Direct binding of the MHC class I molecule H-2Ld to CD8: interaction with the amino terminus of a mature cell surface protein

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.     

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.     

Histophysiological observations on the external auditory meatus, middle, and inner ear of the Weddell seal (Leptonychotes weddelli)

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.     

Immune recognition of viral antigens

The response exhibited by the immune system to viral and other foreign antigens consists of antibody-mediated and T cell-mediated immunity. Structural and molecular biological studies have shown that the antibody response is tailored to provide exquisite specificity by generating binding pockets that are complementary in shape as well as in charge to the antigen. On the other hand, the cellular response uses T-cell receptors (TCRs) and the major histocompatibility complex (MHC) antigens. Structural information on the TCRs is not yet available, but the crystal structures of several MHC class I molecules have shown how one MHC molecule can bind many different peptide sequences that share only the common anchor residue positions that determine allele specificity. MHC class I interactions with the peptide backbone at the N and C termini explain the high specificity of the binding groove for peptide ligands and suggest a universal mode of recognition for peptides to MHC class I molecules. Peptide-MHC class II interactions are less well understood, although recent structural work has shown important differences in the binding clefts of MHC class I and II that lead to longer peptides being bound to class II molecules. Detailed analysis at the molecular level has indicated that conformational changes in both antibodies and MHC molecules occur upon antigen binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.     

Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection

The crystal structure of the mouse major histocompatibility complex (MHC) class I molecule H-2Dd with an immunodominant peptide, designated P18-I10 (RGPGRAFVTI), from human immunodeficiency virus envelope glycoprotein 120 was determined at 3.2 A resolution. A novel orientation of the alpha3 domain of Dd relative to the alpha1/alpha2 domains results in significantly fewer contacts between alpha3 and beta2-microglobulin compared with other MHC class I proteins. Four out of ten peptide residues (P2 Gly, P3 Pro, P5 Arg and P10 Ile) are nearly completely buried in the Dd binding groove. This is consistent with previous findings that Dd exploits a four-residue binding motif comprising a glycine at P2, a proline at P3, a positively charged residue at P5, and a C-terminal hydrophobic residue at P9 or P10. The side-chain of P5 Arg is directed toward the floor of the predominantly hydrophobic binding groove where it forms two salt bridges and one hydrogen bond with Dd residue Asp77. The selection of glycine at P2 appears to be due to a narrowing of the B pocket, relative to that of other class I molecules, caused by Arg66 whose side-chain folds down into the binding cleft. Residue P3 Pro of P18-I10 occupies part of pocket D, which in Dd is partially split by a prominent hydrophobic ridge in the floor of the binding groove formed by Trp97 and Trp114. Residues P6 through P9 form a solvent-exposed bulge, with P7 Phe protruding the most from the binding groove and thereby probably constituting a major site of interaction with T cell receptors. A comparison of H-2Dd/P18-I10 with other MHC class I/peptide complexes of known structure provides insights into the possible basis for the specificity of the natural killer cell receptor Ly-49A for several related class I molecules.     

Overlapping peptide-binding specificities of HLA-B27 and B39: evidence for a role of peptide supermotif in the pathogenesis of spondylarthropathies

OBJECTIVE: Previous studies indicated the increase of HLA-B39 among HLA-B27 negative patients with spondylarthropathies (SpA). This study was performed to examine whether the natural ligands of HLA-B27 are capable of binding to HLA-B39. METHODS: Peptides were synthesized according to the sequences of known natural ligands of HLA-B27 or B39 and were tested for their binding to HLA-B*3901 and B*2705 by quantitative peptide binding assay, using a TAP-deficient RMA-S cell line transfected with human beta2-microglobulin and HLA class I heavy chain genes. RESULTS: Four of the 10 HLA-B27 binding peptides significantly bound to HLA-B*3901. All 4 peptides had hydrophobic/aromatic amino acids (Leu or Phe) at the C-terminus. In contrast, peptides with basic residues (Lys, Arg) or Tyr at the C-terminus did not bind to B*3901. In parallel experiments, 1 of the 2 natural ligands of HLA-B*3901 was found to bind to B*2705. CONCLUSION: A subset of natural HLA-B27 ligands was capable of binding to B*3901. In addition to Arg at position 2 (Arg2), hydrophobic/aromatic C-terminal residues, such as Leu or Phe, seemed to be crucial for the cross-specificity. These results suggested that HLA-B27 and B39 recognize overlapping peptide repertoires, supporting the hypothesis that the peptides presented by both of these class I antigens play a role in the pathogenesis of SpA.     

A case of congenital mumps infection complicated with persistent pulmonary hypertension

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.     

Possible assortment of a1 and a2 region gene segments in human MHC class I molecules

Using pair-wise comparison of aligned nucleotide sequences of distinct and complete human MHC class I molecules, we have constructed triangular tables to study the similarities and differences of various a1 (exon 2) and a2 (exon 3) region sequences. There are two HLA-A (A*6901 and A*6601) and 13 HLA-B (B*4201, B*8101, B*4102, B*4801, B*4007, B*4001, B*4802, Dw53, B*4406, B*4402, B*3901, B*1514 and B*3702) sequences that have identical a1 sequences with other known MHC class I molecules, while their a2 sequences are the same as those of different ones. Of these 15, A*6901, B*4001 and B*4802 have previously been suggested as the results of recombination between A*6801 and A*0201, B*4101 and B*8101, and B*4801 and B*3501, respectively. However, many other sequences can also be used to generate them by recombination. Furthermore, their reciprocal products have never been identified. Thus, gene conversion has subsequently been suggested as an alternative. Another possible genetic mechanism for generating these nucleotide sequence similarities can be assortment, or that some gene segments can be duplicated or multiplicated to be used in different human MHC class I molecules. Interestingly, this genetic mechanism is probably absent for the generation of different mouse MHC class I molecules.     

How HLA-DM affects the peptide repertoire bound to HLA-DR molecules

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.     

Speaking behavior and speech sound characteristics in acute schizophrenia

Analysis of peptides derived from HLA class I molecules indicates that thousands of unique peptides are bound by a single molecular type, and sequence examination of the pooled constituents yields a motif which collectively defines the peptides bound by a given class I molecule. Motifs resulting from pooled sequencing are then used to infer whether particular viral and tumor protein fragments might serve as class I-presented peptide therapeutics. Still undetermined from a pooled motif is the breadth or range of peptides in the population which are brought together to form the pooled motif, and it is therefore not yet known how representative of the population a pooled motif is. By employing hollow fiber bioreactors for large-scale production of HLA class I molecules, sufficient peptides are produced to investigate individual subsets of peptides comprising a motif. Edman sequencing and mass spectrometric analysis of peptides eluted from HLA-B*1501 reveal that many peptide sequences fail to align with either the N- or C-terminal anchors predicted for the B*1501 peptide motif through whole pool sequencing. These analyses further reveal auxiliary anchors not previously detected and peptides significantly larger and smaller than the predicted nonamer, ranging from 6 to 12 amino acids in length. These results demonstrate that constituents of the B*1501 peptide pool vary markedly in comparison with one another and therefore in comparison with previously established B*1501 motifs, and such complexity indicates that many of the peptide ligands presented to CTL cannot be predicted using class I consensus motifs as search criteria.     

A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle

Alloreactive T cells are often specific for individual peptides that are bound to allogeneic major histocompatibility complex (MHC) molecules. Other alloreactive T cells are reported to be peptide-independent or to recognize MHC conformational changes that are induced by multiple peptides. We tested 12 anti-HLA-B7 alloreactive cytotoxic T lymphocyte (CTL) clones that bind a restricted region of HLA-B7, including three CTL clones that were generated in a protocol designed to stimulate peptide-independent T cells. All 12 CTLs recognized multiple point mutations in the HLA-B7 peptide-binding groove. Eleven of the 12 CTLs recognized specific peptides that eluted in one or two fractions on high-performance liquid chromatography (HPLC). None of the CTLs promiscuously recognized 16 HLA-B7-binding synthetic peptides, although one CTL recognized minor by-products in one synthetic peptide preparation. CTL clone KID-9 cross-reacted with allogeneic HLA-B7 and HLA-B27 molecules and recognized a distinct peptide bound to each MHC molecule. CTL clone KD-11 recognized peptides that eluted in two HPLC fractions and recognized HLA-B7-transfected peptide antigen processing defective T2 cells. These results indicate that CTL allorecognition is peptide-specific whether the allogeneic MHC molecules are expressed on normal cells or antigen processing-deficient cells.     

Binding of H-2Kb-specific peptides to TAP and major histocompatibility complex class I in microsomes from wild-type, TAP1, and beta2-microglobulin mutant mice

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.     

Computational determination of side chain specificity for pockets in class I MHC molecules

We show that a rapidly executable computational procedure provides the basis for a predictive understanding of antigenic peptide side chain specificity, for binding to class I major histocompatibility complex (MHC) molecules. The procedure consists of a combined search to identify the joint conformations of peptide side chains and side chains comprising the MHC pocket, followed by conformational selection, using a target function, based on solvation energies and modified electrostatic energies. The method was applied to the B pocket region of five MHC molecules, which were chosen to encompass the full range of specificities displayed by anchors at peptide position 2. These were a medium hydrophobic residue (Leu or Met) for HLA-A*0201, a basic residue (Arg or Lys) for HLA-B*2705; a small hydrophobic residue (Val) for HLA-A*6801, an acidic residue (Glu) for HLA-B*4001 and a bulky residue (Tyr) for H-2K(d). The observed anchors are correctly predicted in each case. The agreement for HLA-B40 and H-2K(d) is especially promising, since their structures have not yet been determined experimentally. Because the experimental determination of motifs by elution is difficult and these calculations take only hours on a high speed workstation, the results open the possibility of routine determination of motifs computationally.     

Glu227-->Lys substitution in the acidic loop of major histocompatibility complex class I alpha 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions

Cytotoxic T lymphocyte (CTL) activation requires specific T cell receptor (TCR)-class I major histocompatibility complex (MHC) antigen complex interactions as well as the participation of coreceptor or accessory molecules on the surface of CTL. CD8 can serve as a coreceptor in that it binds to the same MHC class I molecules as the TCR to facilitate efficient TCR signaling. In addition, CD8 can be "activated" by TCR stimulation to bind to class I molecules with high avidity, including class I not recognized by the TCR as antigenic complexes (non-antigen [Ag] class I), to augment CTL responses and thus serve an accessory molecule function. A Glu/Asp227-->Lys substitution in the class I alpha 3 domain acidic loop abrogates lysis of target cells expressing these mutant molecules by alloreactive CD8-dependent CTL. Lack of response is attributed to the destruction of the CD8 binding site in the alpha 3 domain which is likely to disrupt CD8 coreceptor function. The relative importance of the class I alpha 3 domain acidic loop Glu227 in coreceptor as opposed to accessory functions of CD8 is unclear. To address this issue, we examined CTL adhesion and degranulation in response to immobilized class I-peptide complexes formed in vitro from antigenic peptides and purified class I molecules containing wild-type or Glu227-->Lys substituted alpha 3 domains. The alpha 3 domain mutant class I-peptide complexes were bound by CTL and triggered degranulation, however to much lower levels than wild-type class I-peptide complexes. In further experiments, it is directly demonstrated that the alpha 3 domain mutant class I molecules, which lack the Glu227 CD8 binding site, still serve as TCR-activated, avidity-enhanced CD8 accessory ligands. However, mutant class I peptide Ag complexes failed to effectively serve as CD8 coreceptor ligands to initiate TCR-dependent signals required to induce avidity-enhanced CD8 binding to coimmobilized non-Ag class I molecules. Thus the Glu227-->Lys mutation effectively distinguishes CD8 coreceptor and avidity-enhanced CD8 accessory functions.     

Diminished class II-associated Ii peptide binding to the juvenile dermatomyositis HLA-DQ alpha 1*0501/DQ beta 1*0301 molecule

HLA class II molecules bind and present peptide Ags to T cells, binding specific sets of peptides due to polymorphism in the peptide binding groove. Class II proteins associate with the invariant chain (Ii chain) and its derived class II-associated Ii peptide (CLIP). Ii chain association is important for normal trafficking of class II proteins to the peptide loading vesicles and for blocking premature access of peptides to HLA class II molecules during maturation. We have previously shown that juvenile dermatomyositis is associated with the HLA-DQA1*0501 allele. There is limited information available about the interaction of any DQ molecule with the Ii chain and little information about binding of individual peptides to HLA-DQalpha1*0501/DQbeta1*0301. We sequenced peptides eluted from the juvenile dermatomyositis-associated class II allele HLA-DQalpha1*0501/DQbeta1*0301. Surprisingly, we found no Ii chain or CLIP. Further examination of peptide binding to the HLA-DQalpha1*0501/DQbeta1*0301 molecule demonstrated poor CLIP binding. However, newly synthesized HLA-DQalpha1*0501/DQbeta1*0301 molecules do associate with intact Ii chain. Molecular modeling suggests that CLIP binds differently to HLA-DQalpha1*0501/DQbeta1*0301 than to DR molecules. The lack of CLIP association suggests that HLA-DQalpha1*0501/DQbeta1*0301 has access to peptides earlier in the processing pathway and so might encounter novel peptides that induce autoimmunity.