Laurent Gauthier

Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1s binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.

Kinetics of T-cell Receptor Binding by Bivalent HLA-DR·Peptide Complexes That Activate Antigen-specific Human T-cells

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2·myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2·MBP peptide complexes. The binding of bivalent HLA-DR2·peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t 1 2 of 2.1 to 4.6 min. Soluble bivalent HLA-DR2·MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR·CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2·MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR·peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

Anergy Induction by Dimeric TCR Ligands

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.

Binding of conserved islet peptides by human and murine MHC class II molecules associated with susceptibility to type I diabetes

The major histocompatibility complex (MHC) is the most important susceptibility locus for type I diabetes in humans and NOD mice. NOD mice express a single MHC class II molecule (I‐Ag7) which carries a unique β chain sequence. In humans, DQ alleles that encode DQ8 and DQ2 confer the highest risk for the disease. Soluble DQ8 and I‐Ag7 were used to directly compare the binding specificity of these MHC molecules. Peptides from three islet antigens – insulin, GAD 65 and HSP 60 – bound to both CQ8 and I‐Ag7. These peptides included epitopes that are immunodominant in NOD mice, namely insulin (9u2009–u200923), GAD (206u2009–u2009220) and HSP 60 (441u2009–u2009460). All of these peptide sequences are highly conserved between the human and murine antigens. The binding specificity of DQ8 and I‐Ag7 was similar, but not identical, since two peptides eluted from splenocytes of NOD mice did not bind to DQ8. DQ8 formed long‐lived complexes with the majority of these peptides, indicating that DQ8 is not a poor peptide binder. These results demonstrate functional similarities between human and murine MHC class II molecules that confer susceptibility to type I diabetes.

Cutting Edge: Characterization of Allorestricted and Peptide-Selective Alloreactive T Cells Using HLA-Tetramer Selection

The vast majority of alloreactive T cells recognize foreign MHC molecules in a peptide-dependent manner. A subpopulation of these peptide-dependent alloreactive T cells is peptide-specific and contains T cells that are of interest for tumor immunotherapy. Allorestricted T cells (i.e., peptide-specific and alloreactive) specific for tumor-associated Ags can be raised in vitro. However, it is technically difficult to distinguish between peptide-specific and peptide-nonspecific alloreactive T cells by functional assays in vitro. Here we show for the first time that allorestricted T cells specifically bind HLA-peptide tetrameric complexes, as nominal Ag-specific T cells would do. In consequence, fluorescent HLA-peptide tetrameric complexes can be used for sorting and cloning of allorestricted CTLs specific for a peptide of interest. We also show by the mean of HLA-peptide tetramers the existence of peptide-selective alloreactive T cells that recognize a conformation on the foreign-MHC brought about by some but not all peptides bound.

Structural Basis for Galectin-1-dependent Pre-B Cell Receptor (Pre-BCR) Activation.

Background: Galectin-1 (GAL1) is a ligand for the pre-BCR which is involved in the proliferation and differentiation of normal pre-BII cells. Results: GAL1-dependent pre-BCR clustering is driven mainly by hydrophobic contacts. Conclusion: Constitutive and ligand-induced pre-BCR activation can occur in a complementary manner. Significance: This is the first molecular snapshot of a pre-BCR/ligand interaction that helps pre-BCR clustering and activation. During B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Igμ) and a surrogate light chain composed of the invariant λ5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of λ5 (λ5-UR). Here, we investigated the solution structure of a minimal λ5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the λ5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner.

The D0 Ig-like Domain Plays a Central Role in the Stronger Binding of KIR3DL2 to B27 Free H Chain Dimers

We proposed that the killer cell Ig-like receptor KIR3DL2 binding more strongly to HLA-B27 (B27) β2-microglobulin free H chain (FHC) dimers than other HLA–class I molecules regulates lymphocyte function in arthritis and infection. We compared the function of B27 FHC dimers with other class I H chains and identified contact residues in KIR3DL2. B27 FHC dimers interacted functionally with KIR3DL2 on NK and reporter cells more strongly than did other class I FHCs. Mutagenesis identified key residues in the D0 and other Ig-like domains that were shared and distinct from KIR3DL1 for KIR3DL2 binding to B27 and other class I FHCs. We modeled B27 dimer binding to KIR3DL2 and compared experimental mutagenesis data with computational “hot spot” predictions. Modeling predicts that the stronger binding of B27 dimers to KIR3DL2 is mediated by nonsymmetrical complementary contacts of the D0 and D1 domains with the α1, α2, and α3 domains of both B27 H chains. In contrast, the D2 domain primarily contacts residues in the α2 domain of one B27 H chain. These findings provide novel insights about the molecular basis of KIR3DL2 binding to B27 and other ligands and suggest an important role for KIR3DL2–B27 interactions in controlling the function of NK cells in B27+ individuals.