Distinct antigen MHC class II complexes generated by separate processing pathways.

The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (Ak). These complexes were also detected on the surface of antigen-presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS-stable, long-lived Ak complexes containing *HEL peptides and, unexpectedly, full-length *HEL. Both SDS-stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA-DM or proteases. SDS-stable *HEL-Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS-unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full-length protein as a product of antigen processing.     

MHC class II invariant chains in antigen processing and presentation

Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing.     

Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.     

Intracellular rate-limiting steps in MHC class I antigen processing.

Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CD8+ CTL were analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing beta-galactosidase as model Ag. Only the amount of starting Ag was varied, leaving other factors unaltered. Below a certain level of Ag synthesis, increasing protein amounts led to a sharp rise in recognition by CTL. Higher levels of Ag expression led to a saturation point, which intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition. The rate-limiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this event.     

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

The proteasome and MHC class I antigen processing

By generating peptides from intracellular antigens, which are then presented to T cells, the ubiquitin/26S proteasome system plays a central role in the cellular immune response. Under the control of interferon-gamma the proteolytic properties of the proteasome are adapted to the requirements of the immune system. Interferon-gamma induces the formation of immunoproteasomes and the synthesis of the proteasome activator PA28. Both alter the proteolytic properties of the proteasome complex and enhance proteasomal function in antigen presentation. Thus, a combination of several of regulatory events tunes the proteasome system for maximal efficiency in the generation of MHC class I antigens.     

Regulation of B cell receptor-mediated MHC class II antigen processing by FcgammaRIIB1

The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (FcgammaRIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the FcgammaRIIB1 on BCR-mediated Ag processing and show that coligating the FcgammaRIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the FcgammaRIIB1 was blocked by a FcgammaRII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express FcgammaRIIB1 and in splenic B cells treated with a FcgammaRII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the FcgammaRIIB1 is a negative regulator of the BCR-mediated Ag-processing function.     

Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages

Infection of murine bone-marrow-derived macrophages with viable Mycobacterium tuberculosis (MTB) H37Ra inhibited surface expression of MHC class II (MHC-II) molecules and processing of exogenous antigens for presentation to CD4(+) T hybridoma cells. The inhibition was not dependent on bacterial viability, since it was also produced by exposure to dead bacilli and MTB cytosol preparations, suggesting that it was initiated by a constitutively expressed bacterial component. Northern blot analysis demonstrated that MTB bacilli or cytosol decreased MHC-II mRNA, and immunoprecipitation of biosynthetically labeled molecules confirmed that MHC-II protein synthesis was diminished. Exposure to MTB or MTB cytosol also decreased expression of H2-DM, but H2-DM expression was still sufficient to catalyze conversion of MHC-II to SDS-stable dimers, a measure of MHC-II peptide loading. Thus, infection with MTB decreased both MHC-II and H2-DM expression, but diminished MHC-II synthesis provided the major limitation to antigen processing.     

Reconstruction of a pathway of antigen processing and class II MHC peptide capture

Endocytosed antigens are proteolytically processed and small amounts of peptides captured by class II MHC molecules. The details of antigen proteolysis, peptide capture and how destruction of T-cell epitopes is avoided are incompletely understood. Using the tetanus toxin antigen, we show that the introduction of 3-6 cleavage sites is sufficient to trigger a partially unfolded conformation able to bind to class II MHC molecules. The known locations of T-cell epitopes and protease cleavage sites predict that large domains of processed antigen (8-35 kDa) are captured under these conditions. Remarkably, when antigen is bound to the B-cell antigen receptor (BCR), processing can trigger a concerted 'hand-over' reaction whereby BCR-associated processed antigen is captured by neighbouring class II MHC molecules. Early capture of minimally processed antigen and confinement of the processing and class II MHC loading reaction to the membrane plane may improve the likelihood of T-cell epitope survival in the class II MHC pathway and may help explain the reciprocal relationships observed between B- and T-cell epitopes in many protein antigens and autoantigens.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.     

Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.     

Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4(+) T cells.     

Hydrophobicity as a driver of MHC class I antigen processing

The forces that drive conversion of nascent protein to major histocompatibility complex (MHC) class I-restricted peptides remain unknown. We explored the fundamental property of overt hydrophobicity as such a driver. Relocation of a membrane glycoprotein to the cytosol via signal sequence ablation resulted in rapid processing of nascent protein not because of the misfolded luminal domain but because of the unembedded transmembrane (TM) domain, which serves as a dose-dependent degradation motif. Dislocation of the TM domain during the natural process of endoplasmic reticulum-associated degradation (ERAD) similarly accelerated peptide production, but in the context of markedly prolonged processing that included nonnascent species. These insights into intracellular proteolytic pathways and their selective contributions to MHC class I-restricted peptide supply, may point to new approaches in rational vaccine design.     

Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments

Newly synthesized class II molecules of the major histocompatibility complex must be transported to endosomal compartments where antigens are processed for presentation to class II-restricted T cells. The invariant chain (Ii), which assembles with newly synthesized class II alpha- and beta-chains in the endoplasmic reticulum, carries one or more targeting signals for transport to endosomal compartments where Ii dissociates from alpha beta Ii complexes. Here we show that the transport route of alpha beta Ii complexes is regulated selectively by two forms of Ii (p33 and p35) that are generated by the use of alternative translation initiation sites. Using a novel quantitative surface arrival assay based on labeling with [6-3H]-D-galactose combined with biochemical modification at the cell surface with neuraminidase, we demonstrate that newly synthesized alpha beta Ii molecules containing the Ii-p33 isoform can be detected on the cell surface shortly after passage through the Golgi apparatus/trans-Golgi network. A substantial amount of these alpha beta Ii complexes are targeted to early endosomes either directly from the trans-Golgi network or after internalization from the cell surface before their delivery to antigen processing compartments. The fraction of alpha beta Ii complexes containing the p35 isoform of Ii with a longer cytosolic domain was not detected at the cell surface as determined by iodination of intact cells and the lack of susceptibility to neuraminidase trimming on ice. However, treatment with neuraminidase at 37 degrees C did reveal that some of the alpha beta Ii-p35 complexes traversed early endosomes. These results demonstrate that a fraction of newly synthesized class II molecules arrive at the cell surface as alpha beta Ii complexes before delivery to antigen processing compartments and that class II alpha beta Ii complexes associated with the two isoforms of Ii are sorted to these compartments by different transport routes.     

Allele-selective effect of PA28 in MHC class I antigen processing

PA28 is an IFN-gamma-inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28alpha decreased expression of K(d) (D(d)) while it increased K(b) and L(d) on the cell surface. By contrast, overexpression of PA28alphaDeltaC5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28alpha and beta by small-interfering RNA profoundly augmented expression of K(d) and D(d), but not of L(d), on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of K(d), although correct C-terminal flankings were removed, which in turn hampered production of K(d) ligands. Our results indicate that whereas PA28 negatively influences processing of K(d) (D(d)) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces K(b) (L(d)) epitopes, leading to up-regulation of the MHC molecules.     

MHC class II molecules: transport pathways for antigen presentation

During biosynthesis, MHC class II molecules travel through the endocytic pathway and interact with antigenic peptides before their stable insertion in the plasma membrane. The process of class II association with these peptides and their final deposition at the cell surface are essential steps in boosting specific antibody responses. Therefore, the study of class II molecules is important in understanding how cell-biological events can direct an immune response.     

Macrophages present pinocytosed exogenous antigen via MHC class I whereas antigen ingested by receptor-mediated endocytosis is presented via MHC class II

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.