Dominique A. Weber

Enhanced Dissociation of HLA-DR-Bound Peptides in the Presence of HLA-DM

Human leukocyte antigen (HLA)-DM is a critical participant in antigen presentation that catalyzes the release of class II-associated invariant chain-derived peptides (CLIP) from newly synthesized class II histocompatibility molecules, freeing the peptide-binding site for acquisition of antigenic peptides. The mechanism for the selective release of CLIP but not other peptides is unknown. DM was found to enhance the rate of peptide dissociation to an extent directly proportional to the intrinsic rate of peptide dissociation from HLA-DR, regardless of peptide sequence. Thus, CLIP is rapidly released in the presence of DM, because its intrinsic rate of dissociation is relatively high. In antigen presentation, DM has the potential to markedly enhance the rate of peptide exchange, favoring the presentation of peptides with slower intrinsic rates of dissociation.

Analysis of HLA-E peptide-binding specificity and contact residues in bound peptide required for recognition by CD94/NKG2

The MHC class Ib molecule HLA-E is the primary ligand for CD94/NKG2A-inhibitory receptors expressed on NK cells, and there is also evidence for TCR-mediated recognition of this molecule. HLA-E preferentially assembles with a homologous set of peptides derived from the leader sequence of class Ia molecules, but its capacity to bind and present other peptides remains to be fully explored. The peptide-binding motif of HLA-E was investigated by folding HLA-E in vitro in the presence of peptide libraries derived from a nonameric leader peptide sequence randomized at individual anchor positions. A high degree of selectivity was observed at four of five total anchor positions, with preference for amino acids present in HLA-E-binding peptides from class Ia leader sequences. Selectivity was also observed at the nonanchor P5 position, with preference for positively charged amino acids, suggesting that electrostatic interactions involving the P5 side chain may facilitate assembly of HLA-E peptide complexes. The observed HLA-E peptide-binding motif was strikingly similar to that previously identified for the murine class Ib molecule, Qa-1. Experiments with HLA-E tetramers bearing peptides substituted at nonanchor positions demonstrated that P5 and P8 are primary contact residues for interaction with CD94/NKG2 receptors. A conservative replacement of Arg for Lys at P5 completely abrogated binding to CD94/NKG2. Despite conservation of peptide-binding specificity in HLA-E and Qa-1, cross-species tetramer-staining experiments demonstrated that the interaction surfaces on CD94/NKG2 and the class Ib ligands have diverged between primates and rodents.

Regulated Expression of Human Histocompatibility Leukocyte Antigen (HLA)-DO During Antigen-dependent and Antigen-independent Phases of B Cell Development

Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that ∼50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II–associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin.

The phenotype and development of T cells from transgenic mice expressing a T cell receptor with specificity for insulin presented by the MHC class Ib molecule Qa-1(b) was investigated. Peripheral T cells from the transgenic mice express CD8 and, after activation, kill Qa-1(b)-positive lymphoid target cells in the presence of soluble insulin. Thymic selection requires expression of Qa-1(b) but not the dominant Qa-1-associated peptide, Qdm. In contrast to conventional T cells, selection is at least as efficient when the selecting ligand is expressed only on hematopoietic lineage cells as compared to expression on epithelial cells in the thymus. Our findings suggest that there is a dedicated population of Qa-1-restricted T cells that are selected by interaction with Qa-1 and that the cellular requirements for selection may differ from conventional T cells.

Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

Peptide exchange in MHC molecules

Summary: Major histocompatibihty complex (MHC)‐encoded glycoproteins bind peptide antigens through non‐covalent interactions to generate complexes that are displayed on tbe surface of antigen‐presenting cells (APC) for recognition by T ceils, Peptide‐binding site occupancy is necessary for stable assembly of newly synthesized MHC proteins and export from the endoplasmic reticulum (ER), The MHC class II antigen‐processing pathway provides a mechanism for presentation of peptides generated in the endosomal pathway of APC, The chaperone protein, invariant chain, includes a surrogate peptide that stahilizes newly synthesized class II molecules during transport to endosomal compartments. The invariant chain‐derived peptide must be replaced through a peptide exchange reaction that is promoted by acidic pH and the MHC‐encoded co‐factor HLA‐DM, Peptide exchange reactions are not required for presentation of antigens by MHC class I molecules because they bind antigens during initial assembly in the ER, However, exchange reactions may play an important role in editing the repertoire of peptides presented by both class II and class I molecules, thus influencing the specificity of immunity and tolerance.

HLA-DM and the MHC class II antigen presentation pathway

The MHC class II antigen processing pathway provides a mechanism to selectively present peptides generated in the endosomal compartments of antigen presenting cells to CD4+ T cells. Transport of newly synthesized class II molecules to the endosomal pathway requires the function of an accessory protein, invariant chain, which contains a region that interacts directly with the class II peptide binding site. Release of invariant chain and peptide loading by class II molecules are facilitated by a second accessory protein, HLA-DM. This MHC-encoded membrane protein catalyzes peptide exchange reactions, influencing the repertoire of peptides that are available for recognition by T cells.

Guanylate-binding protein-1 is expressed at tight junctions of intestinal epithelial cells in response to interferon-γ and regulates barrier function through effects on apoptosis

Guanylate-binding protein-1 (GBP-1) is an interferon inducible large GTPase involved in endothelial cell proliferation and invasion. In this report, expression and function of GBP-1 were investigated in vitro in intestinal epithelia after exposure to interferon-γ and in human colonic mucosa from individuals with inflammatory bowel disease (IBD). Interestingly, in contrast to other epithelia, GBP-1 distributed to the plasma membrane in intestinal epithelial cells where it colocalized with the tight junction protein coxsackie- and adenovirus receptor. In addition, expression of GBP-1 was upregulated in colonic epithelia of individuals with IBD. Downregulation of GBP-1 by siRNA resulted in enhanced permeability that correlated with increased apoptosis. Indeed, inhibition of caspase activity prevented the inhibition of barrier formation induced by the loss of GBP-1. These data suggest that GBP-1 is a novel marker of intestinal mucosal inflammation that may protect against epithelial apoptosis induced by inflammatory cytokines and subsequent loss of barrier function.

Cutting Edge: HLA-DM Functions through a Mechanism That Does Not Require Specific Conserved Hydrogen Bonds in Class II MHC-Peptide Complexes

HLA-DM catalyzes peptide dissociation and exchange in class II MHC molecules through a mechanism that has been proposed to involve the disruption of specific components of the conserved hydrogen bond network in MHC-peptide complexes. HLA-DR1 molecules with alanine substitutions at each of the six conserved H- bonding positions were expressed in cells, and susceptibility to DM catalytic activity was evaluated by measuring the release of CLIP. The mutants αN62A, αN69A, αR76A, and βH81A DR1 were fully susceptible to DM-mediated CLIP release, and βN82A resulted in spontaneous release of CLIP. Using recombinant soluble DR1 molecules, the amino acid βN82 was observed to contribute disproportionately in stabilizing peptide complexes. Remarkably, the catalytic potency of DM with each β-chain mutant was equal to or greater than that observed with wild-type DR1. Our results support the conclusion that no individual component of the conserved hydrogen bond network plays an essential role in the DM catalytic mechanism.

Qa-1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity

Qa-1, a nonclassical class I histocompatibility molecule expressed in mice, predominantly assembles with a single nonameric peptide, Qdm, derived from the signal sequence of certain class Ia molecules. The Qa-1/Qdm complex is the primary ligand for CD94/NKG2A inhibitory receptors expressed on a major fraction of natural killer (NK) cells. cells become susceptible to killing by NK cells under conditions where surface expression of the Qa-1/Qdm inhibitory ligand is reduced. The CD94/NKG2 “missingself” recognition system serves as mechanism for removing cells that have abnormalities in the intracellular machinery required for assembly and expression of class I-peptides complexes, as a consequence of viral infection, for example. Despite its highly focused peptide-binding specificity, Qa-1 also has a capacity to act as an antigen-presentation molecule for CD8+ T cells. It appears that a small subpopulation of these T cells undergoes positive selection by interaction with Qa-1 in the thymus, and they maintain their specificity for Qa-1 after maturation. The role of these unusual T cells in adaptive immune responses remains to be defined.