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.     

The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules

The endosomes and lysosomes of antigen-presenting cells host the processing and assembly reactions that result in the display of peptides on major histocompatibility complex (MHC) class II molecules and lipid-linked products on CD1 molecules. This environment is potentially hostile for T cell epitope and MHC class II survival, and the influence of regulators of protease activity and specialized chaperones that assist MHC class II assembly is crucial. At present, evidence indicates that individual proteases make both constructive and destructive contributions to antigen processing for MHC class II presentation to CD4 T cells. Some features of CD1 antigen capture within the endocytic pathway are also discussed.     

Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation

Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 T(h) cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MAT(T)), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.     

Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Ibeta and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.     

Effective antigen presentation by dendritic cells is NF-kappaB dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines

Antigen presentation is a key rate-limiting step in the immune response. Dendritic cells (DC) are the most potent antigen-presenting cells for naive T cells, due to their high expression of MHC and co-stimulatory molecules, but little is known about the biochemical pathways that regulate this function. We here demonstrate that monocyte-derived mature DC can be infected with adenovirus at high efficiency (>95%) and that this procedure can be used to dissect out which pathways are essential for inducing DC antigen presentation to naive T cells. Using adenoviral transfer of the endogenous inhibitor of NF-kappaB, IkappaBalpha, we show that DC antigen presentation is NF-kappaB dependent. The mechanism for this is that NF-kappaB is essential for three aspects of antigen-presenting function: blocking NF-kappaB coordinately down-regulates HLA class II, co-stimulatory molecules like CD80, CD86 and CD40, and immuno-stimulatory cytokines like IL-12 and tumor necrosis factor-alpha. In contrast adhesion molecules are up-regulated after infection with the adenovirus transferring IkappaBalpha, indicating that NF-kappaB also regulates the duration of T cell-DC interaction. These results establish NF-kappaB as an effective target for blocking DC antigen presentation and inhibiting T cell-dependent immune responses, and this finding has potential implications for the development of therapeutic agents for use in allergy, autoimmunity and transplantation.     

Calmodulin kinase II regulates the maturation and antigen presentation of human dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells, which activate the adaptive immune system. Upon receiving a danger signal, they undergo a maturation process, which increases their antigen presentation capacity, but the responsible regulatory mechanisms remain incompletely understood. A Ca2+-calmodulin (Cam)-Cam kinase II (CamK II) pathway regulates phagosome maturation in macrophages, and this pathway is inhibited by pathogenic microbes. Our hypothesis is that signal transduction events which control phagosome maturation also regulate antigen presentation. Stimulation of primary human DC or the human DC line KG-1, with particulate antigen, resulted in the activation of CamK II and its localization to the phagosome and plasma membrane. Two mechanistically distinct inhibitors of CamK II significantly reduced DC maturation, as determined by up-regulation of surface costimulatory and major histocompatibility complex (MHC) class II molecules and secretion of cytokines. Confocal microscopy demonstrated that the CamK II inhibitors blocked the antigen-induced increase in total cellular MHC class molecules as well as their trafficking to the plasma membrane. Inhibition of CamK II was associated with decreased presentation of particulate and soluble MHC class II-restricted antigen, with a greater effect on the former. These data support a model in which CamK II regulates critical stages of the maturation and antigen presentation capacity of human DC, particularly in response to stimulation via phagocytosis.     

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.     

Rapid constitutive generation of a specific peptide-MHC class II complex from intact exogenous protein in immature murine dendritic cells

MHC class II molecules present peptides, derived largely from exogenous antigens, to CD4+ T cells. Complex-generation occurs mainly in the specialized late endosomal MHC class II-rich compartment (MIIC) vesicles of antigen-presenting cells (APC). Dendritic cells (DC) have been reported to store intact antigen in MIIC until the receipt of an activation signal, when they process it into peptide-MHC class II complexes. However, constitutive migration of DC from the periphery to secondary lymphoid organs has been observed, and antigen presentation by nonactivated DC is proposed to play a role in the induction of tolerance to peripheral antigens. Thus, constitutive peptide-MHC class II complex generation must also occur in DC in immunologically quiescent situations. We have used a monoclonal antibody that detects a specific peptide-MHC class II complex to directly demonstrate constitutive complex generation in immature murine DC. Protein-derived peptide-MHC class II complexes were detected by flow cytometry at the DC surface within 1 h of antigen exposure in the absence of an exogenous activation signal, and could be detected by confocal microscopy in the MIIC within 5 min of antigen exposure. This processing activity was endotoxin independent. These data provide evidence for constitutive peptide-MHC class II complex generation in immature DC, and thus support a role for this activity in the induction of peripheral tolerance.     

The geometry of synthetic peptide-based immunogens affects the efficiency of T cell stimulation by professional antigen-presenting cells

In the pathway leading to antibody production there are two points at which CD4(+) T(h) cells need to be recruited. The first of these is priming of T cells by their interaction with dendritic cells (DC) bearing antigen presented on MHC class II molecules and the second is the collaborative interaction of these primed T cells with B cells presenting the same antigen. We have previously shown that the configuration of T and B cell determinants within synthetic peptide immunogens can greatly influence the amount of immunogen required to produce an antibody response. Here we investigate whether the difference in potency of different immunogens is related to their ability to be presented by either DC or B cells. We show that determinants in a branched configuration, which are the most efficient at eliciting antibody in vivo, are presented to T cell clones by splenic CD8(-) DC 10-fold more efficiently than the corresponding determinants in a tandem linear arrangement. B cells also showed preferential presentation of branched immunogens to one T cell clone but in contrast to DC, not to a second T cell clone, indicating differences between the two antigen-presenting cell types. We also show that branched immunogens have a greater stability in serum compared to linear peptides, which may further enhance the differences in their in vivo potency.     

Specific immune responses restored by alteration in carbohydrate chains of surface molecules on antigen-presenting cells

Two class I major histocompatibility (MHC) mutant mouse strains, H-2bm14 and H-2bm6, differ from the strain of origin C57BL/6 (B6, H-2b) in one and two amino acids of the H-2Db and H-2Kb molecule, respectively. The bm14 Db mutation results in specific failure of female bm14 mice to generate a cytotoxic T lymphocyte (Tc) response to the male-specific antigen H-Y. The allospecific Tc response of CD8+ B6T cells against bm6 Kb mutant spleen cells, in contrast to that against other Kb mutants, is absolutely CD4+ T helper cell dependent. Purified CD8+ T cells completely fail to respond. We now report that the inability to mount these specific immune responses is restored by the use of dendritic cells (DC) as antigen-presenting cells (APC). Comparison of MHC expression on various types of APC by cytofluorimetry and quantitative immunoprecipitation showed very high expression of class I and class II MHC molecules on DC. Strikingly, examination of class I and class II molecules by isoelectric focusing revealed qualitative differences as well. We show that the surface MHC class I molecules of DC are present in greater quantity and carry on average fewer sialic acids than the same molecules isolated from other APC types such as spleen cells, lipopolysaccharide blasts or concanavalin A blasts. That sialic acids on cell surface molecules, including MHC, may play a role in antigen presentation is suggested by our finding that removal of sialic acids, by neuraminidase, can restore specific responses to nonresponder APC as well.     

Detection of aberrant association of DM with MHC class II subunits in the absence of invariant chain

Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM alphabeta heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii-/- mice.     

Cross-presentation of antigens by dendritic cells

T lymphocytes recognize antigen presented on the surface of antigen-presenting cells byMHC class I and class II molecules. Classically, MHC class I molecules present self- or pathogen-derived antigens that are synthesized within the cell, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. It is becoming increasingly clear that some dendritic cells are also specialized to process exogenous antigens into the MHC class I pathway for presentation to CD8+ T cells. This process is known as cross-presentation. It provides a mechanism that can drive dendritic cells to generate either tolerance to self-antigens or immunity to pathogens. The cells responsible for, and mechanisms underlying, this decision between tolerance and immunity via cross-presentation has become the focus of intense study to determine how various dendritic cell subsets effect the different outcomes.     

Human duodenal epithelial cells constitutively express molecular components of antigen presentation but not costimulatory molecules

Constitutive expression of major histocompatibility complex (MHC) class II molecules by duodenal epithelial cells (EC) suggests that they can present antigen to CD4(+) T cells. However, other molecular components including invariant chain (Ii), HLA-DM, and costimulatory molecules CD80, CD86 and CD40, are required for efficient T-cell activation. We have investigated whether normal human duodenal EC possess these molecules and whether they can mediate MHC class II antigen presentation. EC were isolated from duodenal biopsies from patients in whom pathology was excluded. Freshly-isolated duodenal EC did not stimulate autologous T-cell proliferation against purified protein derivative of tuberculin. Flow cytometry and immunoblot analysis revealed that duodenal EC constitutively express HLA-DR, Ii, and HLA-DM. Surface MHC class II associated invariant chain peptide (CLIP) was not detectable, suggesting that HLA-DM functions normally in CLIP removal. Duodenal EC expressed SDS-stable HLA-DR alphabeta heterodimers, indicating that peptide binding had occurred. Surface expression of CD80, CD86 or CD40 was not detected although mRNA for these costimulatory molecules was present in all samples. These results suggest that nondiseased human duodenal EC can process and present antigen by the MHC class II pathway, but that they may induce anergy, rather than activation, of local T cells.     

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.     

Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II

Antigen-driven interaction of dendritic cells (DC) with CD4(+) T(h) cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates T(h) cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC-T(h) cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up-regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.     

Failure of trafficking and antigen presentation by CD1 in AP-3-deficient cells

Endocytosed microbial antigens are primarily delivered to lysosomal compartments where antigen binding to MHC and CD1 molecules occurs in an acidic and proteolytically active environment. Signal-dependent delivery to lysosomes has been suggested for these antigen-presenting molecules, but molecular interactions with vesicular coat proteins and adaptors that direct their lysosomal sorting are poorly understood. Here CD1b but not other CD1 isoforms bound the AP-3 adaptor protein complex. In AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2 (HPS-2), CD1b failed to efficiently gain access to lysosomes, resulting in a profound defect in antigen presentation. Since MHC class II traffics normally in AP-3-deficient cells, defects in CD1b antigen presentation may account for recurrent bacterial infections in HPS-2 patients.     

Proteases involved in MHC dass II antigen presentation

Summary: Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen-presenting cells must be broken down into antigenic peptides. Second, class II tnolecules are synthesized with their peptide-binding site blocked by invariant chain (li), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified tnice deficietit in single lysosomal proteases has allowed us to determine their role in these processes, Cathepsins (Cat) B and D. previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immtine responses and for T-cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co-exist in the same antigen-presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.     

Inhibitory p41 isoform of invariant chain and its potential target enzymes cathepsins L and H in distinct populations of macrophages in human lymph nodes

Activation of the CD4(+) T-cell mediated immune response relies on the proteolytic capacity of enzymes involved in modulating major histocompatibility complex (MHC) II-associated antigen presentation in antigen-presenting cells (APC). The MHC II-associated chaperone molecule p41 isoform of invariant chain (inhibitory p41 Ii) has been suggested to regulate stability and activity of cathepsin L in these APC. In the present study the human lymph node distribution of non-inhibitory p31 Ii and inhibitory p41 Ii have been compared by differential labelling, using two specific monoclonal antibodies. The distribution of p41 Ii, but not p31 Ii, matched the distribution of cathepsins L and H in subcapsular and cortical sinuses and germinal centres. Co-localization of p41 Ii with cathepsin H was confirmed in strongly CD68(+) sinus-lining macrophages, acting as APC. Furthermore, p41 Ii was determined together with cathepsins L and H in tingible body macrophages, highly phagocytic, but not antigen-presenting cells inside germinal centres. With respect to the physiological function that these two populations of macrophages have in human lymph nodes, our results support a regulatory function of p41 Ii towards cathepsins L and H in human macrophages, associated with the processes of phagocytosis rather than antigen presentation.     

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.