Ni Lee

The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association

The class Ib antigen HLA-G is expressed as a membrane-bound protein like classical class Ia molecules (M.HLA-G) but, unlike typical class I, is also expressed as a soluble protein (S.HLA-G) with a unique C terminus. Our results show that, similar to classical class I proteins, the membrane-bound form of HLA-G associated with TAP, as evidenced by the ability to immunoprecipitate HLA-G class I heavy chain with TAP antisera. In contrast, the soluble G protein did not appear to associate with TAP in the same manner, since similar immunoprecipitation experiments failed to detect soluble G complex. A detailed analysis of peptides bound to the soluble and membrane HLA-G proteins expressed in the B lymphoblastoid cell line 721.221 showed that, like class Ia complexes, both HLA-G proteins consist of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. The two proteins bind essentially the same set of peptides, which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptides contain Leu at the C terminus and Pro or small hydrophobic amino acids in position 3 followed by Pro or Gly in position 4. The complexity of the bound peptides is lower than that found for some class Ia complexes, but is more similar to class Ia than to the limited repertoire of some murine class Ib molecules.

HLA-E-BOUND PEPTIDES INFLUENCE RECOGNITION BY INHIBITORY AND TRIGGERING CD94/NKG2 RECEPTORS : PREFERENTIAL RESPONSE TO AN HLA-G-DERIVED NONAMER

The HLA‐E class Ib molecule constitutes a major ligand for the lectin‐like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence‐derived nonapeptides bind to endogenous HLA‐E molecules in the HLA‐defective cell line 721.221, inducing HLA‐E surface expression, and promote CD94/NKG2A‐mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA‐E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA‐E‐bound peptides influences CD94/NKG2‐mediated recognition, beyond their ability to stabilize surface HLA‐E. Further, CD94/NKG2A+ NK clones appeared more sensitive to the interaction with most HLA‐E‐peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA‐E loaded with the HLA‐G‐derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA‐E/G‐nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2‐mediated recognition of HLA‐E expressed on extravillous cytotrophoblasts plays an important role in maternal‐fetal cellular interactions.

Protein Expression and Peptide Binding Suggest Unique and Interacting Functional Roles for HLA-E, F, and G in Maternal-Placental Immune Recognition

In this study we focused on the structure and expression of the HLA-E, F, and G class I complexes in placental tissue. Structural analysis included an examination of the peptides bound to soluble and membrane forms of the HLA-G complex isolated directly from placenta. An important distinction was observed from HLA-G bound peptides previously isolated from transfectant cells. Thus, the number of distinct moieties bound to placental-derived proteins was substantially lower than that bound to transfectant-derived HLA-G. Indeed, a single peptide species derived from a cytokine-related protein alone accounted for 15% of the molar ratio of HLA-G bound peptide. To further examine HLA-E and its potential to bind peptide, notably that derived from HLA-G, we combined new Abs to examine expression in placental tissues for all the known forms of the nonclassical class I molecules. Whereas membrane HLA-G was found in extravillous trophoblasts, soluble HLA-G was found in all placental trophoblasts, including villous cytotrophoblasts and syncitiotrophoblasts. Further, HLA-E was found in all cells that expressed either form of HLA-G, consistent with HLA-E being complexed with the HLA-G signal sequence-derived nonamer in these cells. Finally, using new reagents specific for HLA-F, a restricted pattern of expression was observed, primarily on extravillous trophoblasts that had invaded the maternal decidua. Comparative staining indicated that HLA-F was on the surface of these cells, defining them as the first to demonstrate surface expression of this Ag and the first cell type identified to express all three nonclassical HLA class I Ags simultaneously.

HLA-E Allelic Variants CORRELATING DIFFERENTIAL EXPRESSION, PEPTIDE AFFINITIES, CRYSTAL STRUCTURES, AND THERMAL STABILITIES

Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (ER) is replaced by a glycine in HLA-E*0103 (EG). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-EG complexed with two distinct peptides were determined, and both were compared with the HLA-ERstructure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.

HLA-F Surface Expression on B Cell and Monocyte Cell Lines Is Partially Independent from Tapasin and Completely Independent from TAP

In this study we examined HLA-F expression in normal cells and cell lines, with a particular focus on identifying cells that express surface protein. While HLA-F protein was expressed in a number of diverse tissues and cell lines, including bladder, skin, and liver cell lines, no surface expression could be detected in the majority of them. However, surface expression was observed on EBV-transformed lymphoblastoid cell lines and on some monocyte cell lines. Expression on B lymphoblastoid cell lines was observed, while no surface expression on normal B cells or on any peripheral blood lymphocytes could be detected. Surface expression correlated with the presence of a limited amount of endoglycosidase H (Endo H)-resistant HLA-F. However, clearly not all surface-expressed HLA-F was fully glycosylated. We further examined the requirement of HLA-F surface expression for functional TAP and tapasin molecules and identified a clear departure from the dependence shown by other class I molecules on TAP. In contrast, of the two surface glycosylation forms expressed, an Endo H-sensitive form was tapasin independent, while an Endo H-resistant form was clearly tapasin dependent. Finally, we tested whether HLA-F could be stabilized for surface expression without peptide by using the classical cold treatment for surface stabilization of empty class I. Of several cell lines tested, only MHC deletion mutant 721.221 demonstrated a typical class I phenotype, indicating that control of surface stabilization may have a genetic basis resident in the MHC.

HLA-F and MHC Class I Open Conformers Are Ligands for NK Cell Ig-like Receptors

Killer Ig-like receptors (KIRs) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, whereas other KIR receptor–ligand relationships, including those associated with nonclassical MHC-I, have yet to be clearly defined. We report in this article that KIR3DL2 and KIR2DS4 and the nonclassical Ag HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. These interactions extend to include classical MHC-I open conformers as ligands, defining new relationships between KIR receptors and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers—including prototypical HLA-F—and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.

HLA-F Complex without Peptide Binds to MHC Class I Protein in the Open Conformer Form

HLA-F has low levels of polymorphism in humans and is highly conserved among primates, suggesting a conserved function in the immune response. In this study, we probed the structure of HLA-F on the surface of B lymphoblastoid cell lines and activated lymphocytes by direct measurement of peptide binding to native HLA-F. Our findings suggested that HLA-F is expressed independently of bound peptide, at least in regard to peptide complexity profiles similar to those of either HLA-E or classical MHC class I (MHC-I). As a further probe of native HLA-F structure, we used a number of complementary approaches to explore the interactions of HLA-F with other molecules, at the cell surface, intracellularly, and in direct physical biochemical measurements. This analysis demonstrated that HLA-F surface expression was coincident with MHC-I H chain (HC) expression and was downregulated upon perturbation of MHC-I HC structure. It was further possible to directly demonstrate that MHC-I would interact with HLA-F only when in the form of an open conformer free of peptide and not as a trimeric complex. This interaction was directly observed by coimmunoprecipitation and by surface plasmon resonance and indirectly on the surface of cells through coincident tetramer and MHC-I HC colocalization. These data suggest that HLA-F is expressed independently of peptide and that a physical interaction specific to MHC-I HC plays a role in the function of MHC-I HC expression in activated lymphocytes.

HLA‐F is a surface marker on activated lymphocytes

Of the three nonclassical class I antigens expressed in humans, HLA‐F has been least characterized with regard to expression or function. In this study, we examined HLA‐F expression focusing on lymphoid cells, where our previous work with homologous cell lines had demonstrated surface HLA‐F expression. HLA‐F protein expression was observed by Western blot analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes, all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA‐F intracellularly showed an induction of surface HLA‐F protein. An examination of peripheral blood from individuals genetically deficient for TAP and tapasin expression demonstrated the same activation expression profiles for HLA‐F, but with altered kinetics post‐activation. Further analysis of CD4+CD25+ Treg showed that HLA‐F was not upregulated on the major fraction of these cells when they were activated, whereas CD4+CD25− T cells showed strong expression of surface HLA‐F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA‐F and its potential clinical use as a marker of an activated immune response.

HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1

Based on previous findings supporting HLA-F as a ligand for KIR3DL2 and KIR2DS4, we investigated the potential for MHC-I open conformers (OCs) as ligands for KIR3DS1 and KIR3DL1 through interactions measured by surface plasmon resonance. These measurements showed physical binding of KIR3DS1 but not KIR3DL1 with HLA-F and other MHC-I OC while also confirming the allotype specific binding of KIR3DL1 with MHC-I peptide complex. Concordant results were obtained with biochemical pull-down from cell lines and biochemical heterodimerization experiments with recombinant proteins. In addition, surface binding of HLA-F and KIR3DS1 to native and activated NK and T cells was coincident with specific expression of the putative ligand or receptor. A functional response of KIR3DS1 was indicated by increased granule exocytosis in activated cells incubated with HLA-F bound to surfaces. The data extend a model for interaction between MHC-I open conformers and activating KIR receptors expressed during an inflammatory response, potentially contributing to communication between the innate and adaptive immune response.

HLA-F and MHC-I Open Conformers Cooperate in a MHC-I Antigen Cross-Presentation Pathway

Peptides that are presented by MHC class I (MHC-I) are processed from two potential sources, as follows: newly synthesized endogenous proteins for direct presentation on the surface of most nucleated cells and exogenous proteins for cross-presentation typically by professional APCs. In this study, we present data that implicate the nonclassical HLA-F and open conformers of MHC-I expressed on activated cells in a pathway for the presentation of exogenous proteins by MHC-I. This pathway is distinguished from the conventional endogenous pathway by its independence from TAP and tapasin and its sensitivity to inhibitors of lysosomal enzymes, and further distinguished by its dependence on MHC-I allotype-specific epitope recognition for Ag uptake. Thus, our data from in vitro experiments collectively support a previously unrecognized model of Ag cross-presentation mediated by HLA-F and MHC-I open conformers on activated lymphocytes and monocytes, which may significantly contribute to the regulation of immune system functions and the immune defense.