Activation of type B T cells after protein immunization reveals novel pathways of in vivo presentation of peptides

Type B T cells recognize a peptide-MHC conformer generated in recycling endosomes and eliminated by H2-DM in late endosomes; as a result, they recognize exogenous peptide, but fail to respond to the identical epitope generated from the native protein. To investigate the behavior of these cells in vivo, we generated mice transgenic for a type B TCR recognizing the 48-62 epitope of hen egg white lysozyme (HEL) presented by I-A(k). Type B T cells responded only to peptide ex vivo, but responded in vivo to immunization with either protein or peptide in the presence of Freund's adjuvant or LPS. Presentation of the type B conformer was MyD88-independent, evident within 24 h after HEL immunization, and restricted to the CD11b/c(+) APC subset. Immunization with listeriolysin O, a potent inducer of cell death, also primed type B T cells in vivo, and transfer of HEL-bearing allogeneic dendritic cells activated type B T cells. We conclude that a number of conditions in vivo, some of which induce inflammation and cell death, lead to peptide presentation through mechanisms distinct from the classical pathways involving H-2DM molecules.     

Presentation of type B peptide-MHC complexes from hen egg white lysozyme by TLR ligands and type I IFNs independent of H2-DM regulation

In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8α(+) DC or CD8α(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.     

Segregation of open Major Histocompatibility Class I conformers at the plasma membrane and during endosomal trafficking reveals conformation-based sorting in the endosomal system

Fully conformed Major Histocompatibility Class I molecules are complexes of heavy chain non-covalently associated with the peptide and beta-2-microglobulin. Conformational change in the extracellular domain of heavy chain leads to their disassembly and formation of open conformers, a process that physiologically occurs in normal cells and results in their presence at the cell surface. In this study we characterized endosomal trafficking of open conformers of a murine class I allele in order to examine whether conformational change in the extracellular domain of a membrane glycoprotein determines its endosomal sorting. Open conformers segregated from their fully conformed counterparts at the plasma membrane and in endosomes by sequestration in lipid-organized membrane environment. Consequently, open conformers constitutively internalized via distinct clathrin-independent endocytic carriers and converged into "classical" early endosomes together with transferrin receptor and cholera-toxin B subunit. In early endosomes, open conformers were excluded from recycling and diverted towards late endosomes. Due to lack of recycling, open conformers were constitutively internalized at a higher rate than full conformed proteins. Concanamycin A, methyl-β-cyclodextrin and sphingomyelinase treatment prevented segregation of open conformers in vacuolar early endosomes indicating that acidic endosomal environment and membrane composition are critical for the maintenance of the sorting mechanism. In the absence of endosomal acidification open conformers partitioned into lipid disordered membrane composition of early endosomes. Thus, our data suggest for the existence of a lipid-dependent mechanism in the endosomal system that distinguish membrane proteins based on conformation of their extracellular domain.     

Unexpected reactivities of T cells selected by a single MHC-peptide ligand.

In H2-DM mutant mice, most MHC class II molecules are bound by a single peptide, CLIP, derived from the class II-associated invariant chain. Previous studies showed that H2-DM- cells are defective in presenting synthetic peptides to class II-restricted T cells. In sharp contrast, however, the same peptides elicited strong CD4+ T cell responses in H2-DM- animals. We now provide an explanation for this apparent discrepancy. Peptide-specific CD4+ T cells from wild-type mice were efficiently stimulated by H2-DM+, but not by H2-DM- cells pulsed with the cognate peptide. In sharp contrast, CD4+ T cells from mutant animals specific for the same MHC-peptide combination recognized peptide-pulsed H2-DM+ and H2-DM- cells equally well. In addition, unlike Ag-specific T cells from wild-type animals, the reactivities of peptide-specific T cells from mutant animals could not be efficiently blocked by Abs specific for the cognate MHC class II-peptide combination. We further demonstrated that the distinct reactivities of CD4+ T cells from H2-DM+ and H2-DM- mice are due to differences in thymic selection. Collectively, these findings indicate that the CD4+ T cell repertoires of H2-DM+ and H2-DM- mice are remarkably different.     

Interpretation of 1H-NMR spectra of the cyclic analog of kallidin in solutions

1H-NMR resonances of [cyclo (10-1 epsilon)]kallidin (cyclo-KL) in (CD3)2SO and H2O have been assigned by combined analysis of two-dimensional phase-sensitive COSY and NOESY spectra. The presence of three slowly interchangeable conformers cyclo-KL I, cyclo-KL II, and cyclo-KL III, has been established, their population in (CD3)2SO being 25, 35 and 40%, respectively. Cyclo-KL I conformer possesses trans-configuration of all peptide bonds, but in the cyclo-KL II and cyclo-KL III conformers the Pro3-Pro4 and Arg2-Pro3 peptide bonds, respectively, have cis-configuration. In----solution, the following exchange occurs between the conformers: cyclo-KL II----cyclo-KL I----cyclo-KL III. The assignment of 1H-NMR signals of all the three cyclo-KL conformers has been carried out in H2O by gradual titration with (CO3)2SO. The conformer populations in H2O are 45, 25 and 30%, respectively.     

An insertion mutant in DQA1*0501 restores susceptibility to HLA-DM: implications for disease associations

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of α-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three α-chain mutations (α+53G, α+53R, or αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.     

Predominant torsional forms adopted by dipeptide conformers in solution: parameters for molecular recognition.

The present paper describes the predominant conformational forms adopted by dipeptides in aqueous solution. More than 50 dipeptides were subjected to conformational analysis using SYBYL Random Search. The resultant collections of conformers for individual dipeptides, for small groups with related side chain residues and for large groups of about 50 dipeptides were visualized graphically and analysed using a novel three-dimensional pseudo-Ramachandran plot. The distribution of conformers, weighted according to the percentage of each in the total conformer pool, was found to be restricted to nine main combinations of backbone psi (psi) and phi (phi) torsion angles. The preferred psi values were in sectors A7 (+150 degrees to +/-180 degrees), A10 (+60 degrees to +90 degrees) and A4 (-60 degrees to -90 degrees), and these were combined with preferred phi values in sectors B12 (-150 degrees to +/-180 degrees), B9 (-60 degrees to -90 degrees) and B2 (+30 degrees to +60 degrees). These combinations of psi and phi values are distinct from those found in common secondary structures of proteins. These results show that although dipeptides can each adopt many conformations in solution, each possesses a profile of common conformers that is quantifiable. A similarly weighted distribution of dipeptide conformers according to distance between amino-terminal nitrogen and carboxyl-terminal carbon shows how the preferred combinations of backbone torsional angles result in particular N-C geometries for the conformers. This approach gives insight into the important conformational parameters of dipeptides that provide the basis for their molecular recognition as substrates by widely distributed peptide transporters. It offers a basis for the rational design of peptide-based bioactive compounds able to exploit these transporters for targeting and delivery.     

Multiple active site conformers in the carbon monoxide complexes of trematode hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.     

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.     

Prolonged antigen persistence within nonterminal late endocytic compartments of antigen-specific B lymphocytes

Although Ag-specific B lymphocytes can process Ag and express peptide-class II complexes as little as 1 h after Ag exposure, it requires 3-5 days for the immune system to develop a population of Ag-specific effector CD4 T lymphocytes to interact with these complexes. Presently, it is unclear how B cells maintain the expression of cell surface antigenic peptide-class II complexes until effector CD4 T lymphocytes become available. Therefore, we investigated B cell receptor (BCR)-mediated Ag processing and presentation by normal B lymphocytes to determine whether these cells have a mechanism to prolong the cell surface expression of peptide-class II complexes derived from the processing of cognate AG: Interestingly, after transit of early endocytic compartments, internalized Ag-BCR complexes are delivered to nonterminal late endosomes where they persist for a prolonged period of time. In contrast, Ags internalized via fluid phase endocytosis are rapidly delivered to terminal lysosomes and degraded. Moreover, persisting Ag-BCR complexes within nonterminal late endosomes exhibit a higher degree of colocalization with the class II chaperone HLA-DM/H2-M than with the HLA-DM/H2-M regulator HLA-DO/H2-O. Finally, B cells harboring persistent Ag-BCR complexes exhibit prolonged cell surface expression of antigenic peptide-class II complexes. These results demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of Ag-BCR complexes within nonterminal late endosomes and suggest that this intracellular Ag persistence allows for the prolonged cell surface expression of peptide-class II complexes derived from the processing of specific AG:     

Cis-trans isomerization of an angiotensin I-converting enzyme inhibitor. An enzyme kinetic and nuclear magnetic resonance study

The angiotensin I-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) inhibitor, ramiprilat (2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-Ala]-(1S,3S,5S)-2- azabicyclo[3.3.0]octane-3-carboxylic acid), is shown to exist in tow conformational isomers, cis and trans, which interconvert around the amide bond. The two conformers were separated by reversed-phase high-performance liquid chromatography. The conformers were identified by nuclear Overhauser effect measurements. From line shape analysis the isomerization rate constants were determined to be kcis----trans = 15 s-1 and ktrans----cis = 5 s-1 at 368 K in [2H]phosphate buffer (p2H 7.5). By enzyme kinetic studies using 3-(2-furylacryloyl)-L-Phe-Gly-Gly as substrate, the trans conformer was found to be the most potent enzyme inhibitor, whereas the cis conformer had a very low inhibitory effect. A new inhibition mechanism is presented for this type of slow, tight-binding inhibitors that contain an amide bond. This mechanism involves an equilibrium between the two conformers and the enzyme-bound inhibitor complex.     

Primary structure of histone H2B from gonads of the starfish Asterias rubens. Identification of an N-dimethylproline residue at the amino-terminal

The complete amino acid sequence (121 residues) of histone H2B from gonads of the starfish Asterias rubens has been established from structural data obtained essentially from large fragments generated by cleavage of histone H2B at aspartyl residues and by limited hydrolysis of the dimer H2A-H2B with mouse submaxillary gland protease. No real sequence homology can be found between the amino-terminal sequence (residues 1-21) of starfish and calf H2B. One non-conservative substitution (serine-32 in calf----lysine-28 in starfish) leads to the presence of a cluster of eight basic residues (sequence 23-30) and to the disappearance of a potential site of phosphorylation. A particular structural feature of starfish histone H2B is the presence of N-dimethylproline at its amino-terminal end. By comparison with N-terminal acetylation, which is commonly found in histones, N-terminal methylation is rarely observed. At the present time the functional significance of the N-terminal methylation as well as that of the proline-rich nature of the amino-terminal sequence of the starfish histone H2B remain to be defined.     

Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., B?chinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs. These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.     

Conformational preferences and binding to neurophysins of oxytocin analogs with sarcosine or N-methylalanine in position 7

The 600 MHz proton n.m.r. spectra of (sarcosyl7)-oxytocin and (N-methylalanyl7) oxytocin in 2H2O solution have been recorded and completely assigned. In each case the spectrum indicates the presence of two slowly interconverting conformers, which are the cis-trans isomers about the peptide bond between residues six and seven. The trans isomer is energetically favored in both cases. When neurophysin is added to a solution of (N-methylalanyl7) oxytocin or (sacrosyl7)-oxytocin at pH 3.0, the proportion of minor conformer remains constant, indicating that the cis and trans conformers are equally tightly bound to the protein.     

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2⁺ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2⁺ I-A^k conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2⁺ versus Ia.2⁻ I-A^k class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.     

Structural analysis of peptides that interact with Newcastle disease virus

A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide-virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive beta-turns around T2-L3-T4-T5 and K6-L7-Y8-C9. The other conformer exhibited a beta-hairpin bend type of structure with a bend around L3-T4-T5-K6.     

Phenylisoserine in the gas-phase and water: Ab initio studies on neutral and zwitterion conformers

The conformational landscape of phenylisoserine (PhIS) was studied. Trial structures were generated by allowing for all combinations of single-bond rotamers. Based on the B3LYP/aug-cc-pVDZ calculations 54 conformers were found to be stable in the gas phase. The six most stable conformers were further optimized at the B3LYP/aug-cc-pVTZ and MP2/aug-cc-pVDZ levels for which characteristic intramolecular hydrogen bond types were classified. To estimate the influence of water on PhIS conformation, the IEF-PCM/B3LYP/aug-cc-pVDZ calculations were carried out and showed 51 neutral and six zwitterionic conformers to be stable in water solution. According to DFT calculations, the conformer equilibrium in the gas phase is dominated by one conformer, whereas the MP2 calculations suggest three PhIS structures to be significantly populated. Comparison of DFT and MP2 energies of all 57 structures stable in water indicates that, in practice, one zwitterionic and one neutral conformer determine the equilibrium in water. Based on the AIM calculations, we found that for the neutral conformers in vacuum and in water, d(H...B) is linearly correlated with Laplacian at the H-bond critical point.     

Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin

We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate.     

DM peptide-editing function leads to immunodominance in CD4 T cell responses in vivo

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158-173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM-/-) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33-48 and 261-276). DM-/- B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.     

Editing of an immunodominant epitope of glutamate decarboxylase by HLA-DM

HLA-DM stabilizes peptide-receptive class II alphabeta dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273-285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.