Effect of pulmonary surfactant on innate immune responses in influenza virus infected human airway epithelial cells

Overwhelming inflammatory responses leading to neutrophil invasion are hypothesised to be the main cause of mortality in influenza virus induced acute respiratory distress syndrome (ARDS). Previously, pulmonary surfactant has been shown to modulate inflammatory responses to bacterial agents. The aim of the present study was to investigate the effect of pulmonary surfactant on innate immune responses in an in vitro model of influenza virus infected human airway epithelial cells. Human lung type II alveolar epithelial cells A549 and BEAS-2B human bronchial epithelial cells were infected with influenza A virus H1N1 strains A/Swine/1976/31, A/WSN/33 and A/PR/8/34. Poly I:C, Escherichia coli Ol 11 :B4 LPS and measles virus strain Edmonston were used as cytokine stimulation controls. The effect of pulmonary surfactant was compared to that of dexamethasone. This in vitro study showed that physiological concentrations (up to 500 ug/ml) of clinically approved SP-A and SP-D depleted surfactant preparations (i) were non-toxic in BEAS-2B cells, (ii) had no effect on influenza virus infectivity, and (iii) reduced influenza virus induced cytokine production comparable to dexamethasone. Porcine Curosurf* significantly inhibited IL-8 and RANTES production in A/WSN/33 infected cells, by 30 and 35% respectively (p<0.05). Bovine Survanta* had a less pronounced effect. In luciferase reporter assays pulmonary surfactant, in contrast to dexamethasone, non-specifically inhibited both TLR3/RIG-I mediated NF-kappaB promoter activation and IFN-beta promoter activation. Our results indicate that SP-A and SP-D depleted surfactant preparations attenuate pro-inflammatory responses in influenza A virus infected human airway epithelial cells, but inhibitory effects on IFN-beta promoter activity were also observed. This suggests that pulmonary surfactant may be of clinical benefit in reducing pro-inflammatory responses in virus induced ARDS, however, a weakening of IFN-beta mediated anti-viral responses can not be excluded

Structural basis for ineffective T-cell responses to MHC anchor residue-improved 'heteroclitic' peptides

MHC anchor residue-modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery

TCR/pMHC optimized protein crystallization screen

The interaction between the clonotypic αβ T cell receptor (TCR), expressed on the T cell surface, and peptide-major histocompatibility complex (pMHC) molecules, expressed on the target cell surface, governs T cell mediated autoimmunity and immunity against pathogens and cancer. Structural investigations of this interaction have been limited because of the challenges inherent in the production of good quality TCR/pMHC protein crystals. Here, we report the development of an ‘intelligently designed’ crystallization screen that reproducibly generates high quality TCR/pMHC complex crystals suitable for X-ray crystallographic studies, thereby reducing protein consumption. Over the last 2 years, we have implemented this screen to produce 32 T cell related protein structures at high resolution, substantially contributing to the current immune protein database. Protein crystallography, used to study this interaction, has already extended our understanding of the molecular rules that govern T cell immunity. Subsequently, these data may help to guide the intelligent design of T cell based therapies that target human diseases, underlining the importance of developing optimized approaches for crystallizing novel TCR/pMHC complexes