Involvement of CD91 and scavenger receptors in Hsp70‐facilitated activation of human antigen‐specific CD4+ memory T cells

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress‐inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen‐specific CD4⁺ T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen‐specific cells were detected combining HLA‐DRB1^*0401 or HLA‐DRB1^*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen‐specific CD4⁺ T cell responses.     

Improved methods for predicting peptide binding affinity to MHC class II molecules

Major histocompatibility complex class II (MHC‐II) molecules are expressed on the surface of professional antigen‐presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC‐II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T‐cell epitopes. We here present updated versions of two MHC–II–peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC–peptide binding affinity data obtained from the Immune Epitope Database covering HLA‐DR, HLA‐DQ, HLA‐DP and H‐2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2 .     

70-kDa heat shock proteins: specific interactions with HLA-DR molecules and their peptide fragments

Heat shock protein 70 (HSP70):peptide complexes are involved in MHC class I and class II-restricted antigen presentation enabling enhanced activation of antigen-specific T cells. Here, we investigated the potential of bacterial and mammalian HSP70 molecules to interact with peptide fragments from HLA-DR and the corresponding complete HLA-DR molecules. Peptide fragments were found to interact with DnaK, the HSP70 homologue from E. coli, but less with stress-inducible human Hsp70. Only a peptide sequence exclusively found in rheumatoid arthritis-protective HLA-DR molecules did not interact with DnaK. Subsequently, we investigated the interaction of complete HLA-DR molecules with HSP70 and detected a specific HSP70:HLA-DR interaction, with highest affinity for human stress-inducible Hsp70. In contrast to the peptide fragments, no allele-specific differences in Hsp70 affinity were detected with complete HLA-DR molecules. Interaction with HLA-DR molecules was increased at lowered pH values, whereas HSP70-chaperoned peptides were released at acidic pH, thus HSP70 could serve as scanner and carrier for antigenic peptides of self or foreign origin and transfer chaperoned peptides onto MHC class II molecules in acidic late endosomal compartments. Our findings indicate that direct interaction between mammalian HSP70 and HLA-DR molecules could be involved in the HSP70-mediated enhancement of MHC class II-restricted peptide presentation and CD4(+) T cell activation.     

Distinct binding sites for the ATPase and substrate-binding domain of human Hsp70 on the cell surface of antigen presenting cells

Hsp70 has high potential as an immune-adjuvant molecule: it mediates cytokine expression and maturation of antigen presenting cells (APCs) and also elicits a cytotoxic T-lymphocyte (CTL) response to antigenic peptides. How Hsp70 interacts with APCs is only poorly understood. Various surface proteins have been implicated in binding Hsp70 but their role in antigen presentation has remained controversial. The specific aim of this work was to determine the binding and uptake of human full-length Hsp70 as well as its separate ATPase (N70) and substrate-binding domains (C70) by APCs. Using laser scanning microscopy and FACS analysis, we established the existence of at least two distinct receptors for Hsp70, which are localized to distinct microdomains of the APC membrane. These receptors interact with the N70 and C70 domains of Hsp70, respectively. This observation was supported by the finding of a substantial portion of Hsp70 and C70, but not N70, in a detergent resistant membrane fraction. Accordingly, C70 and N70 did not compete with each other for binding. The bound proteins were rapidly internalized, with N70 and C70 localizing to separate endosomal compartments. Similarly, internalized free and peptide-loaded Hsp70 segregated rapidly within the cell. Efficient cross presentation of antigenic peptide bound to Hsp70 or C70 was demonstrated with the B3Z read out system. Consequently, the interaction of C70 with its putative receptor seems to be responsible for Hsp70-mediated cross presentation. Future studies should make use of C70 in identifying the uptake receptor of Hsp70-peptide complexes. In addition we could observe a stimulation of uptake of free peptide by preincubation with Hsp70 and N70, but not C70, whereas an Hsp-dependent cytokine secretion could not be detected. Consequently, by employing the individual domains it may be possible to distinguish between the different outcomes of Hsp70 treatment, like immune stimulation, DC maturation and antigen-specific responses.     

T‐cell receptors: Tugging on the anchor for a tighter hold on the tumor‐associated peptide

Although it has been shown that human tumor‐associated, HLA anchor residue modified “heteroclitic” peptides may induce stronger immune responses than wild‐type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor‐expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584–591] highlight a novel twist on T‐cell receptor (TCR) recognition of HLA–peptide complexes. Tumor‐associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T‐cell cross‐reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan‐A/MART‐1_26‐35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8⁺ T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA‐A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide “lifts” its amino‐terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue “optimized” heteroclitic peptide, which cannot be “pulled” from the HLA groove.     

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

Summary: In antigen‐presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen‐binding sites in pre‐endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii‐degrading proteases, the peptide exchange factor, human leukocyte antigen‐DM (HLA‐DM), and its modulator, HLA‐DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen‐binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post‐Golgi mechanisms; these factors include MHC class II‐associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.     

Binding of Ras Oncogene Peptides to Purified HLA-DQ(αl*0102, ß1*0602) and -DR(α, ß1*0101) Molecules

Mutated oncogene peptides may be presented to T cells by HLA molecules. To be able to design the optimal peptides for stimulation of T cells in individuals with different HLA molecules, it is important to analyse the binding characteristics of oncogene peptides to HLA. HLA-DQ6 (DQ(α1*0102, ß1*0602)) and HLA-DRI (DR(α, ßl*0101)) molecules were purified from lysates of homozygous EBV-transformed cell lines. Purified HLA molecules were then tested for their ability to bind synthetic peptides in gel filtration assays. A _p21 ras oncogene peptide (previously found to stimulate DQ6-restricted T-cell clones) and an influenza matrix peptide were labelled with ¹²⁵I and served as indicator peptides for binding to DQ6 and DR1 respectively. Binding of homologous truncated and mutated _p21 ras peptides and unrelated peptides was then evaluated by their capacity to inhibit binding of the indicator peptides. _p21 ras-derived peptides were found to bind to both DQ6 and DR1 molecules indicating the existence of a promiscuous binding motif in these peptides. The binding affinities seemed to vary between the different peptides, but the amino acid substitutions resulting from natural mutations were not critical for binding. Notably, the results obtained for DQ6 in the biochemical peptide binding assay correlated well with results obtained in a functional assay using T-cell clones as probes.     

Composition of the HLA‐DR‐associated human thymus peptidome

Major histocompatibility complex class II (MHC‐II) molecules bind to and display antigenic peptides on the surface of antigen‐presenting cells (APCs). In the absence of infection, MHC‐II molecules on APCs present self‐peptides and interact with CD4⁺ T cells to maintain tolerance and homeostasis. In the thymus, self‐peptides bind to MHC‐II molecules expressed by defined populations of APCs specialised for the different steps of T‐cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC‐II‐associated peptides from five fresh human thymus samples. The data show a diverse self‐peptide repertoire, mostly consisting of predicted MHC‐II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA‐II‐bound peptidome and provide insight into how this peptidome is generated and how it drives T‐cell repertoire formation.     

Heat shock protein 70 moderately enhances peptide binding and transport by the transporter associated with antigen processing

Hsp70 molecules are capable of binding antigenic peptides and eliciting CTL responses to the bound peptide. However, the precise mechanism for the induction of CTL has not been determined. One possibility is that hsp molecules can directly shuttle peptides in the MHC class I antigen processing and presentation pathway, as previously postulated. Here, we have addressed this issue by testing the effect of purified hsp70 molecules on peptide binding and transport by the transporter associated with antigen processing (TAP). Our results indicate that purified hsp70 molecules moderately enhance TAP function. In addition, we detect a physical association between hsp70 molecules and TAP, as well as the homologous drug transporter P-glycoprotein. We conclude that while hsp70 molecules may not be directly involved in the delivery of peptide to TAP, they may play an important role in TAP transport by binding to TAP and promoting its function.     

The CD4+ T‐cell epitope‐binding register is a critical parameter when generating functional HLA‐DR tetramers with promiscuous peptides

Detection of CD4⁺ T cells specific for tumor‐associated antigens is critical to investigate the spontaneous tumor immunosurveillance and to monitor immunotherapy protocols in patients. We investigated the ability of HLA‐DR^*1101 multimers to detect CD4⁺ T cells specific for three highly promiscuous MAGE‐A3 derived peptides: MAGE‐A3_191–205 (p39), MAGE‐A3_281–295 (p57) and MAGE‐A3_286–300 (p58). Tetramers stained specific CD4⁺ T cells only when loaded with p39, although all peptides activated the specific T cells when presented by plastic‐bound HLA‐DR^*1101 monomers. This suggested that tetramer staining ability was determined by the mode rather than the affinity of peptide binding to HLA‐DR^*1101. We hypothesized that peptides should bear a single P1 anchor residue to bind all arms of the multimer in a homogeneous register to generate peptide‐HLA‐DR conformers with maximal avidity. Bioinformatics analysis indicated that p39 contained one putative P1 anchor residue, whereas the other two peptides contained multiple ones. Designing p57 and p58 analogues containing a single anchor residue generated HLA‐DR^*1101 tetramers that stained specific CD4⁺ T cells. Producing HLA‐DR^*1101 monomers linked with the optimized MAGE‐A3 analogues, but not with the original epitopes, further improved tetramer efficiency. Optimization of CD4⁺ T‐cell epitope‐binding registers is thus critical to generate functional HLA‐DR tetramers.     

Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules

The expression of major histocompatibility complex class II (MHC II) molecules is post‐translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II‐degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent‐solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)‐DR isolated from both HLA‐DM‐expressing and DM‐null cells. Even following CatG cleavage, peptide binding was retained by pre‐loaded, soluble recombinant HLA‐DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane‐proximal β2 domain. All allelic variants of HLA‐DR tested and murine I‐A^g7 class II molecules were susceptible, whereas murine I‐E^k and HLA‐DM were not, consistent with their altered sequence at the P1’ position of the CatG cleavage site. CatG effects were reduced on HLA‐DR molecules with DRB mutations in the region implicated in interaction with HLA‐DM. In contrast, addition of CatG to intact B‐lymphoblastoid cell lines (B‐LCLs) did not cause degradation of membrane‐bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen‐presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context‐dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.     

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T‐cell recognition

While Burkitt lymphoma (BL) has a well‐known defect in HLA class I‐mediated antigen presentation, the exact role of BL‐associated HLA class II in generating a poor CD4⁺ T‐cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4⁺ T cells via the HLA class II pathway. This defect in CD4⁺ T‐cell recognition was not associated with low levels of co‐stimulatory molecules on BL cells, as addition of external co‐stimulation failed to elicit CD4⁺ T‐cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL‐associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II‐mediated antigen presentation and CD4⁺ T‐cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase‐like molecule that enhances class II‐mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II‐mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.     

Structural basis for ineffective T‐cell responses to MHC anchor residue‐improved “heteroclitic” peptides

MHC anchor residue‐modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild‐type peptide. The best‐studied system to date is the decamer MART‐1/Melan‐A_26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)‐A*0201 anchoring. The resulting EL AGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the EL AGIGILTV peptide can fail to recognize the natural tumor‐expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA‐A*0201‐EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA‐A*0201‐EL AGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced‐fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.     

Machine learning reveals a non‐canonical mode of peptide binding to MHC class II molecules

MHC class II molecules play a fundamental role in the cellular immune system: they load short peptide fragments derived from extracellular proteins and present them on the cell surface. It is currently thought that the peptide binds lying more or less flat in the MHC groove, with a fixed distance of nine amino acids between the first and last residue in contact with the MHCII. While confirming that the great majority of peptides bind to the MHC using this canonical mode, we report evidence for an alternative, less common mode of interaction. A fraction of observed ligands were shown to have an unconventional spacing of the anchor residues that directly interact with the MHC, which could only be accommodated to the canonical MHC motif either by imposing a more stretched out peptide backbone (an 8mer core) or by the peptide bulging out of the MHC groove (a 10mer core). We estimated that on average 2% of peptides bind with a core deletion, and 0·45% with a core insertion, but the frequency of such non‐canonical cores was as high as 10% for certain MHCII molecules. A mutational analysis and experimental validation of a number of these anomalous ligands demonstrated that they could only fit to their MHC binding motif with a non‐canonical binding core of length different from nine. This previously undescribed mode of peptide binding to MHCII molecules gives a more complete picture of peptide presentation by MHCII and allows us to model more accurately this event.     

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides.

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)-DR1 and HLA-DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the beta chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306-318 (IC50). Among the high-affinity peptides (IC50 < or = 1 microM), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty-two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1-specific or DR103-specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR-binding peptides are able to outcompete specific T-cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T-cell response and mutation at position 71 abolishes completely the T-cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T-cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele-specific peptides and also influence the conformation of promiscuous peptides.     

HLA class I polymorphism has a dual impact on ligand binding and chaperone interaction

This article will describe coordinated analyses of how amino acid substitutions in the HLA class I antigen binding groove modify chaperone interaction and peptide ligand presentation. By parallel testing of ligand presentation and chaperone interaction with a series of natural HLA-B subtypes, this study has discovered that position 116 of the HLA-B15 class I heavy chain is pivotal in both peptide selection and control of interaction between the assembly complex and the class I heavy chain. Correlated with these qualitative differences in peptide selection and chaperone association are quantitative differences in the expression levels of the HLA molecules at the cell surface. These parallel studies, therefore, demonstrate that particular HLA class I polymorphisms can simultaneously influence ligand presentation and interaction with intracellular chaperones.     

The interplay between HLA‐B27 and ERAP1/ERAP2 aminopeptidases: from anti‐viral protection to spondyloarthritis

The human leukocyte antigen class I gene HLA‐B27 is the strongest risk factor for ankylosing spondylitis (AS), a chronic inflammatory arthritic disorder. More recently, the Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and 2 genes have been identified by genome wide association studies (GWAS) as additional susceptibility factors. In the ER, these aminopeptidases trim the peptides to a length suitable to fit into the groove of the major histocompatibility complex (MHC) class I molecules. It is noteworthy that an epistatic interaction between HLA‐B27 and ERAP1, but not between HLA‐B27 and ERAP2, has been highlighted. However, these observations suggest a paramount centrality for the HLA‐B27 peptide repertoire that determines the natural B27 immunological function, i.e. the T cell antigen presentation and, as a by‐product, elicits HLA‐B27 aberrant behaviours: (i) the misfolding leading to ER stress responses and autophagy and (ii) the surface expression of homodimers acting as ligands for innate immune receptors. In this context, it has been observed that the HLA‐B27 carriers, besides being prone to autoimmunity, display a far better surveillance to some viral infections. This review focuses on the ambivalent role of HLA‐B27 in autoimmunity and viral protection correlating its functions to the quantitative and qualitative effects of ERAP1 and ERAP2 polymorphisms on their enzymatic activity.     

Cellular mechanisms of artificial peptides binding to HLA

On the basis of the consideration that cell-free models cannot precisely mimic the complexity of the intracellular environment, we used a system to investigate the mechanisms that enable antigen-presenting cells (APC) to bind exogenous peptides through their human leukocyte antigen (HLA) molecules. We evaluated the uptake of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein by B-EBV cell lines, under various conditions. The results can be summarized as follows: a) the kinetics of peptide binding and release are very fast in living, fully competent cells; b) the peptide-HLA complexes are short-living and the DR molecules continuously undergo peptidic exchange; c) using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. The data suggest that in APC, cellular mechanisms are operative that increase the efficiency of both loading and unloading of Class II HLA with exogenous peptides. This is likely to be related to the recycling of Class II molecules to intracellular compartments, were binding takes place. The observation that the HLA-peptide complex is a dynamic structure, suggests the possibility of replacing natural peptides with synthetic ones at this level, in order to regulate the immune response.     

Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation

Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 T(h) cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MAT(T)), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.     

Peptidomic analysis of type 1 diabetes associated HLA‐DQ molecules and the impact of HLA‐DM on peptide repertoire editing

HLA‐DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII‐bound peptides in human embryonic kidney 293T cells expressing HLA‐DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM‐resistant, DM‐dependent, or DM‐sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ‐peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T‐cell epitopes to DM editing.