Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/− or Cat D−/− antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/− splenocytes, as it did in Cat D−/− cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Structural requirements for antigen presentation by mouse CD1

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid αgalactosylceramide (α-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the α helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and α-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that α-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A′ pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) α chains and different β chains respond differently to α-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR β in defining natural killer T cell specificity, despite the more restricted diversity of the α chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR α and β chains survey a surface composed of both mCD1.1 and the carbohydrate portion of α-GalCer.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

Extracellular antigen processing and presentation by immature dendritic cells

In antigen presentation to CD4+ T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.

Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld).

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon γ (IFN-γ)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-γ but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-γ-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing d amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.