Pathways for antigen cross presentation

Dendritic cells (DCs) have the unique ability to capture cellular tissue antigens, and to present them on MHC class I molecules to antigen-specific CD8⁺ T lymphocytes after migration to the draining lymph nodes. This process, called cross presentation can lead either to the tolerization or activation of antigen-specific CD8⁺ T cells. Antigen capture is believed to occur by phagocytosis of antigen-bearing dead cells. Recent studies suggest that the antigen transferred from the phagocytosed cell to the DC during cross presentation is a proteasome substrate, rather than a proteasomal degradation product. In most cases, the formation of the peptide-MHC class I complexes in DCs requires the export of protein antigens from phagosomes to the cytosol, where they undergo proteasomal degradation. The resulting peptides are then translocated by TAP to the lumen of a cross presentation-loading compartment, for association to MHC class I under the control of chaperones and oxido-reductases. This loading compartment may be either the endoplasmic reticulum (ER) or a mix phagosome-ER compartment. MHC class I egress from the loading compartment to cell surface remains to be analyzed.     

Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.     

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

EpsinR,a target for pyrenocine B,role in endogenous MHC‐II‐restricted antigen presentation

While the presentation mechanism of antigenic peptides derived from exogenous proteins by MHC class II molecules is well understood, relatively little is known about the presentation mechanism of endogenous MHC class II‐restricted antigens. We therefore screened a chemical library of 200 compounds derived from natural products to identify inhibitors of the presentation of endogenous MHC class II‐restricted antigens. We found that pyrenocine B, a compound derived from the fungus Pyrenochaeta terrestris, inhibits presentation of endogenous MHC class II‐restricted minor histocompatibility antigen IL‐4 inducible gene 1 (IL4I1) by primary dendritic cells (DCs). Phage display screening and surface plasmon resonance (SPR) analysis were used to investigate the mechanism of suppressive action by pyrenocine B. EpsinR, a target molecule for pyrenocine B, mediates endosomal trafficking through binding of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). Lentiviral‐mediated short hairpin (sh) RNA downregulation of EpsinR expression in DCs resulted in a decrease in the responsiveness of CD4⁺ T cells. Our data thus suggest that EpsinR plays a role in antigen presentation, which provides insight into the mechanism of presentation pathway of endogenous MHC class II‐restricted antigen.     

Reassessing the role of HLA‐DRB3 T‐cell responses: Evidence for significant expression and complementary antigen presentation

In humans, several HLA‐DRB loci (DRB1/3/4/5) encode diverse β‐chains that pair with α‐chains to form DR molecules on the surface of APC. While DRB1 and DRB5 have been extensively studied, the role of DRB3/4 products of DR52/DR53 haplotypes has been largely neglected. To clarify the relative expression of DRB3, we quantified DRB3 mRNA levels in comparison with DRB1 mRNA from the same haplotype in both B cells and monocytes, observing quantitatively significant DRB3 synthesis. In CD19⁺ cells, DRB1*03/11/13 was 3.5‐fold more abundant than DRB3, but in CD14⁺ this difference was only two‐fold. Monocytes also had lower overall levels of DR mRNA compared with B cells, which was confirmed by cell surface staining of DRB1 and DRB3. To evaluate the functional role of DRB3, tetramer‐guided epitope mapping was used to detect T cells against tetanus toxin and several influenza antigens presented by DRB3*0101/0202 or DRB1*03/11/13. None of the epitopes discovered were shared among any of the DR molecules. Quantitative assessment of DRB3‐tetanus toxin specific T cells revealed that they are present at similar frequencies as those observed for DRB1. These results suggest that DRB3 plays a significant role in antigen presentation with different epitopic preferences to DRB1. Therefore, DRB3, like DRB5, serves to extend and complement the peptide repertoire of DRB1 in antigen presentation.     

Localization of peptide/MHC class II complexes in macrophages following antigen processing of viable Streptococcus pyogenes

The subcellular localization of peptide/MHC complexes was investigated during processing of the surface M5 protein from Streptococcus pyogenes. Bone marrow-derived macrophages were pulsed with viable S. pyogenes for 20 min followed by various periods of chase. T hybridoma cells detected complexes of one epitope, M5(17-31) with E(d) on the surface of macrophages within 30 min of chase. In contrast, complexes with another epitope, M5(308-319) with A(d) peaked later. Intracellular localization of peptide/MHC-II complexes was studied by subcellular fractionation and detection of complexes in fractions by T hybridoma cells. M5(17-31)/E(d) complexes were detected in light membrane fractions containing plasma membrane and early endosomes by 10-30 min. M5(308-319)/A(d) complexes were detected in these light membranes after 3 h of chase. Thus, the time course of M5(308-319)/A(d) presentation was delayed relative to M5(17-31)/E(d). However, neither type of complex was detected at any time in fractions containing phagosomes. Both species of peptide/MHC complexes localized to endocytic compartments, indicating a role for endosomes in presentation of antigens from phagocytosed bacteria.     

MGL-mediated internalization and antigen presentation by dendritic cells: a role for tyrosine-5

Professional antigen-presenting cells are essential for the initiation of adaptive immune responses; however, they also play a vital role in the maintenance of tolerance towards self-antigens. C-type lectins can function as antigen receptors by capturing carbohydrate ligands for processing and presentation. Here, we focused on the dendritic cell (DC)-expressed macrophage galactose-type lectin (MGL), a C-type lectin with a unique specificity for terminal GalNAc residues, such as the tumor-associated Tn antigen. Soluble model antigens are efficiently internalized by MGL and subsequently presented to responder CD4+ T cells. The tyrosine-5 residue in the YENF motif, present in the MGL cytoplasmic domain, was essential for the MGL-mediated endocytosis in CHO cells. In conclusion, MGL contributes to the antigen processing and presentation capacities of DC and may provide a suitable target for the initiation of anti-tumor immune responses.     

Viral immune evasion: Lessons in MHC class I antigen presentation

The MHC class I antigen presentation pathway enables cells infected with intracellular pathogens to signal the presence of the invader to the immune system. Cytotoxic T lymphocytes are able to eliminate the infected cells through recognition of pathogen-derived peptides presented by MHC class I molecules at the cell surface. In the course of evolution, many viruses have acquired inhibitors that target essential stages of the MHC class I antigen presentation pathway. Studies on these immune evasion proteins reveal fascinating strategies used by viruses to elude the immune system. Viral immunoevasins also constitute great research tools that facilitate functional studies on the MHC class I antigen presentation pathway, allowing the investigation of less well understood routes, such as TAP-independent antigen presentation and cross-presentation of exogenous proteins. Viral immunoevasins have also helped to unravel more general cellular processes. For instance, basic principles of ER-associated protein degradation via the ubiquitin-proteasome pathway have been resolved using virus-induced degradation of MHC class I as a model. This review highlights how viral immunoevasins have increased our understanding of MHC class I-restricted antigen presentation.     

Chaperone BAG6 is dispensable for MHC class I antigen processing and presentation

Antigen processing for direct presentation on MHC class I molecules is a multistep process requiring the concerted activity of several cellular complexes. The essential steps at the beginning of this pathway, namely protein synthesis at the ribosome and degradation via the proteasome, have been known for years. Nevertheless, there is a considerable lack of factors identified to function between protein synthesis and degradation during antigen processing. Here, we analyzed the impact of the chaperone BAG6 on MHC class I cell surface expression and presentation of virus-derived peptides. Although an essential role of BAG6 in antigen processing has been proposed previously, we found BAG6 to be dispensable in this pathway. Still, interaction of BAG6 and the model antigen tyrosinase was enhanced during proteasome inhibition pointing towards a role of BAG6 in antigen degradation. Redundant chaperone pathways potentially mask the contribution of BAG6 to antigen processing and presentation.     

Major histocompatibility complex class I-intercellular adhesion molecule-1 association on the surface of target cells: implications for antigen presentation to cytotoxic T lymphocytes

Polarization and segregation of the T-cell receptor (TCR) and integrins upon productive cytotoxic T-lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen-A2 (HLA-A2) MHC-I and ICAM-1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA-A2 and ICAM-1, induced the accumulation of membrane rafts, and beads loaded with ICAM-1-specific antibodies caused the selective recruitment of HLA-A2 MHC-I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA-A2 and ICAM-1 from the raft fraction, abatement of HLA-A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca(2+) flux in virus-specific CTLs. These data suggest that productive engagement of ICAM-1 on target cells facilitates the polarization of MHC-I at the CTL-target cell interface, augmenting presentation of cognate peptide-MHC (pMHC) complexes to CTLs. We propose that ICAM-1-MHC-I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation.     

LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4⁺ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4⁺ T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.     

MHC class II antigen presentation and immunological abnormalities due to deficiency of MHC class II and its associated genes

Antigen presentation by Major Histocompatibility Complex (MHC) class II molecules plays an important role in controlling immunity and autoimmunity. Multiple co-factors including the invariant chain (Ii), HLA-DM and HLA-DO are involved in this process. While the role for Ii and DM has been well defined, the biological function of DO remains obscure. Our data indicate that DO inhibits presentation of endogenous self-antigens and that developmentally-regulated DO expression enables antigen presenting cells to preferentially present different sources of peptide antigens at different stages of development. Disruption of this regulatory mechanism can result in not only immunodeficiency but also autoimmunity. Despite the fact that deletion of each of the three genes in experimental animals is associated with profound immunological abnormalities, no corresponding human diseases have been reported. This discrepancy suggests the possibility that primary immunodeficiencies due to a genetic defect of Ii, DM and DO in humans are under diagnosed or diagnosed as “common variable immunodeficiency”, a category of immunodeficiency of heterogeneous or undefined etiology. Clinical tests for any of these potential genetic defects are not yet available. We propose the use of multi-color flow cytometry in conjunction with intracellular staining to detect expression of Ii, DM, DO in peripheral blood B cells as a convenient reliable screening test to identify individuals with defects in antigen presentation.     

The tetramer model: a new view of class II MHC molecules in antigenic presentation to T cells

Crystallographic studies suggest a plausible divalent interaction between T-cell receptor (TCR) and MHC class II molecules. In addition, biochemical data suggest that these divalent MHC molecules are preformed at the membrane of the antigen-presenting cell. The tetramer model is based on these preformed tetrameric class II molecules that can be loaded with identical or different peptides in their two grooves. This enables divalent class II molecules to deliver two different messages to T cell: 1) a two-peptide message, in which the tetramer with two identical peptides is able to cross-link two TCRs triggering full activation of a T cell. At the thymic level we propose that this message induces negative selection; or 2) a one-peptide message: only one of the peptides loaded in the class II tetramer is able to interact with that TCR. This message would be involved in triggering partial activation phenomena in mature lymphocytes, whereas in thymocytes this message would mediate positive selection. Since high concentrations of a peptide would favor the load of tetramers with identical peptides, the tetramer could therefore be viewed as a quantitative-qualitative transducer that would trigger different responses depending on the concentration of antigenic peptides.     

Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation

The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-β. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.     

Concerted antigen presentation by dendritic cells and B cells is necessary for optimal CD4 T-cell immunity in vivo

The relative contributions of different types of antigen presenting cells to T-cell activation, expansion and induction of effector functions are still not fully understood. In order to evaluate the roles of dendritic versus B cells during these phases of a CD4 T-cell response in vivo, we adoptively transferred major histocompatibility complex class II restricted, T-cell receptor-transgenic CD4+ T cells into transgenic mice expressing selectively the T-cell restricting class II molecules on either dendritic cells, B cells or both. Upon immunization with peptide antigen, we observed that dendritic cells were sufficient to induce activation, expansion, interleukin-2 production and germinal centre migration of antigen-specific T cells, independently of other antigen-presenting cells. In contrast, neither resting nor activated B cells had similar antigen-presenting capacities in vivo. However, in double transgenic mice where both B cells and dendritic cells were capable of presenting antigen, T cells showed increased proliferation, expansion and cytokine production in vivo. Moreover, higher antigen-specific CD4 T-cell numbers accumulated in germinal centres. Our data demonstrate that dendritic cells are sufficient to activate naive CD4 T cells in vivo, but B cells subsequently can enhance CD4 T-cell expansion further.     

Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin

Targeting exogenous antigen into the MHC class I-restricted presentation pathway is a prerequisite for the induction of cytotoxic T lymphocytes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections and tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin derived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins carrying an active or inactive endoplasmic reticulum retrieval signal (the C-terminal peptides KDEL or KDELGL, respectively) could be presented by peripheral blood mononuclear cells in an MHC class I-restricted manner to Mage 1-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In further analysis, we showed that treatment with brefeldin A or paraformaldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracellular processing of the antigen. Immunofluorescence analysis also revealed that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct antigen into the exogenous class I-restricted pathway makes it an attractive non-living and non-toxic vaccine vector.     

Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery

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