Anne B. Vogt

Editing of the HLA-DR-peptide repertoire by HLA-DM

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non‐classical MHC class II molecule HLA‐DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self‐peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA‐DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self‐peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high‐performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR‐associated self‐peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high‐stability MHC class II‐peptide complexes by kinetic proofreading before these complexes are presented to T cells.

HLA-DM Acts as a Molecular Chaperone and Rescues Empty HLA-DR Molecules at Lysosomal pH

HLA-DM (DM) is a nonclassical MHC class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (Ii)-derived CLIP peptides, as well as other peptides bound with low kinetic stability. Here we provide evidence that in lysosomal compartments of B cells a considerable fraction of DM is stably associated with empty DR alphabeta dimers, thereby preventing their functional inactivation and aggregation. Upon encounter with cognate peptide, the DM-associated DR molecules can be rapidly loaded and no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone. In view of the suggested shortage of DM-resistant self-peptides in the loading compartment, empty class II molecules that are chaperoned by DM may enable the antigen-processing system to respond promptly to the challenge by newly entering antigens.

How HLA-DM edits the MHC class II peptide repertoire: Survival of the fittest?

Loading of classical major histocompatibility complex (MHC) class II molecules with antigen-derived peptides is fast, efficient and highly selective in vivo, quite in contrast to in vitro findings with isolated class II proteins and synthetic peptides. Do accessory proteins speed up the loading process in antigen-presenting cells? Here, a model is presented in which the nonclassical MHC class II molecule HLA-DM plays a pivotal role as a chaperone, catalyst and editor during epitope selection.

A ROLE FOR HLA-DO AS A CO-CHAPERONE OF HLA-DM IN PEPTIDE LOADING OF MHC CLASS II MOLECULES

In B cells, the non‐classical human leukocyte antigens HLA‐DO (DO) and HLA‐DM (DM) are residents of lysosome‐like organelles where they form tight complexes. DM catalyzes the removal of invariant chain‐derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR–DM–DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM–DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high‐stability peptides. Thus, DO appears to act as a co‐chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.

Self-Release of CLIP in Peptide Loading of HLA-DR Molecules

The assembly and transport of major histocompatibility complex (MHC) class II molecules require interaction with the invariant chain. A fragment of the invariant chain, CLIP, occupies the peptide-binding groove of the class II molecule. At endosomal pH, the binding of CLIP to human MHC class II HLA-DR molecules was counteracted by its amino-terminal segment (residues 81 to 89), which facilitated rapid release. The CLIP(81–89) fragment also catalyzed the release of CLIP(90–105) and a subset of other self-peptides, probably by transient interaction with an effector site outside the groove. Thus, CLIP may facilitate peptide loading through an allosteric release mechanism.

The impact of the non‐classical MHC proteins HLA‐DM and HLA‐DO on loading of MHC class II molecules

Summary: Peptide binding to classical major histocompatibility complex (MHC) class II molecules is known to be determined by the properties of the class ii peptide binding groove but recently it turned out to be co‐controlled by the activity of the non‐classical MHC molecules HLA‐DM and HLA‐DO: HLA‐DM functions as a mediator of peptide exchange. In addition, HLA‐DM is a chaperone for MHC class II molecules in endosomal and lysosomal loading compartments because it stabilizes the empty MHC class Ii peptide binding groove and keeps it receptive for peptide loading until appropriate peptide ligands are captured. Since HLA‐DM favors the generation of high‐stability peptide‐MHC class Ii complexes by releasing low‐stability peptide ligands, DM activity affects the peptide repertoire presented on the ceil surface of antigen‐presenting cells. HLA‐DO is expressed mainly in B cells and binds tightly to HLA‐DM thereby modulating its activity Together, HLA‐DM and HLA‐DO are critical factors in shaping the MHC class Il‐associated self or foreign peptide repertoire of antigen presenting cells and, hence, govern initiation or prevention of an immune response.

T cell response to two immunodominant proteolipid protein (PLP) peptides in multiple sclerosis patients and healthy controls

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system in which autoimmune T lymphocytes reacting with myelin antigens are believed to play a pathogenic role. Since HLA binding is involved in the selection of T cell responses, we have examined PLP peptide binding to HLA DR2, an HLA allele frequently found in MS patients. Both PLP 40–60 and PLP 89–106 show significant, high affinity binding to HLA DR2. We then tested whether responses to PLP peptides 40–60 and 89–106 are elevated in multiple sclerosis patients compared to matched controls. We also analysed T cell responses to MBP 87–106, which is considered to be the immunodominant region of MBP in humans. Here we demonstrate heterogenous T cell responses to PLP 40–60, PLP 89–106 and MBP 87–106 in both MS patients and controls. The overall number of TCL and the HLA restriction of those TCL did not vary significantly in the two groups. PLP 40–60 specific cytolytic TCL were increased in MS patients, whereas healthy controls had increased percentages of cytolytic TCL responding to PLP 89–106 and MBP 87–106. Although the data presented here shows heterogenous responses in T cell numbers, differences in numbers and specificity of cytolytic cells could be involved in the pathogenesis of autoimmune demyelinating disease.

Identification of destabilizing residues in HLA class II-selected bacteriophage display libraries edited by HLA-DM

HLA‐DM (DM) functions as a peptide editor by catalyzing the release of class II‐associated invariant chain peptides (CLIP) and other unstable peptides, thus supporting the formation of stable class II‐peptide complexes for presentation. To investigate the general features that determine the DM susceptibility of HLA‐DR1/peptide complexes, we generated a large DM‐ensitive peptide repertoire from an M13 bacteriophage display library using a novel double selection protocol: we selected bacteriophage capable of binding to DR1 molecules and, subsequently, we enriched DR1‐bound bacteriophage susceptible to elution by purified DM molecules. Sequence and mutational analyses of the DR1/DM double‐selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as CLIP 89 – 101, HA 307  –  319 and bovine collagen II (CII) 261–273. Our results demonstrate that DM susceptibility does not only depend on the number and nature of anchor residues, or the peptide length. Instead, less obvious sequence characteristics play a major role in the DM editing process and ultimately in the composition of peptide repertoires presented to T cells.

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.

A Novel Strategy for the Discovery of MHC Class II–Restricted Tumor Antigens: Identification of a Melanotransferrin Helper T-Cell Epitope

CD4+ helper T cells play a critical role in orchestrating host immune responses, including antitumor immunity. The limited availability of MHC class II-associated tumor antigens is still viewed as a major obstacle in the use of CD4+ T cells in cancer vaccines. Here, we describe a novel approach for the identification of MHC class II tumor-associated antigens (TAAs). By combining two-dimensional liquid chromatography and nanoelectrospray ionization tandem mass spectrometry, we developed a highly sensitive method for the detection of human leukocyte antigen (HLA)-DR-associated peptides of dendritic cells upon exposure to necrotic tumor cells. This approach led to the identification of a novel MHC class II-restricted TAA epitope derived from melanotransferrin. The epitope stimulated T cells derived from melanoma patients and healthy individuals and displayed promiscuity in HLA-DR restriction. Moreover, the same peptide was also presented by MHC class II-positive melanoma cells. This strategy may contribute to increase the number of tumor epitopes presented by MHC class II molecules and may support the development of more efficacious vaccines against cancer.