The murine liver-specific nonclassical MHC class I molecule Q10 binds a classical peptide repertoire

The biological properties of the nonclassical class I MHC molecules secreted into blood and tissue fluids are not currently understood. To address this issue, we studied the murine Q10 molecule, one of the most abundant, soluble class Ib molecules. Mass spectrometry analyses of hybrid Q10 polypeptides revealed that alpha1alpha2 domains of Q10 associate with 8-9 long peptides similar to the classical class I MHC ligands. Several of the sequenced peptides matched intracellularly synthesized murine proteins. This finding and the observation that the Q10 hybrid assembly is TAP2-dependent supports the notion that Q10 groove is loaded by the classical class I Ag presentation pathway. Peptides eluted from Q10 displayed a binding motif typical of H-2K, D, and L ligands. They carried conserved residues at P2 (Gly), P6 (Leu), and Pomega (Phe/Leu). The role of these residues as anchors/auxiliary anchors was confirmed by Ala substitution experiments. The Q10 peptide repertoire was heterogeneous, with 75% of the groove occupied by a multitude of diverse peptides; however, 25% of the molecules bound a single peptide identical to a region of a TCR V beta-chain. Since this peptide did not display enhanced binding affinity for Q10 nor does its origin and sequence suggest that it is functionally significant, we propose that the nonclassical class I groove of Q10 resembles H-2K, D, and L grooves more than the highly specialized clefts of nonclassical class I Ags such as Qa-1, HLA-E, and M3.     

C-terminal anchoring of a peptide to class II MHC via the P10 residue is compatible with a peptide bulge.

The binding of antigenic peptide to class II MHC is mediated by hydrogen bonds between the MHC and the peptide, by salt bridges, and by hydrophobic interactions. The latter are confined to a number of deeper pockets within the peptide binding groove, and peptide side chains that interact with these pockets are referred to as anchor residues. T cell recognition involves solvent-accessible peptide residues along with minor changes in MHC helical pitch induced by the anchor residues. In class I MHC there is an added level of epitope complexity that results from binding of longer peptides that bulge out into the solvent-accessible, T cell contact area. Unlike class I MHC, class II MHC does not bind peptides of discrete length, and the possibility of peptide bulging has not been clearly addressed. A peptide derived from position 24-37 of integrin beta(3) can either bind or not bind to the class II MHC molecule HLA DRB3*0101 based on a polymorphism at the P9 anchor. We show that the loss of binding can be compensated by changes at the P10 position. We propose that this could be an example of a class II peptide bulge. Although not as efficient as P9 anchoring, the use of P10 as an anchor adds another possible mechanism by which T cell epitopes can be generated in the class II presentation system.     

The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus

The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.     

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.     

Cooperativity of hydrophobic anchor interactions: evidence for epitope selection by MHC class II as a folding process

Peptide binding to MHC class II (MHCII) molecules is stabilized by hydrophobic anchoring and hydrogen bond formation. We view peptide binding as a process in which the peptide folds into the binding groove and to some extent the groove folds around the peptide. Our previous observation of cooperativity when analyzing binding properties of peptides modified at side chains with medium to high solvent accessibility is compatible with such a view. However, a large component of peptide binding is mediated by residues with strong hydrophobic interactions that bind to their respective pockets. If these reflect initial nucleation events they may be upstream of the folding process and not show cooperativity. To test whether the folding hypothesis extends to these anchor interactions, we measured dissociation and affinity to HLA-DR1 of an influenza hemagglutinin-derived peptide with multiple substitutions at major anchor residues. Our results show both negative and positive cooperative effects between hydrophobic pocket interactions. Cooperativity was also observed between hydrophobic pockets and positions with intermediate solvent accessibility, indicating that hydrophobic interactions participate in the overall folding process. These findings point out that predicting the binding potential of epitopes cannot assume additive and independent contributions of the interactions between major MHCII pockets and corresponding peptide side chains.     

Structural basis of diverse peptide accommodation by the rhesus macaque MHC class I molecule Mamu-B*17: insights into immune protection from simian immunodeficiency virus

The MHC class I molecule Mamu-B*17 has been associated with elite control of SIV infection in rhesus macaques, akin to the protective effects described for HLA-B*57 in HIV-infected individuals. In this study, we determined the crystal structures of Mamu-B*17 in complex with eight different peptides corresponding to immunodominant SIV(mac)239-derived CD8(+) T cell epitopes: HW8 (HLEVQGYW), GW10 (GSHLEVQGYW), MW9 (MHPAQTSQW), QW9 (QTSQWDDPW), FW9 (FQWMGYELW), MF8 (MRHVLEPF), IW9 (IRYPKTFGW), and IW11 (IRYPKTFGWLW). The structures reveal that not only P2, but also P1 and P3, can be used as N-terminal anchor residues by Mamu-B*17-restricted peptides. Moreover, the N-terminal anchor residues exhibit a broad chemical specificity, encompassing basic (H and R), bulky polar aliphatic (Q), and small (T) residues. In contrast, Mamu-B*17 exhibits a very narrow preference for aromatic residues (W and F) at the C terminus, similar to that displayed by HLA-B*57. Flexibility within the whole peptide-binding groove contributes to the accommodation of these diverse peptides, which adopt distinct conformations. Furthermore, the unusually large pocket D enables compensation from other peptide residues if P3 is occupied by an amino acid with a small side chain. In addition, residues located at likely TCR contact regions present highly flexible conformations, which may impact TCR repertoire profiles. These findings provide novel insights into the structural basis of diverse peptide accommodation by Mamu-B*17 and highlight unique atomic features that might contribute to the protective effect of this MHC I molecule in SIV-infected rhesus macaques.     

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.     

I-Ep-bound self-peptides: identification, characterization, and role in alloreactivity

T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64-76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.     

Differences between the lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale membrane domains.

Plasma membranes of many cells appear to be divided into domains, areas whose composition and function differ from the average for an entire membrane. We have previously used fluorescence photo-bleaching and recovery to demonstrate one type of membrane domain, with dimensions of micrometers (Yechiel, E., and M. Edidin. 1987, J. Cell Biol. 105: 755-760). The presence of membrane domains is inferred from the dependence of the apparent mobile fraction of labeled molecules on the size of the membrane area probed. We now find that by this definition classical class I MHC molecules, H-2Db, are concentrated in domains in the membranes of K78-2 hepatoma cells, while the nonclassical class I-related molecules, Qa-2, are free to pass the boundaries of these domains. The two proteins are highly homologous but differ in their mode of anchorage to the membrane lipid bilayer. H-2Db is anchored by a transmembrane peptide, while Qa-2 is anchored by a glycosylphosphatidylinositol (GPI) anchor. A mutant class I protein with its external portion derived from Qa-2 but with transmembrane and cytoplasmic sequences from a classical class I molecule shows a dependence of its mobile fraction on the area of membrane probed, while a mutant whose external portions are a mixture of classical and nonclassical class I sequences, GPI-linked to the bilayer, does not show this dependence and hence by our definition is not restricted to membrane domains.     

Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.     

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.     

Protective immunity against disparate tumors is mediated by a nonpolymorphic MHC class I molecule

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.     

The crystal structure of H-2D(b) complexed with a partial peptide epitope suggests a major histocompatibility complex class I assembly intermediate

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

Preimplantation embryo development (<Emphasis Type="Italic">Ped</Emphasis>) gene copy number varies from 0 to 85 in a population of wild mice identified as <Emphasis Type="Italic">Mus musculus domesticus</Emphasis>

The preimplantation embryo development (Ped) gene regulates the rate of preimplantation embryonic cleavage division and subsequent embryo survival. In the mouse, the Ped gene product is Qa-2 protein, a nonclassical MHC class I molecule encoded by four tandem genes, Q6/Q7/Q8/Q9. Most inbred strains of mice have all four genes on each allelic chromosome, making a total of eight Qa-2 encoding genes, but there are a few strains that are missing all eight genes, defining a null allele. Mouse strains with the presence of the Qa-2 encoding genes express Qa-2 protein and produce embryos with a faster rate of preimplantation embryonic development and a greater chance of embryo survival compared to mouse strains with the null allele. There is extensive evidence that the human homolog of Qa-2 is HLA-G. HLA-G in humans, like Qa-2 in mice, is associated with enhanced reproductive success. The human population is an outbred population. Therefore, for a better comparison to the human population, we undertook an investigation of the presence of the genes encoding Qa-2 in an outbred population of mice. We used Real-Time Quantitative PCR to quantify the number of Qa-2 encoding genes in a population of 32 wild mice identified as Mus musculus domesticus both by morphologic assessment and by PCR analysis of their DNA. We found great variability in the number of Qa-2 encoding genes in the wild mice tested. The wild mouse with the highest number of Qa-2 encoding genes had 85 such genes, whereas we discovered one wild mouse without any Qa-2 encoding genes. Evolutionary implications of a range of Qa-2 encoding gene numbers in the wild mouse population are discussed, as well as the relevance of our findings to humans.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.     

Identification of two major histocompatibility complex class Ib genes, Q7 and Q9, as the Ped gene in the mouse

The Ped (preimplantation embryonic development) gene influences the rate of preimplantation embryonic development and subsequent embryonic survival. The protein product of the Ped gene, the Qa-2 protein, is a major histocompatibility complex (MHC) class Ib protein. There are two alleles of the Ped gene, fast (Qa-2 [+]) and slow (Qa-2 [-]). Qa-2 is encoded by four very similar MHC class Ib genes: Q6, Q7, Q8, and Q9. Recent research in our laboratory has shown that the Ped phenotype is potentially encoded by the Q7 and/or Q9 gene because the Q7 and Q9 genes, but not the Q6 or Q8 gene, are expressed during preimplantation mouse embryonic development. In this study we utilized microinjection of transgenes to assess the functional roles of both the Q7 and Q9 genes in control of the rate of preimplantation development. The Q7 gene, the Q9 gene, and a combination of the Q7 and Q9 genes were microinjected into Ped slow zygotes, and the Ped phenotype and cell surface expression of Qa-2 protein were assayed after a 72-h or 96-h incubation period. We found that the microinjected individual Q7 and Q9 genes increased the rate of preimplantation development. Simultaneous injection of the Q7 and Q9 genes did not have a synergistic effect on the Ped phenotype. Microinjection of the Q7 and/or Q9 genes resulted in protein expression in 10-25% of the microinjected embryos. These results show that both the Q7 and Q9 genes encode the mouse Ped phenotype.     

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.     

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.