Antony N. Antoniou

Control of antigen presentation by a single protease cleavage site.

Protein antigens require limited proteolytic processing to generate peptides for binding to class II MHC molecules, but the proteases and processing sites involved are largely unknown. Here we analyze the effect of eliminating the three major asparagine endopeptidase (AEP)-processing sites in the microbial antigen tetanus toxin C fragment. The mutant antigen is highly resistant to proteolysis by AEP and crude lysosomal extracts and is dramatically impaired in its ability to be processed and presented to T cells. Remarkably, processing at a single asparagine residue (1219) is obligatory for optimal presentation of many T cell epitopes in this antigen. These studies demonstrate that cleavage at a single processing site can be crucial for effective antigen presentation.

Anergic T cells act as suppressor cells in vitro and in vivo.

The potential suppressive effects of allospecific anergic T cells were investigated both in vitro and in vivo. Allospecific T cells were rendered unresponsive in vitro using immobilized anti‐CD3 mAb. These anergic T cells profoundly inhibited proliferation of responsive T cells in an antigen‐specific manner. The observed inhibition did not appear to be due to the release of inhibitory cytokines in that secretion of IL‐2, IFN‐γ, IL‐4, IL‐10 and TGF‐β was greatly reduced following the induction of anergy, and neutralizig mAb specific for IL‐4, IL‐10 and TGF‐β failed to reverse the inhibition. Furthermore, the suppression mediated by anergic T cells required cell to cell contact. In vivo, adoptive transfer of anergic T cells into recipients of allogeneic skin grafts led to prolonged skin graft survival. Consistent with the lack of inhibitory cytokine production by the anergic cells, prolongation of skin allograft rejection was not influenced by the simultaneous administration of a neutralizing anti‐IL‐4 antibody. These results indicate that anergic T cells can function as antigen‐specific suppressor cells both in vitro and in vivo.

Multistep Autoactivation of Asparaginyl Endopeptidase in Vitro and in Vivo

Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently discovered lysosomal cysteine protease that specifically cleaves after asparagine residues. How this unusually specific lysosomal protease is itself activated is not fully understood. Using purified recombinant pro-enzyme, we show that activation is autocatalytic, requires sequential removal of C- and N-terminal pro-peptides at different pH thresholds, and is bimolecular. Removal of the N-terminal propeptide requires cleavage after aspartic acid rather than asparagine. Cellular processing, either of exogenously added AEP precursor or of pulse-labeled endogenous precursor, introduces at least one further cleavage to yield the final mature lysosomal enzyme. We also provide evidence that in living cells, there is clear compartmental heterogeneity in terms of AEP activation status. Moreover, we show that human monocyte-derived dendritic cells harbor inactive proforms of AEP that become activated upon maturation of dendritic cells with lipopolysaccharide.

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone‐assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide‐loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N‐terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation.

Assembly and export of MHC class I peptide ligands

During the assembly of MHC class I molecules with peptide, a series of transient interactions are made with endoplasmic reticulum-resident chaperones and MHC class I-specific accessory molecules. These interactions culminate in the trafficking of MHC class I molecules to the cell surface and presentation of peptide to CD8+ T lymphocytes. Recent studies have revealed just how important these early interactions are, and how they influence the quality control of assembly and the optimisation of peptide–ligand binding.

Formation of HLA-B27 homodimers and their relationship to assembly kinetics

The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with β2-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove. Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2 by slowing its assembly rate by incubation of cells at 26 °C, suggesting that homodimer formation in the endoplasmic reticulum may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27 molecules and immunoglobulin-binding protein at the cell surface.

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

Antibody modulation of antigen presentation: positive and negative effects on presentation of the tetanus toxin antigen via the murine B cell isoform of FcγRII

Tetanus toxin has been a valuable model antigen to study the MHC class II‐restricted antigen processing pathway and is also frequently used to provide T helper determinants in vaccine formulations. To date most basic studies on the processing of this antigen have utilized human T and B cell clones. As a first step towards extending studies on this antigen into the murine system we have generated a panel of T cell clones and mAb in H‐2b and H‐2d mice. We investigated the presentation of tetanus toxin C fragment (TTCF) by the murine B cell lines LB27.4 (H‐2dxb), A20 (H‐2d) and IIA1.6 (H‐2d) and the extent to which this could be modulated by the addition of mAb. One mAb, 10G5, induced strikingly enhanced presentation of T cell determinants located in the N‐terminal region of TTCF while other antibodies inhibited presentation of these and other epitopes. The enhancing effects of the 10G5 antibody were blocked by the anti‐FcR antibody 2.4G2 and were not observed in the FcR‐negative IIA1.6 cell line. Interestingly, both FcγRIIB1 and FcγRIIB2 isoforms of FcγRII were able to restore antibody enhanced presentation in IIA1.6 cells but only if the cytoplasmic tails were intact. These results show that the B cell isoform of FcγRII (FcγRIIB1) can mediate capture and presentation of some antigen/antibody complexes and might play a role in BCR‐independent antigen presentation in vivo.

Membrane ruffling, macropinocytosis and antigen presentation in the absence of gelsolin in murine dendritic cells

Previous studies have shown that mice lacking the actin‐severing and capping protein gelsolin have defects in leukocyte and platelet function. Moreover, dermal fibroblasts from gelsolin knockout (Gsn–) mice showed substantially reduced motility, membrane ruffling and pinocytosis. We have generated dendritic cells (DC) from spleens of Gsn– mice to investigate the importance of gelsolin in antigen endocytosis and processing. We show here that Gsn– DC produce apparently normal membrane ruffles which can resolve to form large macropinosomes. Moreover, presentation of exogenous antigens on both MHC class II and class I molecules was equivalent in Gsn– and wild‐type DC. Thus the major rearrangements of the actin cytoskeleton needed for DC antigen uptake and presentation can proceed in the absence of a major actin filament regulatory protein.

Modulation by epitope-specific antibodies of class II MHC-restricted presentation of the tetanus toxin antigen

Summary: Above a certain affinity the dissociation rate of monovalent antigen from antibody becomes slower than the time taken for antigen capture, endocytosis and processing by professional antigen presenting cells. Thus, when high affinity antibodies drive antigen uptake, either directly via B‐cell membrane immunoglobulin or indirectly via Fc receptors, the substrate for processing may frequently be an antigen/antibody complex. Here we review studies using the tetanus toxin antigen which show that bound antibodies can dramatically affect proteolytic processing, dependent on the epitope specificity and multiplicity of antibodies bound. Certain antibodies protect or ‘footprint’ specific domains of the antigen during processing in B‐cell clones resulting in modulation of loading of class II MHC‐restricted T‐cell epitopes, Processing and class II MHC loading of some T‐cell epitopes within the footprinted region was hindered, as might be expected, but. surprisingly, presentation of other T‐cell epitopes was boosted considerably These studies show that protein/protein complexes can be processed in an unpredictable fashion by antigen presenting cells and indicate a possible mechanism whereby cryptic T‐cell epitopes might be revealed in autoimmune disease.