Adnane Achour

Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers.

Murine natural killer (NK) cells are inhibited from killing their targets by the interaction between inhibitory, C‐type lectin like Ly49 receptors and major histocompatibility complex (MHC) class I molecules. The receptors have overlapping specificity, and it has been difficult to analyze specific aspects of the interaction between different Ly49 receptors and their respective ligands. We have addressed this problem using tetramers of bacterially expressed, non‐glycosylated, MHC class I molecules refolded with different peptides. Our results indicate that this technology is useful for analysis of Ly49 receptor specificity as well as for monitoring of NK cell subsets, with the following major conclusions emerging from this study: (1) tetramers of H‐2Dd bound the Ly49A receptor; the MHC associated glycan, previously suggested to be involved in recognition by this receptor, is thus not required for Ly49A receptor binding; (2) in support and extension of a recent report indicating peptide selectivity in the recognition of H‐2Kb by Ly49C+ cells, H‐2Kb tetramer binding to Ly49C receptors was strongly influenced by the peptide presented by the MHC class I molecule; (3) tetramer binding allowed visualization of interactions that have not previously been detected in functional studies, such as the recognition of H‐2Db by Ly49A and Ly49C.

PEPTIDE DEPENDENCY AND SELECTIVITY OF THE NK CELL INHIBITORY RECEPTOR LY-49C

MHC class I molecules can prevent NK cell‐mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin‐superfamily receptors, we have studied a system based on the murine NK receptor Ly‐49C in the lectin‐superfamily. Loading of TAP‐deficient RMA‐S cells with the H‐2Kb‐restricted, ovalbumin‐derived peptide OVA257u2009–u2009264 (pOVA) induced their ability to bind Ly‐49C‐transfected reporter cells, and also protected them from killing by Ly‐49C+ NK cells. Other peptides that bound and stabilized H‐2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C‐terminal parts of the α1 helix (73u2009–u200977) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73u2009–u200977. In conclusion, protection mediated by the murine C‐type lectin receptor Ly‐49C is peptide dependent and selective.

Malondialdehyde modification of myelin oligodendrocyte glycoprotein leads to increased immunogenicity and encephalitogenicity

Self proteins may become autoantigenic through structural modification. We studied malondialdehydation of recombinant rat (rr) myelin oligodendrocyte glycoprotein (MOG), an autoantigen in multiple sclerosis. Malondialdehyde (MDA) modification changed protein weight and charge, the location of these adducts being mapped by Fourier transform ion cyclotron resonance. Molecular modelling revealed significant differences in the MDA‐rrMOG three‐dimensional structure. DBA/1 mice immunised with MDA‐rrMOG developed greater proliferative responses and more severe experimental autoimmune encephalomyelitis than mice immunised with unmodified rrMOG. MDA‐rrMOG was taken up more effectively by antigen‐presenting cells (APC), at least partially through scavenger receptors. Exposure to MDA‐rrMOG led to increased expression of IL‐23, IL‐12 and IL‐12R, indicating a role not only for increased antigen uptake but also for activation of APC. We thus provide biochemical, structural, immunological and clinical data that suggest that the post‐translationally modified form of this myelin autoantigen is a more relevant form of the molecule.

A Structural Basis for LCMV Immune Evasion: Subversion of H-2Db and H-2Kb Presentation of gp33 Revealed by Comparative Crystal Structure Analyses

LCMV infection of H-2(b) mice generates a CD8(+) CTL response mainly directed toward three immunodominant epitopes. One of these, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. The virus can escape immune recognition in the context of both these MHC class I molecules through single mutations of the peptide. In order to understand the underlying structural mechanism, we determined the crystal structures of both complexes. The structures reveal that the peptide is presented in two diametrically opposed manners by H-2D(b) and H-2K(b), with residues used as anchor positions in one MHC class I molecule interacting with the TCR in the other. Importantly, the peptides N-terminal residue p1K protrudes from the binding cleft in H-2K(b). We present structural evidence that explains the functional consequences of single mutations found in escape variants.

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge.

Background Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. Principal Findings HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4+ T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. Conclusions Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.

MHC Class I Recognition by NK Receptors in the Ly49 Family Is Strongly Influenced by the β2-Microglobulin Subunit

NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the α1, α2, α3, and β2-microglobulin (β2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the β2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the β2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine β2m to human β2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human β2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of β2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the α1 and α2 domains of the heavy chain.

Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j

Human cytomegalovirus (HCMV) down‐regulates cell surface expression of HLA classu2004I molecules (HLA‐I). UL18, an HCMV‐encoded HLA‐I homologue, has been proposed to protect virus‐infected cells against NK cell recognition by engaging the inhibitory receptor leukocyte Ig‐like receptor (LIR)‐1, which also binds a broad spectrum of HLA‐I alleles, including HLA‐G1. Because genetic and biological differences exist among HCMV strains, we characterized laboratory (AD169) and clinical (4636, 13B, 109B) strain‐derived UL18 proteins. Compared to the known AD169‐derived UL18, mutations were found in clinical strain‐derived UL18. They were clustered in the α3u2004domain (13B), previously shown to be critical for LIR‐1 binding, or in the α1u2004domain (4636). In cytotoxicity assays, pretreatment of LIR‐1+ NKL with soluble 4636‐UL18 completely abolished LIR‐1‐dependent protection from NK lysis, conferred by the expression of HLA‐G1 on target cells (721.221‐HLA‐G1+). Similarly, flow cytometry, Biacore and ELISA experiments showed 4636‐UL18 and 13B‐UL18 to have the strongest binding capacity to LIR‐1. Our results suggest the importance of two independent UL18 regions for LIR‐1 binding, one localized on the tip of the α3u2004domain, and another composed of two loops that emerge from the α1u2004domain. Strain variations in these domains may result in different UL18‐mediated effects on LIR‐1+ cells during the course of HCMV infection.

Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition.

Lymphocytic choriomeningitis virus infection of H-2b mice generates a strong CD8+ CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2Db and H-2Kb MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2Db and H-2Kb MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2Db and H-2Kb. Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.

The identification of a common pathogen‐specific HLA class I A*0201‐restricted cytotoxic T cell epitope encoded within the heat shock protein 65

Bacterial antigens recognized by CD8+ T cells in the context of MHC classu2004I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA‐A*0201‐restricted CD8+ T cell responses against six new high‐affinity HLA‐A*0201‐binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock proteinu200465 (hsp65). One of these epitopes, Mhsp65(9369), is identical in a large number of pathogenic bacteria, and is recognized in a CD8‐independent fashion. Mhsp65(9369) could be presented by either mycobacterial hsp65‐pulsed target cells or BCG‐infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high‐affinity HLA classu2004I‐binding epitopes that are naturally processed and are recognized efficiently by MHC class I‐restricted CD8+ T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.

Murine class I major histocompatibility complex H–2Dd: expression, refolding and crystallization

A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system. It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein. Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions. The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution.