Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Noise Reduction and Image Quality Improvement of Low Dose and Ultra Low Dose Brain Perfusion CT by HYPR-LR Processing

To evaluate image quality and signal characteristics of brain perfusion CT (BPCT) obtained by low-dose (LD) and ultra-low-dose (ULD) protocols with and without post-processing by highly constrained back-projection (HYPR)–local reconstruction (LR) technique.Simultaneous BPCTs were acquired in 8 patients on a dual-source-CT by applying LD (80 kV,200 mAs,14×1.2 mm) on tube A and ULD (80 kV,30 mAs,14×1.2 mm) on tube B. Image data from both tubes was reconstructed with identical parameters and post-processed using the HYPR-LR. Correlation coefficients between mean and maximum (MAX) attenuation values within corresponding ROIs, area under attenuation curve (AUC), and signal to noise ratio (SNR) of brain parenchyma were assessed. Subjective image quality was assessed on a 5-point scale by two blinded observers (1:excellent, 5:non-diagnostic).Radiation dose of ULD was more than six times lower compared to LD. SNR was improved by HYPR: ULD vs. ULD+HYPR: 1.9±0.3 vs. 8.4±1.7, LD vs. LD+HYPR: 5.0±0.7 vs. 13.4±2.4 (both p<0.0001). There was a good correlation between the original datasets and the HYPR-LR post-processed datasets: r = 0.848 for ULD and ULD+HYPR and r = 0.933 for LD and LD+HYPR (p<0.0001 for both). The mean values of the HYPR-LR post-processed ULD dataset correlated better with the standard LD dataset (r = 0.672) than unprocessed ULD (r = 0.542), but both correlations were significant (p<0.0001). There was no significant difference in AUC or MAX. Image quality was rated excellent (1.3) in LD+HYPR and non-diagnostic (5.0) in ULD. LD and ULD+HYPR images had moderate image quality (3.3 and 2.7).SNR and image quality of ULD-BPCT can be improved to a level similar to LD-BPCT when using HYPR-LR without distorting attenuation measurements. This can be used to substantially reduce radiation dose. Alternatively, LD images can be improved by HYPR-LR to higher diagnostic quality

Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation

Modulation of NKp30- and NKp46-Mediated Natural Killer Cell Responses by Poxviral Hemagglutinin

Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection

Multiple receptors trigger human NK cell-mediated cytotoxicity against porcine chondrocytes.

Xenotransplantation of genetically engineered porcine chondrocytes may provide a therapeutic solution for the repair of cartilage defects of various types. However, the mechanisms underlying the humoral and cellular responses that lead to rejection of xenogeneic cartilage are not well understood. In this study, we investigated the interaction between human NK cells and isolated porcine costal chondrocytes (PCC). Our data show that freshly isolated NK cells adhere weakly to PCC. Consequently, PCC were highly resistant to cytolysis mediated by freshly isolated NK cells. However, the presence of human natural Abs in the coculture was often sufficient to trigger cytotoxicity against PCC. Furthermore, IL-2 stimulation of NK cells or activation of PCC with the proinflammatory cytokines TNF-alpha or IL-1 alpha resulted in increased adhesion, which was paralleled by increased NK cell-mediated lysis of PCC. NK cell adhesion to PCC could be blocked by Abs against human LFA-1 and porcine VCAM-1. NKG2D and NKp44 were involved in triggering cytotoxicity against PCC, which expressed ligands for these activating NK cell receptors. Our data further suggest that NKp30 and NKp46 may contribute to the activation of NK cells by PCC under certain conditions. Finally, comparative studies confirmed that PCC are more resistant than porcine aortic endothelial cells to human NK cell-mediated lysis. Thus, the data demonstrate that human NK cells can kill pig chondrocytes and may therefore contribute to rejection of xenogeneic cartilage. In addition, we identify potential targets for intervention to prevent the NK cell response against pig xenografts