Heterodimer HLA-DM Fused with Constant Fragment of the Heavy Chain of the Human Immunoglobulin Accelerates Influenza Hemagglutinin HA306-318 Loading to HLA-DR1.

Major histocompatibility complex class II (MHC II) plays an important role not only in the adaptive immune responses to foreign pathogens, but also in the development of some autoimmune diseases. Non-classical MHC, HLA-DM is directly involved in MHC II loading with the peptide. To study this process, we synthesized recombinant proteins HLA-DR1 and HLA-DM. α/ß-Chains of DR1 heterodimer contained C-terminal leucine domains of the fos and jun factors, respectively. Each DM chain contained constant fragment of human antibody heavy chain fused via a long linker domain. In addition, DM α-chain carried N165D substitution suppressing potential glycosylation at this site. We observed significant acceleration of DR1 peptide loading with influenza HA306-318 hemagglutinin in the presence of DM, which indicates functionality of recombinant DR1-DM protein couple. Our results can be used to study the presentation of other viral and self-antigens and can become the basis for the development of new drug modeling.

Endogenous HLA class II epitopes that are immunogenic in vivo show distinct behavior toward HLA-DM and its natural inhibitor HLA-DO.

CD4(+) T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4(+) T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4(+) T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II-expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4(+) T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.

The kinetic basis of peptide exchange catalysis by HLA-DM.

The mechanism by which the peptide exchange factor HLA-DM catalyzes peptide loading onto structurally homologous class II MHC proteins is an outstanding problem in antigen presentation. The peptide-loading reaction of class II MHC proteins is complex and includes conformational changes in both empty and peptide-bound forms in addition to a bimolecular binding step. By using a fluorescence energy transfer assay to follow the kinetics of peptide binding to the human class II MHC protein HLA-DR1, we find that HLA-DM catalyzes peptide exchange by facilitating a conformational change in the peptide-bound complex, and not by promoting the bimolecular MHC-peptide reaction or the conversion between peptide-receptive and -averse forms of the empty protein. Thus, HLA-DM serves essentially as a protein-folding or conformational catalyst.

Receptor-mediated endocytosis of antigens overcomes the requirement for HLA-DM in class II-restricted antigen presentation.

HLA-DM- APC are unable to present soluble Ags to T cells in the context of class II DR molecules. This defect in DM- APC can be overcome by receptor-mediated delivery of Ag into cells. Ag conjugated to ligands for cell surface receptors, such as transferrin or goat anti-human Ig, was processed and presented by DM- T2.DR4 cells. Intracellular processing of Ag conjugates was required, as receptor cross-linking alone did not restore presentation by DM- APC. Ag conjugates targeted by transferrin receptors to endosomes or via surface Ig to endosomal and lysosomal compartments, were each efficiently presented by DM- cells. These Ag conjugates were predominantly localized in light density endosomes in T2.DR4 cells. This study demonstrates that the facilitated uptake and sorting of exogenous Ag by cell surface receptors allow efficient class II-restricted presentation even in the absence of HLA-DM.

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 class II molecule signal transduction leads to either apoptosis or activation via two different pathways.

The class II human leukocyte antigens (HLA class II) are constitutively expressed on antigen presenting cells (APC) and are essential for peptide presentation to helper T lymphocytes. Signal transduction by HLA class II molecules on B lymphocytes has been described and has been shown in many cases to induce cellular proliferation. However, since signalling via HLA class II can also lead to apoptosis, it has not been clear how the outcome of the signals is determined. We have distinguished two separate HLA class II-initiated pathways leading to either proliferation or apoptosis of primary human B lymphocytes. Proliferation requires new gene transcription and activation of src family tyrosine kinases. In contrast, apoptosis is significantly increased in the absence of transcription/translation. It is dependent on serine/threonine phosphatases and cytoskeletal mobility. An extracellular source of calcium was essential for apoptosis, suggesting the need for sustained high level of intracellular calcium. Activation of iso-enzymes of the protein kinase C family was needed for both pathways. We therefore conclude that HLA class II molecules can initiate two distinct signalling pathways leading to either proliferation or apoptosis of APC.

Both L- and D-isomers of allotrap 2702 prolong cardiac allograft survival in mice.

BACKGROUND: Peptides derived from a conserved region of the human leukocyte antigen class I heavy chain (a.a. 75-84) have been shown to have immunomodulatory activity. The peptide 07.75-84, derived from HLA-B7, prolonged Lewis 1.W cardiac allograft survival in Lewis 1.A recipients. In combination with cyclosporine A, permanent heart allograft acceptance was induced in the Lewis to ACI donor/recipient combination. In mice, 2702.75-84, derived from HLA-B2702, prolonged the survival of C57B1/6 skin allograft in CBA recipients. In vitro, 2702.75-84 inhibited NK and cytotoxic T cells. To further evaluate the immunomodulatory activity of both L- and D-isoforms of 2702.75-84, we studied their effects in a mouse cardiac allograft model. METHODS: Peptides consisting of L- and D-amino acids were synthesized and administered to CBA (H-2k) recipients of a C57B1/6 (H-2b) cardiac allografts. In addition to peptide monotherapy, combinations of peptide with cyclosporine A and azathioprine were evaluated. Graft survival was monitored by palpation. Breakdown products of 2702.75-84 were identified by a combination of high-performance liquid chromatography and mass spectrometry. RESULTS: Daily administration of 2702.75-84 to CBA (H-2k) recipients of a C57B1/6 (H-2b) cardiac allograft prolonged graft survival to 11.4 +/- 2.6 days compared with 7.5 +/- 1.2 days in untreated control animals (n = 8; p = 0.0003, Mann-Whitney U Test). Other major histocompatibility complex class I-derived peptides including HLA-G.75-84, HLA-07.75-84, HLA-2705.75-84, H-2Kk.75-84, H-2Dk.75-84, H-Kb.75-84, and H-2Db.75-84 had no effect. In combination with a subtherapeutic dose of cyclosporine A (days 0 to 4), 2702.75-84 prolonged graft survival to at least 70 days (five of eight grafts still beating, p = 0.0002) compared with a median graft survival of 14 days in animals that received cyclosporine A monotherapy. Like other peptides consisting of L-amino acids, 2702.75-84 is subject to degradation by serum proteases. Breakdown products of 2702.75-84 were identified by a combination of high-performance liquid chromatography and mass spectrometry. These compounds were found to be inactive when tested in vivo. In contrast to peptides consisting of L-amino acids, peptides consisting of D-amino acids are resistant to proteolytic degradation. When D2702.75-84 was administered to graft recipients at 5 to 10 mg/kg/day (days 0 to 10), graft survival was prolonged to more than 50 days (5 of 13 grafts still beating, p = 0.0001). In combination with azathioprine, administration of D2702.75-84 induced graft acceptance (> 100 days) in 100% of animals (all grafts still beating). CONCLUSIONS: This study shows that administration of 2702.75-84 consisting of L- or D-amino acids will prolong graft survival in mice. In addition, the data suggest that the D2702 isomer is more potent than the L-isomer in vivo and may allow reduced dosage or frequency of administration.