Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine

Summary One function of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120-specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II-positive/Ii-negative (class II(+)/Ii(-)) antigen-presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4(+) and CD8(+) T cells. Activated CD4(+) T cells locally strengthen the response of CD8(+) CTL, thus enhancing the potency of a DNA vaccine.     

Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules

A way to study the role of intracellular trafficking of an antigen in its presentation to T cells is to target the antigen to various cell compartments of the antigen-presenting cells (APC) and compare the nature of the complexes associating major histocompatibility complex (MHC) molecules and antigenic peptides, expressed on the cell surface. MHC class I+ and MHC class II+ mouse L fibroblasts secreting hen egg lysozyme (HELs cells) or expressing HEL in their cytosol (HELc cells) were obtained after transfection with HEL cDNA and signal sequence-deleted HEL cDNA, respectively. HEL was evidenced in both HELs- and HELc-transfected cells and the former type of transfectant secreted a large amount of HEL. However, HEL produced in the cytosol exhibited a short half-life of less than 5 min. HEL-derived peptides could not be shown biochemically either in HELc- nor in HELs-transfected cells. We then studied the capacity of these cells to present HEL to HEL-specific class I- and class II-restricted T cells. Both cell types could be recognized by the HEL-specific MHC class I-restricted CTL clones. In contrast, MHC class II-HEL peptide complexes, recognized by HEL-specific helper T cell hybridomas, could be detected on MHC class II+ HELs- but not HELc-transfected cells. In vivo experiments showed, however, that HELc-transfected cells could provide host APC with HELc-derived peptides able to associate with MHC class II molecules. This was inferred from the capacity of MHC class II-HELc-transfected cells, unable by themselves to elicit any anti-HEL antibody response, to prime syngeneic and allogeneic mice against HEL. The priming was revealed by the induction of an antibody response after a boost with an amount of HEL unable itself to elicit an antibody response.     

Processing and presentation of hen egg-white lysozyme by macrophages

The processing and presentation by macrophages of the well-defined protein hen egg-white lysozyme (HEL) was analyzed using two HEL-specific T cell hybridomas. The processing studies revealed that both clones required that native HEL be processed, while neither clone required any processing of a tryptic digest of lysozyme. A differential requirement for processing was found for the intact, denatured lysozyme (CM-HEL) with one clone (2A11) requiring processing, and a second clone (3A9) did not require any processing. The determinant on the HEL molecule that both clones recognized was localized to a tryptic fragment containing residues 46 to 61. By testing the immunogenicity of fragments of the 46-61 peptide, mouse lysozyme, and human lysozyme, we were able to localize the T cell determinant to either of two residues, Gly-49 or Leu-56.     

Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain allows antigen presentation upon recycling

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (Δ15Ii) or 23 (Δ23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas Δ15Ii did not affect antigen presentation by class II molecules, Δ23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of Δ23Ii can be internalized, followed by degradation of Δ23Ii and return of free class II αβ heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.     

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.     

Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E.

Proteolytic degradation (processing) of antigen by antigen-presenting cells is a major regulatory step in the activation of a T lymphocyte immune response. However, the enzymes responsible for antigen processing remain largely undefined. In this study we show that cathepsin E, and not the ubiquitous lysosomal cathepsin D, is the major aspartic proteinase in a murine antigen-presenting cell line, A20. This enzyme is localized to a non-lysosomal compartment of the endosomal system in these cells. Functional studies using a highly specific inhibitor of cathepsin E show that this enzyme is essential for the processing of ovalbumin by this cell line. Thus, cathepsin E, whose function was hitherto unknown, may play a major role in antigen processing.     

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.     

A novel major histocompatibility complex class I-related chain allele

We report the identification of a novel major histocompatibility complex class I-related chain (MICB) allele, provisionally designated as MICB-0114 pending the WHO Nomenclature Classification for the MICB locus. This new allele is identical to MICB-0103101v except for a single mutation of G to A in exon 4 that translates into an amino acid substitution from glutamic acid to lysine.     

In the absence of major histocompatibility complex class II molecules,invariant chain is translocated to late endocytic compartments by autophagy

It has been suggested that the cytoplasmic amino-terminal tail of invariant chain (Ii) contains a sorting signal that directs trafficking of the major histocompatibility complex (MHC) class II: Ii oligomeric complex to endocytic compartments. This model is based, in part, on the observation that in the absence of MHC class II molecules, Ii is detectable in lysosomal structures, a phenotype that is dependent on an intact NH₂ terminus. However, the route by which Ii gains access to endosomal compartments in the absence of class II molecules remains uncertain. Here we report a mechanism that localizes Ii in lysosomal compartments independently of class II. We show that murine Ii can be detected by immunofluorescence within late endocytic compartments of stably transfected Ltk^? mouse fibroblasts. Immunochemical studies indicate that degradation of Ii in these cells is sensitive to the lysosomotropic agent ammonium chloride, yet the majority of Ii that undergoes this apparent lysosomal degradation is sensitive to the enzyme endoglycosidase H. This finding suggests that Ii may reach the lysosomal compartment by a route that bypasses the Golgi complex. Consistent with this possibility, we found that in contrast to Ii which is complexed to class II molecules, transport of free Ii to lysosomes is prevented by 3-methyladenine, an inhibitor of the autophagic pathway of protein degradation, a process which involves direct transport from the endoplasmic reticulum to lysosomes. These data suggest the route of transport that leads to endosomal localization of Ii in the absence of class II is distinct from that taken when expressed with class II. This forces a re-evaluation of the concept that the cytosolic tail of Ii contains a dominant Golgi-to-endosomal sorting signal.     

Genes predisposing to autoimmunity augment constitutive major histocompatibility complex class II-associated presentation of the self-antigen IgG2a in vivo

The self-antigen IgG2ab is poorly presented to a gamma2ab 435-451-reactive I-Ad-restricted T-cell hybridoma unless available in high concentrations or targeted to Fcgamma- or complement receptors. Environmental factors, probably the extent of microbial challenge, profoundly influence the constitutive gamma2ab/I-Ad presentation in IgCHb, H-2d mice. Here we report also a strong genetic impact. Constitutive presentation was highly efficient in spleen and thymus of (NZB x BXSB)F1 mice, which inherit a predisposition to develop lupus. Presentation correlated with disease progression and the serum levels of IgG2ab and IgG2ab complement factor 3 complexes. The finding that constitutive presentation was by far most efficient in males indicated that it was augmented by the Y chromosome-linked autoimmune acceleration Yaa gene. In line with previous data for healthy mice, constitutive gamma2ab/I-Ad presentation was most pronounced in the adherent spleen cell fraction and improved by further enrichment for dendritic cells. Notably, however, whereas in normal mice the gamma2ab determinant was undetectable on B cells lacking surface IgG2ab, such B cells contributed considerably to constitutive presentation in (NZB x BXSB)F1 hybrids. Presumably this resulted from complement receptor-mediated internalization of IgG2ab-containing immune complexes formed in lupus. These data add to the evidence that B cells with self-reactive receptors, known to exist in the mature repertoire, may present non-cognate foreign antigen to anti-foreign helper T lymphocytes and thus differentiate into autoantibody-secreting cells, and might likewise account for the polyclonal B-cell activation characteristic of several autoimmune syndromes.     

T and B cell receptors discriminate major histocompatibility complex class II conformations influenced by the invariant chain.

Direct recognition of major histocompatibility complex (MHC) molecules may occur when T cells are positively selected in the thymus and also during recognition of non-self MHC molecules. Since peptide recognition and binding of particular monoclonal antibodies is strongly influenced by the invariant chain (Ii) of the class II molecule, we have asked whether Ii also affects recognition of non-self MHC molecules by T cells. We find that Ii binding alters MHC class II conformation as detected by a monoclonal antibody, and that this alteration is retained in cell surface MHC class II molecules after Ii dissociates. This altered conformation also affects recognition by allogeneic T cells. Normal T cells and T cell clones preferentially recognize MHC class II molecules that have been associated with Ii, suggesting that thymic selection may be influenced by MHC conformation independently of specific peptide binding.     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

A biological function for the XP motif within the N terminus of major histocompatibility complex class II-associated peptides

A high proportion (up to 30%) of major histocompatibility complex (MHC) class II-bound peptides in the mouse and humans contains a proline residue at the N-terminal penultimate position (XP motif). We used a set of ovalbumin (OVA)-specific and hen egg lysozyme (HEL)-specific T cell hybridomas and asked whether the XP motif in MHC class II-associated peptides might influence the stimulation of T cells. We created N-terminally substituted variants of OVA323–339, an H2-A^d restricted OVA epitope and of HEL50–63, a dominant epitope in the context of H2-A^k. Our results show that the N-terminal sequence of MHC class II-bound peptides has a strong impact for the overall stimulation of specific T cells. Proline at the N terminus of antigenic peptides, in contrast to other amino acids, is tolerated or even enhances the recognition of MHC class II-bound peptides significantly.     

MHC class II-associated invariant chain peptide replacement by T cell epitopes: engineered invariant chain as a vehicle for directed and enhanced MHC class II antigen processing and presentation

Proteolysis of the invariant chain (Ii) leads to the generation of abundant MHC class II-associated invariant chain peptides (CLIP), which bind in the MHC class II binding groove via supermotifs in a manner similar to that of antigenic peptides. We have engineered an Ii vector with the capacity to express any antigenic peptide of interest instead of CLIP, for T cell stimulation. When peripheral blood mononuclear cells (PBMC) were pulsed with Ii hybrids encoding T cell epitopes of tetanus toxin or acetylcholine receptor, stimulation of T cells was dramatically enhanced compared to stimulation after priming with either the native or recombinant proteins. Site-specific insertion of antigenic sequences into the CLIP region promoted enhanced antigenicity of Ii hybrids which were shown to be processed intracellularly in a chloroquine-sensitive compartment. Naturally processed T helper epitopes were visualized directly on the surface of PBMC and identified as analogs of CLIP associated with MHC class II molecules. This novel Ii vector provides a flexible and efficient system for the delivery of defined peptide epitopes to T cells which might be useful in the development of specific vaccines and in the study of intracellular processing.     

Surfactant protein D augments bacterial association but attenuates major histocompatibility complex class II presentation of bacterial antigens

Surfactant protein D (SP-D) is a secreted pattern recognition molecule associated with lung surfactant and mediates the clearance of pathogens in multiple ways. SP-D is an established part of the innate immune system, but it also modulates the adaptive immune response by interacting with both antigen-presenting cells and T cells. In a previous study, antigen presentation by bone marrow-derived dendritic cells was enhanced by SP-D. As dendritic cell function varies depending on the tissue of origin, we extended these studies to antigen-presenting cells isolated from mouse lung. Flow cytometric studies showed that SP-D binds calcium dependently and specifically to lung CD11c-positive cells. Opsonization of fluorescently labeled Escherichia coli by SP-D enhanced uptake by lung dendritic cells. SP-D facilitated the association of E. coli and antigen-presenting cells by increasing the frequency of CD11+ cells associated with E. coli by up to 10-fold. In contrast to the effect on bone marrow-derived dendritic cells, SP-D decreased the antigen presentation of ovalbumin, expressed in E. coli, to ovalbumin-specific major histocompatibility complex class II-specific T-cell hybridomas by 30-50%. The reduction of antigen presentation did not depend on whether the dendritic cells were isolated from the lungs of nonstimulated mice or mice that had been exposed to LPS aerosols. Our results show that SP-D increases the opsonization of pathogens, but decreases the antigen presentation by lung dendritic cells, and thereby, potentially dampens the activation of T cells and an adaptive immune response against bacterial antigens--during both steady-state conditions and inflammation.     

Carboxy-terminal residues of major histocompatibility complex class II-associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-A^b cells (which expresses I-A^b that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-A^b-binding peptides. Here we investigate the contribution of I-A^b-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-A^b-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-A^b. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.     

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57–68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8⁺ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s, r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.     

TAP-dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin

Immunization of mice with mixtures of listeriolysin, a pore-forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or β-galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo. Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore-forming activity of listeriolysin. This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway. The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min. Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.     

Antigen processing and presentation of a naturally glycosylated protein elicits major histocompatibility complex class II-restricted,carbohydrate-specific T cells

It is well known that T cells recognize antigen as processed peptides bound to major histocompatibility complex molecules on the surface of antigen-presenting cells. Recently, it has been shown that T cells can specifically recognize synthetic glycopeptides. However, whether glycopeptides are selected for presentation during antigen processing of glycoproteins and eventually elicit carbohydrate-specific T cells is still an open question. In this study, we utilized synthetic glycopeptides to analyze T cell recognition of the naturally glycosylated immunodominant peptide representing type II collagen (CII) residues 256–270. In this peptide, lysines at positions 264 and 270 may be post-translationally modified by hydroxylation and subsequent O-linked glycosylation with β-galactosyl or α-glucosyl-(1 → 2)-β-galactosyl residues. T cell hybridomas established from type II collagen-immunized mice specifically recognized CII 256–270 with either galactose or glucosyl-galactose at position 264. The T cell hybridoma recognizing glucosyl-galactose displayed no cross-reactivity either to galactose or to the structurally different α-galactosyl-(1 → 4)-β-galactose. Furthermore, the T cell hybridoma recognizing galactose did not cross-react to glucosyl-galactose or galactosyl-galactose, indicating that the antigen-presenting cells (bulk spleen cells, lipopolysaccharide-stimulated spleen cells, anti-CD40-stimulated spleen cells, peritoneal exudate cells or CFA-primed lymph node cells) inefficiently processed carbohydrates when the antigen was given as a glycopeptide.     

Molecular mechanisms of class I major histocompatibility complex antigen processing and presentation

The presentation of antigenic peptides by class I major histocompatibility complex molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by CD8+ T lymphocytes which inspect peptides, derived from intracellular proteins, bind to class I molecules on the surface of most cells. The transporter associated with antigen processing selectively translocates cytoplasmically derived peptides of appropriate sequence and length into the lumen of the endoplasmic reticulum where they associate with newly synthesized class I molecules. The translocated peptides are generated by multicatalytic and multisubunit proteasomes which degrade cytoplasmic proteins in a ATP-ubiquitin-dependent manner. This review discusses our current molecular understanding of class I antigen processing and presentation.