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.     

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.     

A Role for MHC Class II Antigen Processing in B Cell Development

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.     

Localization of self antigen: implications for antigen presentation and induction of tolerance

The fifth component of complement (C5) is a self antigen expressed in serum of normal mice at a concentration of about 50 μg/ml. We have previously shown that C5 is constitutively processed and presented by antigen-presenting cells (APC) in normal mice to induce and maintain complete tolerance in major histocompatibility complex (MHC) class II-restricted T cells. This report addresses the question of whether C5 presentation involves exogenous antigen which has been internalized for processing or whether intracellular, biosynthesized C5 is being presented with MHC class II. Macrophages were found to synthesize, but not secrete C5 in bone marrow chimeras made from irradiated C5-deficient [C5(?)] hosts reconstituted with C5-sufficient [C5(+)] bone marrow [C5(+) ← C5(?)]. In these mice, macrophages are the only source of C5. [C5(+) ← C5(?)] chimeras are not tolerant of C5 and generate C5-specific T and B cell responses upon immunization indistinguishable from those of C5(-) mice. Macrophages from [C5(+) ← C5(-)] chimeras are unable to activate C5-specific T cell hybrids in vitro unlike macrophages from a C5(?) strain that has matured in a C5-expressing environment [C5(?) ← C5(+) chimeras]. This shows that under physiological conditions in vivo intracellular C5 does not get access to the class II presentation pathway and thus, does not induce tolerance in class II-restricted T cells.     

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.     

Polyubiquitination of lysine‐48 is an essential but indirect signal for MHC class I antigen processing

Peptides presented on major histocompatibility complex (MHC) class I molecules are generated via cytosolic proteolysis. However, the nature of the endogenous peptide precursors and the intracellular processing steps preceding protein degradation remain poorly defined. Here, we assessed whether ubiquitination is an essential signal for proteasomal cleavage of antigen substrates in human cells. Conversion into antigenic peptides occurred in the absence of any detectable N‐terminal ubiquitination of the model antigens, and did not require the presence of any of the four types, nor a minimum number of ubiquitinatable amino acids within the antigen substrate. However, the knockdown of ubiquitin, expression of a lysine 48 (K48) ubiquitin mutant, or inhibition of proteasome‐associated deubiquitinases significantly impaired antigen presentation. The results presented here are consistent with a model in which the binding of the antigen substrate by an adaptor protein leads to its K48‐polyubiquitination and the subsequent delivery of the antigen cargo for degradation by the 26S proteasome. Altogether, these findings show an important but indirect role of K48‐polyubiquitination in preproteasomal antigen sampling.     

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.     

Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of peptides derived from cytosolic proteins, but the underlying processing and presentation pathways have remained elusive. Here we show that endogenous presentation of an epitope derived from the cytosolic protein neomycin phosphotransferase II (NeoR) on MHC class II is mediated by autophagy. This presentation pathway involves the sequestration of NeoR into autophagosomes, and subsequent delivery into the lytic compartment. These results identify endosomes/lysosomes as the processing compartment for cytosolic antigens and furthermore link endogenous antigen presentation on MHC class II with the process of cellular protein turnover by autophagy.     

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.     

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.     

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.     

Sculpting MHC class II–restricted self and non‐self peptidome by the class I Ag‐processing machinery and its impact on Th‐cell responses

It is generally assumed that the MHC class I antigen (Ag)‐processing (CAP) machinery — which supplies peptides for presentation by class I molecules — plays no role in class II–restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)‐cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4⁺ T‐cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II‐associated self peptides suggesting that the CAP machinery impacts class II–restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4⁺ T‐cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4⁺ T‐cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II–restricted peptidome, impacting the CD4⁺ T‐cell repertoire, and ultimately altering Th‐cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II–restricted epitopes that would otherwise be capable of eliciting functional Th‐cell responses.     

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation

The B cell receptor (BCR) enables antigen-specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasma membrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.     

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.     

Redirecting soluble antigen for MHC class I cross‐presentation during phagocytosis

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen‐presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross‐presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross‐presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross‐presentation pathway.     

Recognition of Salmonella by Dectin‐1 induces presentation of peptide antigen to type B T cells

Type B T cells recognize peptide–MHC class II (pMHCII) isoforms that are structurally distinct from those recognized by conventional type A T cells. These alternative type B conformers result from peptide loading in the absence of HLA‐DM. Type A conformers are more stable than type B pMHCII conformers but bind the same peptide in the same register. Here, we show that interaction of Salmonella Typhimurium with bone marrow derived dendritic cells (BMDCs) isolated from C3H/HeNCr1 mice results in enhanced presentation of peptide Ag to type B T cells. The effect could be mimicked by purified PAMPs, the most potent of which were curdlan and zymosan, β‐(1,3)‐glucan‐containing polymers that are recognized by Dectin‐1. Blocking of Dectin‐1 with Ab and laminarin inhibited the induction of the type B T‐cell response by BMDCs, confirming its role as a PRR for S. Typhimurium. Splenic DCs (sDCs) expressed Dectin‐1 but were refractive to the induction of type B responses by S. Typhimurium and curdlan. Type B T cells have been shown to escape thymic tolerance and to transfer pathology in an autoimmune disease model. The induction of type B responses by gram‐negative bacteria provides a mechanism by which autoreactive T cells may be produced during infection.     

Characterization of MHC-II antigen presentation by B cells and monocytes from older individuals

In this study we examine the effects of aging on antigen presentation of B cells and monocytes. We compared the antigen presentation function of peripheral blood B cells from young and old subjects using a system that specifically measures the B cell receptor (BCR)-mediated MHC-II antigen presentation. Monocytes were studied as well. Overall the mean magnitude of antigen presentation of soluble antigen and peptide was not different in older and younger subjects for both B cells and monocytes. Older subjects, however, showed increased heterogeneity of BCR-mediated antigen presentation by their B cells. The magnitude and variability of peptide presentation, which do not require uptake and processing, were the same between groups. Presentation by monocytes had similar variability between the older and younger subjects. These data suggest that poor B cell antigen processing, which results in diminished presentation in some older individuals may contribute to poor vaccine responses.     

Mycobacterium tuberculosis PPE18 protein inhibits MHC class II antigen presentation and B cell response in mice

PPE18 protein belongs to PE/PPE family of Mycobacterium tuberculosis. We reported earlier that PPE18 protein provides survival advantage to M. tuberculosis during infection. In the current study, we found that PPE18 inhibits MHC class II-mediated antigen presentation by macrophages in a dose-dependent manner without affecting the surface level of MHC class II or co-stimulatory molecules. PPE18 does not affect antigen uptake or presentation of preprocessed peptide by macrophages. Antigen degradation was found to be inhibited by PPE18 protein due to perturbation in phagolysosomal acidification. PPE18-mediated inhibition of MHC class II antigen presentation caused poorer activation of CD4 T cells. Mice infected with M. smegmatis expressing PPE18 exhibited reduced maturation and activation of B cells and had decreased Mycobacteria-specific antibody titers. Thus M. tuberculosis probably utilizes PPE18 to inhibit MHC class II antigen presentation causing poorer activation of adaptive immune responses. This study may be useful in understanding host–pathogen interaction and open up directions of designing novel therapeutics targeting PPE18 to tackle this nefarious pathogen.