Ayub Qadri

Lysophospholipid sensing triggers secretion of flagellin from pathogenic salmonella

Flagellin induces inflammatory and innate immune responses through activation of Toll-like receptor 5. Here we show that proinflammatory monomeric flagellin produced by salmonella during infection of intestinal epithelial cells was not derived from polymeric bacterial cell wall–associated flagellum but instead was synthesized and secreted de novo by the bacterium after direct sensing of host-produced lysophospholipids. Inhibition of lysophospholipid biosynthesis in intestinal epithelial cells reduced flagellin production and release from salmonella. Lysophospholipids induced a cAMP-dependent signaling pathway in salmonella that resulted in production and secretion of active flagellin. The induction of Toll-like receptor ligand synthesis and secretion by a host signal represents a previously unknown regulatory mechanism for inflammation and innate immunity during infection with a bacterial pathogen.

Biophysical and structural studies of TCRs and ligands: implications for T cell signaling

The availability of soluble alphabeta TCRs and the individual chains has now made it possible to carry out structural studies of these molecules and analyze their molecular interactions with peptide-MHC ligands. Recent X-ray crystallographic structures of TCR alpha and beta chains have finally established their structural similarity with the lg molecules. Kinetic measurements of the interaction between TCRs and their ligands have provided strong evidence in favour of an affinity/avidity model for T cell activation in the periphery as well as during development in the thymus.

T Cell Recognition of Distinct Peptide:I-Au Conformers in Murine Experimental Autoimmune Encephalomyelitis

We have used T cells bearing TCRs that are closely related in sequence as probes to detect conformational variants of peptide-MHC complexes in murine experimental autoimmune encephalomyelitis in H-2u mice. The N-terminal epitope of myelin basic protein (MBP) is immunodominant in this model. Our studies have primarily focused on T cell recognition of a position 4 analog of this peptide (MBP1–9[4Y]) complexed with I-Au. Using site-directed mutagenesis, we have mapped the functionally important complementarity determining region residues of the 1934.4 TCR Vα domain. One of the resulting mutants (Tyr95 to alanine in CDR3α, Y95A) has interesting properties: relative to the parent wild-type TCR, this mutant poorly recognizes Ag complexes generated by pulsing professional APCs (PL-8 cells) with MBP1–9[4Y] while retaining recognition of MBP1–9[4Y]-pulsed unconventional APCs or insect cell-expressed complexes of I-Au containing tethered MBP1–9[4Y]. Insect cell expression of recombinant I-Au with covalently tethered class II-associated invariant chain peptide or other peptides which bind relatively weakly, followed by proteolytic cleavage of the peptide linker and replacement by MBP1–9[4Y] in vitro, results in complexes that resemble peptide-pulsed PL-8 cells. Therefore, the distinct conformers can be produced in recombinant form. T cells that can distinguish these two conformers can also be generated by the immunization of H-2u mice, indicating that differential recognition of the conformers is observed for responding T cells in vivo. These studies have relevance to understanding the molecular details of T cell recognition in murine experimental autoimmune encephalomyelitis. They are also of particular importance for the effective use of multimeric peptide-MHC complexes to characterize the properties of Ag-specific T cells.

Sandwich enzyme immunoassays for detection of Salmonella typhi

Enzyme immunoassays were developed using monoclonal antibodies raised against somatic (O), flagellar (H) and capsular (Vi) antigens of Salmonella typhi. The assay based on anti-O monoclonal antibodies could specifically detect S. typhi and soluble lipopolysaccharide (LPS) isolated from S. typhi. Anti-H MoAbs detected motile S. typhi and soluble flagellar antigen. Monoclonal antibodies against capsular polysaccharide could detect Vi-containing S. typhi as well as soluble Vi antigen. The three assays reported here detected S. typhi with 100% sensitivity in blood culture broths obtained from bacteriologically confirmed typhoid patients and were negative with blood specimens containing Salmonella senftenberg, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis or Streptococcus (alpha-hemolytic) derived from patients with pyrexia. The assays, however, did not demonstrate the presence of soluble antigens in sera and urine samples obtained from typhoid patients.

Monoclonal antibodies against two discrete determinants on Vi capsular polysaccharide.

Vi is a linear homopolymer of 1,4 N-acetyl galactosaminuronic acid. It is present in S. typhi and some other members of Enterobacteriaceae. Vi antigen of S. typhi has been associated with the virulence of the organism and a vaccine based upon this antigen has been found to confer immunity against typhoid. In this paper, we report production and characterization of four hybrid cell clones secreting monoclonal antibodies against Vi capsular polysaccharide. Binding analysis using different derivatives of Vi showed that three monoclonal antibodies reacted with the antigenic determinant constituted by O-acetyl group and one recognised the epitope constituted by N-acetyl and carboxyl groups together. All the antibodies bound to Vi positive strains of S. typhi and did not show any significant reactivity with Vi negative strains of S. typhi, S. paratyphi A, S. paratyphi B and E. coli. Besides their utility in studying the sub-specificity of antibodies produced after vaccination with Vi, these antibodies would be helpful in the diagnosis of typhoid fever.

Hemoglobin Transforms Anti-Inflammatory Salmonella typhi Virulence Polysaccharide into a TLR-2 Agonist

Vi capsular polysaccharide is a major virulence determinant of the human typhoid- causing pathogen Salmonella typhi; it is absent in nontyphoidal Salmonella serovars. We show in this study that through its specific interaction with the membrane recognition complex containing the prohibitin family of molecules, Vi can inhibit the production of inflammatory cytokines from mononuclear phagocytes stimulated with Salmonella flagellin. Remarkably, Vi lost this anti-inflammatory capability and switched to a proinflammatory state when cell stimulations were performed in the presence of serum. The serum-transformed proinflammatory form of Vi induced secretion of cytokines from monocytes by specifically engaging TLR-2/TLR-1. The molecule responsible for bringing about this conversion of Vi from an anti-inflammatory to a proinflammatory form was serum-derived hemoglobin. Derivatives of Vi incapable of interacting with hemoglobin did not switch to a proinflammatory state in vitro or in vivo. These findings provide compelling evidence for a role of hemoglobin in transforming the anti-inflammatory S. typhi virulence polysaccharide into an immune activator.

Src kinases central to T-cell receptor signaling regulate TLR-activated innate immune responses from human T cells

TLRs have a fundamental role in immunity. We have recently reported that stimulation of TLR2 and TLR5 in freshly isolated and activated human T cells with microbial ligands without concomitant activation through the TCR brings about secretion of neutrophil chemoattractant, CXCL8, and effector cytokine, IFN-γ, respectively. However, the mechanism of TLR signaling in T cells has not been worked out. Here, we show that the Src family kinases, p56lck (Lck) and p59fyn (Fyn), which are essential for activation of T cells through the TCR, are also critical for signal transduction through TLRs in human T cells. The secretion of CXCL8 following stimulation of the model human T cell line, Jurkat, with the TLR5 ligand, flagellin, was reduced in presence of the Src-kinase inhibitor, PP2 and specific inhibitors of Lck and Fyn. These inhibitors suppressed generation of activated JNK and p38, which were both required for TLR-induced CXCL8 production. The Lck-deficient derivative of Jurkat, JCam1.6, responded poorly to TLR2, TLR5 and TLR7 agonists, and did not generate active signaling intermediates. Lck and Fyn inhibitors also reduced TLR5-induced IFN-γ secretion from the activated T cell phenotype-representing T cell line, HuT78, without modulating JNK and p38 activation. These results reveal that TCR and TLRs share key proximal signaling regulators in T cells.

Identification of specific recognition molecules on murine mononuclear phagocytes and B lymphocytes for Vi capsular polysaccharide: modulation of MHC class II expression on stimulation with the polysaccharide

Vi bacterial polysaccharide is a homopolymer of α1–4 N‐acetyl polygalacturonic acid with variable O‐acetylation at position C‐3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose‐dependent binding to the murine monocyte cell lines WEHI‐274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B‐cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP‐1. Stimulation with Vi led to up‐regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi‐bound molecules from cell surface biotinylated A20 and WEHI‐274.1 revealed two bands with MW of about 32 000 and 36 000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.

A Role for the Region Encompassing the c″ Strand of a TCR Vα Domain in T Cell Activation Events

The distinct strand topology of TCR Vα domains results in a flatter surface in the region encompassing the c″ strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at Vα residue 82. This residue is in proximity to the c″ strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the Vα domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4+ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8hiIFN‐γnull in freshly isolated to CXCL8loIFN‐γhi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4+ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.