Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Identification of autoantigen epitopes in MHC Class II transgenic mice

Summary: The major histocompatibility (MHC) class Ib molecules HLA-E, HIA-F and HLA-G are relatively non-polymorphic compared to class la molecules. Both HLA-E and HLA-G bind peptides and are involved in natural killer (NK)-cell recognition, but the role of HLA-F is unclear. HLA-E binds specifically to the conserved leader sequence peptides from the class la MHC molecules and interacts on the cell surface with the CD94/NKG2 class of NK-cell receptors. The framework structure of HLA-E is similar to that of the MHC class la molecules, but the peptide-binding groove is highly adapted for the specific binding of the leader sequence peptides. This is different from class la molecules, which have highly promiscuous peptide-binding grooves. The HLA-E groove makes full use of all the available pockets and imposes specificity along the entire length of the peptide. HLA-G binds nonamer peptides with leucine or isoleucine at position 2, proline at position 3 and leucine at position 9. Expression of HLA-G inhibits NK cells expressing the CD94/NKG2 class of receptors, though an interaction with these receptors has not been directly demonstrated.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum

The aminopeptidase ERAAP is essential for trimming peptides presented by major histocompatibility complex (MHC) class I molecules. Inhibition of ERAAP by cytomegalovirus results in evasion of the immune response by this virus, and polymorphisms in ERAAP are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b). Antigen-experienced T cells specific for the Qa-1(b)-FL9 complex were frequent in naive mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8(+) T cell response specific for Qa-1(b)-FL9. MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.     

Positive selection of an H2-M3 restricted T cell receptor.

Thymocytes are positively selected for alphabeta T cell antigen receptors (TCR) that recognize antigen in conjunction with self-major histocompatibility complex (MHC) molecules. MHC bound peptides participate in positive selection; however, their role has remained controversial. A TCR transgenic mouse was established using a TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize an NADH dehydrogenase subunit 1 (ND1)-derived peptide as the physiological ligand of positive selection. This peptide bears no apparent sequence homology to the cognate peptide, is expressed ubiquitously, and yet does not interfere with peripheral T cells. Our studies also suggest that positive selection becomes promiscuous at high epitope densities.     

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.     

The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes

Functional MHC class I molecules are expressed on the cell surface in the absence of beta(2)-microglobulin (beta(2)m) light chain that can interact with CD8(+) T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8(+) T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that beta(2)m-free H-2D(b) assembles with short peptides that are approximately 9 amino acid residues in length, akin to ligands associated with completely assembled beta(2)m(+) H-2D(b). Remarkably, a subset of the peptides associated with the beta(2)m-free H-2D(b) has an altered anchor motif. However, they also include peptides that contain a beta(2)m(+)H-2D(b) binding anchor motif. Further, the H-2K(b)- and H-2D(b)-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2(b) class I in beta(2)m-deficient cells and are recognized by epitope-specific CD8(+) T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of beta(2)m with peptides and form CD8(+) T lymphocyte epitopes.     

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

Peptide Binding to MHC Class I is Determined by Individual Pockets in the Binding Groove

H-2K^b and HLA-A2 are MHC⁴ class I molecules with a similar overall structure. Important differences between these two class I molecules reside in the structure of the individual pockets in the antigenic-peptide-binding groove. H-2K^b, which has a deep C pocket, binds specifically peptides with a tyrosine or a phenylalanine at position 5. In contrast, HLA-A2 has a shallow C pocket, which cannot accommodate large side chains at position 5. Site-directed mutagenesis was used to generate a chimera between the murine H-2K^b and the human HLA-A2 [H-2K^b/HLA-A2(C')]. The structure of this chimera is similar to H-2K^b except for the region around the deep C pocket, where residues at positions 9, 97 and 99 were substituted with those bulkier residues from HLA-A2. Peptide binding between this chimera and H-2K^b-binding peptides [VSV (52–59), OVA (257–264), and MCMV pp89 (168–176)], revealed that the deep C pocket of H-2K^b was crucial for high-affinity binding. While a peptide, VSV (52–59), was found to bind with severalfold lower 'affinity' to H-2K^b/HLA-A2(C') than to the wild-type H-2K^b, a VSV analogue with the tyrosine in position 5 (Tyr5) substituted with an alanine was found to bind with a similar 'affinity' to both MHC class I molecules. Computer-aided modelling of the H-2K^b/HLA-A2(C') complex indicates that the VSV (52–59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing away from the groove. These results confirm a role of the individual pockets in determining peptide-binding affinity and specificity and suggest that this may be accomplished by changes in side-chain orientation.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.     

Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide

Designing peptide-based vaccines for therapeutic applications in cancer immunotherapy requires detailed knowledge of the interactions between the antigenic peptide and major histocompatibility complex (MHC) in addition to that between the peptide-MHC complex and the T-cell receptor. Past efforts to immunize with high-affinity tumour-associated antigenic peptides have not been very immunogenic, which may be attributed to the lack of T cells to these peptides, having been deleted during thymic development. For this reason, low-to-medium affinity non-canonical peptides represent more suitable candidates. However, in addition to the difficulty in identifying such antigens, peptide binding to MHC, and hence its ability to induce a strong immune response, is limited. Therefore, to enhance binding to MHC and improve immune responses, anchor modifications of non-canonical tumour-associated peptides would be advantageous. In this study, the non-canonical tumour-associated peptide from MUC1, MUC1-8 (SAPDTRPA), was modified at the MHC anchor residues to SAPDFRPL (MUC1-8-5F8L) and showed enhanced binding to H-2Kb and improved immune responses. Furthermore, the crystal structure of MUC1-8-5F8L in complex with H-2Kb was determined and it revealed that binding of the peptide to MHC is similar to that of the canonical peptide OVA8 (SIINFEKL).     

A pattern search method for putative anchor residues in T cell epitopes

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called “anchors” because of their property of fitting into “pockets” inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues. A large set of MHC class I-restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, K^b, K^d, D^b are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA-A1, All, B8, B14, H-2L^d and for the MHC class II alleles I-A^b and I-A^s. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform. This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.     

Definition of a novel binding site on CD8 cells for a conserved region of the MHC class Ib molecule Qa-1 that regulates IFN-gamma expression

Natural killer (NK) cells and activated CD8 cells both express cytotoxic activity and produce substantial levels of IFN-gamma in response to viral and bacterial infections. In the case of NK cells, cellular activation and IFN-gamma expression are regulated by an interaction between NK receptors and MHC class Ib molecules, including HLA-E/Qa-1. We have used soluble tetrameric complexes of the murine class Ib molecule Qa-1 to define the significance of this interaction for CD8 cells. We find that all CD8 cells express a receptor for Qa-1 and that ligation of this receptor by Qa-1 results in up-regulation of IFN-gamma production.     

An assay for peptide binding to HLA-Cw*0102

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) molecules is based on the ability of peptides to stabilize MHC class I molecules synthesized by transporter associated with antigen processing (TAP)-deficient cell. The TAP-deficient cell line T2 has previously been used in the assembly assay to analyze peptide binding to HLA-A*0201 and -B*5101. In this study, we have extended this technique to assay for peptides binding to endogenous HLA-Cw*0102 molecules. We have analyzed the peptide binding of 20 peptides with primary anchor motifs for HLA-Cw*0102. One-third of the peptides analyzed bound with high affinity, half of the peptides examined did not bind, whereas the remaining peptides displayed intermediate binding activity. Interest in HLA-C molecules has increased significantly in recent years, since it has been shown that HLA-C molecules both can present peptides to cytotoxic T lymphocytes (CTL) and in addition are able to inhibit natural killer (NK)-mediated lysis.     

A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) 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 peptide's 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.     

Motifs in the primary structure of MHC Class I molecules and their use for the design of synthetic T-cell receptor ligands

This paper attempts to describe a great variety of allelic forms of mammalian major histocompatibility gene complex (MHC) Class I molecules as a single "formula" that contains information on motifs in their primary structure. The positions of amino acid residues that are invariant even in highly evolutionarily different species have been found in the structure of MHC Class I molecular domains that are recognizable by T-cell receptors (TCR). The spatial arrangement of invariant residues in the tertiary structure of MHC molecules is indicative of their structure-forming function: they are generally located at the sites of contact of alpha-helices and beta-sheets. A study of homology between the structures of MHC Classes I and II molecules has identified a fragment located in the "kink"-region of both MHC Class I and beta-chains of MHC class II molecules, at which the spectra of possible amino acid substitutions significantly coincide. Since this region can act as an "anchor" for interaction with TCR, the authors have synthesized dendrimers with the direct and inverse sequences of AA158-175 of an H-2Ld molecule, whose biological activity has been investigated in the fetal thymic organ cultures isolated from 14-day embryos of B10.D2 (KdIdDdLd) mice. The findings suggest that synthesized peptides can affect the development and shaping of T-cell repertoire.