Dissecting the structural determinants of the interaction between the human cytomegalovirus UL18 protein and the CD85j immune receptor

UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC class I and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its alpha3 domain with an affinity that is approximately 1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. In this study we first demonstrated that the UL18alpha3 domain is not sufficient per se for binding and that beta2-microglobulin is necessary for UL18-CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the alpha2 and alpha3 domains and specific amino acids within them were identified that are potentially involved in the UL18-CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules.     

Crystal structure of LIR-2 (ILT4) at 1.8 Å: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18

Background

Leukocyte Immunoglobulin-like Receptor-1 (LIR-1) and LIR-2 (also known as ILT2 and ILT4 respectively) are highly related cell surface receptors that bind a broad range of class I MHC molecules with low (μM) affinities. Expressed on monocytic cells and macrophages, both molecules transmit inhibitory signals after binding ligands. In addition to binding host class I MHC, the LIR-1 molecule, which is also expressed on lymphoid tissues, binds with a high (nM) affinity to UL18, a class I MHC homolog encoded by Human Cytomegalovirus (HCMV). In comparison, LIR-2 binds UL18 only weakly (μM K_D). To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.

Results

Recombinant LIR-2 (domains 1 and 2) was produced in E. coli and crystallized using streak seeding to optimize the crystal morphology. A data set complete to 1.8 Å was collected at 100 K from a single crystal in the P4₁2₁2 spacegroup. The structure was solved by molecular replacement, using a search model based on the LIR-1 structure.

Conclusions

The overall structure of LIR-2 D1D2 resembles both LIR-1, and Killer Inhibitory Receptors, in that the A strand in each domain forms hydrogen bonds to both β sheets, and there is a sharp angle between the two immunoglobulin-like domains. However, differences from LIR-1 are observed in each domain, with two key changes apparent in the ligand-binding domain, D1. The region corresponding to the residue 44–57 helix of LIR-1 adopts a topology distinct from that of both LIR-1 and the KIR structures, involving a shortened 3₁₀ helix. Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76–84 loop mainchain is displaced 11 Å with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation. In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.     

Genetic variability of the major histocompatibility complex class I homologue encoded by human cytomegalovirus leads to differential binding to the inhibitory receptor ILT2

Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+ but activates LIR-1- NK cells

The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1(+) NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (DeltaUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-alpha-activated NK bulk cultures derived from a donor with a high frequency of LIR-1(+) NK cells. A single LIR-1(high) NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1(+) NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1(-) NK cells. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent.     

Increased expression of leukocyte Ig-like receptor-1 and activating role of UL18 in the response to cytomegalovirus infection

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-gamma, produced mainly by LIR-1(+) T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus, suggesting a stimulatory role for UL18. However, purified UL18Fc proteins inhibited IFN-gamma production of LIR-1(+) T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1, which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses, this unlikely plays a role in response to infected cells. Instead, our data point to an activating role for viral UL18 during infection, where indirect intracellular effects cannot be excluded.     

The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.     

Crystal Structure of Myeloid Cell Activating Receptor Leukocyte Ig-like Receptor A2 (LILRA2/ILT1/LIR-7) Domain Swapped Dimer: Molecular Basis for Its Non-binding to MHC Complexes

The leukocyte Ig-like receptor (LILR/ILT/LIR) family comprises 13 members that are either activating or inhibitory receptors, regulating a broad range of cells in the immune responses. LILRB1 (ILT2), LILRB2 (ILT4) and LILRA1 (LIR6) can recognize MHC (major histocompatibility complex) class I or class I-like molecules, and LILRB1/HLA-A2, LILRB1/UL18 and LILRB2/HLA-G complex (extracellular domains D1D2) structures have been solved recently. The details of binding to MHC have been described. Despite high levels of sequence similarity among LILRA1, LILRA2 (ILT1), LILRA3 (ILT6) and LILRB1/B2, all earlier experiments showed that LILRA2 does not bind to MHC, but the reason is unknown. Here, we report the LILRA2 extracellular D1D2 domain crystal structure at 2.6 Å resolution, which reveals structural shifts of the corresponding MHC-binding amino acid residues in comparison with LILR B1/B2, explaining its non-binding to MHC molecules. We identify some key residues with great influence on the local structure, which exist only in the MHC-binding receptors. Moreover, we show that LILRA2 forms a domain-swapped dimer. Further work with these key swapping residues yields a monomeric form, confirming that the domain-swapping is primarily amino acid sequence-specific. The structure described here supports the dimer conformation in solution observed earlier, and implies a stress-induced regulation by dimerization, consistent with its function as a heat shock promoter.     

Cell surface expression of MR1B,a splice variant of the MHC class I-related molecule MR1, revealed with antibodies

The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints.     

Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells.

Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage-displayed hexapeptide library, and virus-neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C-terminal segment of the alpha2 domain of the heavy chain of human MHC class I molecules (MHC-I alpha2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST-fused MHC-I alpha2- and FNIII-derived oligopeptides interacted with recombinant fibers in a subgroup-specific manner. In vivo, the MHC-I alpha2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In beta2-microglobulin-transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4x10(10)/M. The results suggested that the conserved region of MHC-I alpha2-domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors.     

Complexes of HLA-G protein on the cell surface are important for leukocyte Ig-like receptor-1 function

The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophoblast cells at the maternal-fetal interface during pregnancy. HLA-G can inhibit the killing mediated by NK cells via interaction with the inhibitory NK cell receptor, leukocyte Ig-like receptor-1 (LIR-1). Comparison of the sequence of the HLA-G molecule to other class I MHC proteins revealed two unique cysteine residues located in positions 42 and 147. Mutating these cysteine residues resulted in a dramatic decrease in LIR-1 Ig binding. Accordingly, the mutated HLA-G transfectants were less effective in the inhibition of NK killing and RBL/LIR-1 induced serotonin release. Immunoprecipitation experiments demonstrated the involvement of the cysteine residues in the formation of HLA-G protein oligomers on the cell surface. The cysteine residue located at position 42 is shown to be critical for the expression of such complexes. These oligomers, unique among the class I MHC proteins, probably bind to LIR-1 with increased avidity, resulting in an enhanced inhibitory function of LIR-1 and an impaired killing function of NK cells.     

Mapping the ligand of the NK inhibitory receptor Ly49A on living cells

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2D(d), H-2D(k), and H-2D(p) but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2(u)) not only bound Ly49A but also inhibited cytolysis by Ly49A(+) effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2D(d)-bearing cells of different lymphoid tissues was proportional to the level of H-2D(d) expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2D(d) complex, suggest a role for amino acid residues at the amino-terminal end of the alpha1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2D(d) mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.     

Crystal structure of the murine cytomegalovirus MHC-I homolog m144

Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/beta2-microglobulin (beta2m) complex at 1.9A resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable alpha1, alpha2, and alpha3 domains. A unique disulfide bond links the alpha1 helix to the beta-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the alpha1 and alpha2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the alpha2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.     

MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-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 alpha1, alpha2, alpha3, and beta2-microglobulin (beta2m) 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 beta2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the beta2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine beta2m to human beta2m 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 beta2m, 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 beta2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the alpha1 and alpha2 domains of the heavy chain.     

Differential binding properties of human pregnancy zone protein- and alpha2-macroglobulin-proteinase complexes to low-density lipoprotein receptor-related protein.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Both alpha-macroglobulins (alpha-Ms) covalently bind proteinases, which is accompanied by the exposure of carboxy terminal receptor recognition domains important for the rapid clearance from the circulation and tissues. It is accepted that the molecule responsible for the clearance of alpha2-M- and PZP-proteinase complexes is the low-density lipoprotein receptor-related protein (LRP). Although both alpha-M-proteinase complexes bind to the same receptor, differences in the binding properties have been reported. In addition, although it is known that the binding of alpha2-M-proteinase complexes to LRP can be blocked by Ni2+, the effect on PZP-proteinase has never been examined. In order to investigate differences in the binding properties of both alpha-Ms to the receptor, we purified LRP from human placenta by affinity chromatography and then analyzed the specificity and affinity of binding of alpha2-M- and PZP-proteinase complexes to the receptor by enzyme immunoassay. Our results clearly established that although both alpha-M-proteinase complexes specifically bind to LRP, PZP-chymotrypsin complexes bind to the receptor with lesser apparent affinity (Kd approximately equal 320 nM) than alpha2-M-chymotrypsin complexes (Kd approximately equal 40 nM). We also demonstrated that Ni2+ blocks the binding of alpha2-M-chymotrypsin complexes, but not PZP-chymotrypsin complexes, to LRP. These data suggest that the binding to LRP involves conformational differences between both alpha-Ms in a region immediately upstream of the carboxy terminal receptor recognition domain. The possibility that PZP-proteinase complexes interact with other receptors not available to alpha2-M-proteinase complexes could be considered.     

PtdIns(4,5)P2 interacts with CaM binding domains on TRPM3 N-terminus

TRPM3 has been reported to play an important role in Ca²⁺ homeostasis, but its gating mechanisms and regulation via Ca²⁺ are unknown. Ca²⁺ binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that another Ca²⁺ binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P₂, interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.