but slip-only bonds for antagonists, thereby amplifying the power of discrimination between altered peptide ligands. These results show that the force-regulated 2D binding kinetics of the 3.L2 TCR for pMHC-II determine functions of CD4(+) T cells.

Two dimensional affinity range correlates with phenotypic response to lipid antigens in iNKT cells (IRC4P.600)

Abstract Invariant natural killer T cells (iNKT) are a unique group of T cells that possess an invariant T cell receptor (TCR) alpha chain paired with a limited repertoire of TCR beta chains that recognize lipid antigens presented by the MHC-I like molecule, CD1d. The micropipette adhesion frequency assay was used to define 2D affinities of iNKT cells for a panel of lipid:CD1d antigens. Despite having a limited diversity of αβ TCRs, NKT cells possess approximately a 100-fold range of affinities for the lipids tested. Our results show that αGalCer, an INFγ inducing lipid, has an approximately 10-fold higher affinity than OCH, an IL-4 skewing lipid in B6 mice. Moreover, thymic B6 iNKT cells, which predominantly produce Th1 cytokines (IFNγ), possessed a 4-fold higher affinity as compared to iNKT cells from Balb/C mice that favor more of a Th2 (IL-4) response. iNKT cell phenotype is also affected by their location as cells from the liver of Balb/C mice, which are predominantly IFNγ producers have a 2-fold increase in affinity for αGalCer compared to IL-4 predominant thymic iNKT cells. In addition to affinity differences related to phenotype, TCR expression levels of thymic derived iNKT cells reveal that Balb/C mice express twice as much TCR as the B6 mice. Taken together, these results indicate that NKT2 cytokine producing iNKT cells possess lower 2D affinity and higher TCR density compared to their NKT1 producing counterparts.

Apolipoprotein Α-IV Is a Novel Ligand of Platelet αIIbβ3 Integrin and an Endogenous Thrombosis Inhibitor: Measurement of Single-Molecular Interactions By Biomembrane Force Probe

Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown.A single-molecule technique, biomembrane force probe (BFP), was employed to detect direct interactions between apoA-IV and platelet αIIbβ3 integrin. The BFP adhesion frequency assay demonstrated that apoA-IV bound to αIIbβ3 integrin on ADP treated platelets. ApoA-IV also bound to purified activated αIIbβ3 integrin or the integrin expressed on Chinese hamster ovary (CHO) cells. In comparison, apoA-IV did not significantly bind to αIIbβ3 integrin on resting platelets, GPIb-complex expressed on CHO cells, αMβ2 integrin expressed on K562 cells, nor purified α5β1 and αvβ3 integrins. Importantly, apoA-IV-αIIbβ3 interactions in these experiments could be completely inhibited by a blocking monoclonal antibody (M1) against β3 integrin. These data clearly demonstrated the specificity of apoA-IV for αIIbβ3 integrin. Furthermore, 2D kinetics measurements revealed that the effective 2D affinity of apoA-IV-αIIbβ3 is 43% of that between fibrinogen and αIIbβ3. The BFP competition assay showed that apoA-IV competitively inhibited fibrinogen-αIIbβ3 interactions at its physiological concentration.Platelet functional studies in vitro showed that recombinant apoA-IV significantly inhibited both mouse and human platelet aggregation following stimulation with various agonists. Consistently, platelet aggregation in platelet rich plasma of apoA-IV deficient mice (apoA-IV-/-) was enhanced. Depletion of human plasma apoA-IV also enhanced ADP-induced human platelet aggregation. In ex vivo perfusion chambers, recombinant apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV in blood enhanced ex vivo thrombus growth under both low and high shear stresses. Using two in vivo intravital microscopy thrombosis models and a carotid artery thrombosis model, we demonstrated that FeCl3- and laser-induced thrombosis were enhanced in apoA-IV-/-mice, while transfusion of recombinant apoA-IV markedly attenuated this process. In addition, we found recombinant apoA-IV significantly decreased platelet P-selectin expression, and consistently more P-selectin expression was observed on ADP treated platelets from apoA-IV-/- mice, suggesting that apoA-IV occupancy may inhibit fibrinogen or other prothrombotic ligands mediated αIIbβ3 outside-in signaling.We further found that the N-terminus of apoA-IV plays a key role in its inhibitory function and the exposure of N-terminus is negatively regulated by its C-terminus. Furthermore, mutation of the two aspartic acid (D) residues at apoA-IV N-terminal 5 and 13 abolished its binding for αIIbβ3 integrin as demonstrated by BFP adhesion frequency assay, resulting in the loss of these inhibitory effects.These findings suggest that D5 and D13 of apoA-IV are the potential binding sites for αIIbβ3 integrin.Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a new link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases. These findings may also contribute to hemostasis, P-selectin mediated postprandial platelet activation and inflammation. DisclosuresNo relevant conflicts of interest to declare.

281: Assembly of IFNAR1/IFNAR2 is regulated by ubiquitin specific protease 18

Type I Interferons (IFN) are key cytokines in the regulation of the innate immune response. All IFNs bind to a shared cell surface receptor comprised of two subunits, IFNAR1 and IFNAR2. Together with IFN the receptors assemble to hetero-trimeric “ternary” complexes which initiate phosphorylation cascades through the intracellularly associated Janus-Kinases Jak1 and Tyk2. Detailed structure–function analysis of IFNs has established that the dynamics and affinities of IFN interactions with the receptor subunits play a critical role for regulating signalling specificities. Here we have explored the IFN-induced assembly of the ternary signalling complex at physiological expression level by dual colour single molecule imaging. To this end, we employed highly specific and efficient orthogonal posttranslational labelling approaches with photostable organic fluorophores combined with TIRF-microscopy. By single molecule co-localization and co-tracking analysis we have mapped lateral distribution and diffusion of the two receptor subunits with very high spatial and temporal resolution in living cells. Thus, we were able to monitor IFN-induced assembly, co-diffusion and dissociation of individual ternary signalling complexes in the plasma membrane. Systematic modulation of the binding affinities towards the receptor subunits revealed that binding affinity of IFNα2 towards IFNAR1 is optimized for efficient ternary complex formation at physiological receptor surface concentrations. Interestingly, the negative feedback regulator ubiquitin-specific protease 18 (USP18) reduces receptor dimerization by IFNα2, but not IFNβ, which binds to IFNAR1 with higher affinity. Strikingly, this USP18-dependent loss of 2D affinity could be mimicked by truncating the receptor’s intracellular domains. These observations suggest that ternary complexes are stabilized by intracellular interactions of Jak1 and Tyk2 and that these interactions are interfered by USP18, thus selectively abrogating signalling by IFNs with low affinity towards IFNAR1.