Class II-peptide Complexes Research Articles

Specific tolerance to an acetylcholine receptor epitope induced in vitro in myasthenia gravis CD4+ lymphocytes by soluble major histocompatibility complex class II-peptide complexes.

In autoimmune disorders, inactivation of pathogenic antigen-specific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (A ChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-A1) with a known peptide epitope (alpha 144-163) and MHC class II restriction (DR4 Dw14.2 or 4.2) by using soluble MHC-class II peptide complexes. Preincubation of PM-A1 T cells with such complexes induced death by apoptosis in < or = 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < or = 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells. This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II: peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo.

Assembly, transport, and function of MHC class II molecules.

MHC class II molecules assemble in the endoplasmic reticulum in a chaperone-mediated fashion to form a nine-chain structure consisting of three alpha beta dimers associated with an invariant chain trimer. This complex is transported through the Golgi apparatus and into the endosomal system. The signal for endosomal targeting resides in the cytoplasmic tail of the invariant chain. Current evidence argues that the segregation of the class II-invariant chain complex from the constitutive pathway of membrane protein transport occurs in the trans-Golgi network. However, class II-invariant chain complexes that reach the cell surface are also rapidly internalized into endosomes. Within the endosomal system, probably in a late endosome/pre-lysosome, the invariant chain is degraded, releasing alpha beta dimers that bind peptides predominantly derived from endocytosed proteins. Evidence suggests that many of these peptides are actually generated in lysosomes. The precise mechanisms involved in forming class II-peptide complexes are unclear, although the existence of antigen-processing mutants argues that additional gene products, at least one of which is encoded in the MHC, are involved. After binding peptides, class II molecules are transported by an unknown route to the cell surface, where their primary function of presenting antigenic peptides to CD4+ T cells is carried out.

Separation of complexes of major histocompatibility class II molecules and known antigenic peptide by metal chelate affinity chromatography

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exist for separation of complexes of a known antigenic epitope and MHC class II from empty MHC class II and complexes MHC class II and endogenously bound peptide. Here we describe an one step metal chelate affinity chromatography method to purify complexes of MHC class II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB1 *1501/DRB5 *0101) and a peptide analog from human myelin basic protein MBP(84–102) containing a 6 histidine tag (6 × His) and a tyrosine residue at the N-terminus end [6 × His-MBP(83–102)Y 83] were prepared and purified. The absence of residual free 6 × His-MBP peptide in the complex preparations were confirmed by gel filtration and TLC analyses. The purified complexes were applied onto Ni 2+· nitrilotriacetic acid (Ni 2+ · NTA)-agarose affinity support and 6 × His-tagged peptide class II complexes were selectively eluted with imidazole-containing buffer. The quantitation of bound peptides in the eluted complexes showed 100% occupancy of HLA-DR2 (DRB1 *1501/DRB5 *0101) with [6×His-MBP(83–102)Y 83] peptide with recovery of 50–75%. The presence of a single peptide entity in the eluted complexes was confirmed by reverse-phase narrobore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni 2+·NTA eluted complexes when analyzed by two-dimensional IEF gel electrophoresis. Finally, we demonstrate that both MBP(84–102) and [6 × His-MBP(83–102)Y 83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous antigen presenting cells (APCs). These results demonstrate that metal chelate affinity chromatography can be used to prepare MHC class II-peptide complexes containing single peptide. Such complexes of class II molecules containing known have significant clinical relevance for antigen-specific therapy of various autoimmune diseases may provide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptors (TRC).

Peptide binding to surface class II molecules is the major pathway of formation of immunogenic class II-peptide complexes for viable antigen presenting cells.

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-class II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules was occurring after endocytosis of peptides as well as by surface binding, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surface complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lymphoblastoid cells (BLCLs). Detection of the bound peptide was made possible by a rabbit anti-serum (alpha HAP) raised against HAP 128-145, which recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significant inhibition in the appearance of these complexes was caused by a decrease in the temperature at which the cells were incubated with peptide. Temperature-specific inhibition was also observed for fixed DRw11-positive APCs and purified DRw11 molecules indicating that the effect of temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such complexes. Moreover, class II molecules have evolved to stably bind peptide optimally at physiologic temperatures, independent of cell metabolism.

Kinetics and specificity of peptide-MHC class II complex displacement reactions.

The peptide-induced acceleration of the dissociation of pre-formed complexes of the detergent-solubilized mouse class II molecules IEd and IEk with fluorescein-labeled peptides was investigated using high-performance size exclusion chromatography. While it is generally believed that functional complexes of MHC class II alpha beta heterodimers and peptides have a 1:1 stoichiometry, the data provide qualitative as well as quantitative kinetic evidence that the enhancement of the release of one peptide by a second peptide is due to a two-peptide intermediate. Different combinations of peptides were tested for their ability to accelerate each other's release from IEd. The importance of positive charge for the interaction with IEd was confirmed by the finding that not only dynorphin 1-13 but also poly-L-lysine (14-19 mer) and a peptide corresponding to a mitochondrial presequence (net charge +6) efficiently enhance the release of pre-bound peptides. SDS-PAGE analysis revealed that the efficiently displacing peptides do not stabilize the IEd alpha beta heterodimer at acidic pH, in contrast to the IEd-restricted antigenic peptide HEL 107-116. The data support a mechanism in which the second peptide binds specifically to the pre-formed class II-peptide complex, which, depending on the properties of the peptides involved, leads to the destabilization of the complex and the release of the first peptide.

Structural Requirements for Recognition of a Type II Collagen Peptide by Murine T Cell Hybridomas

T lymphocytes play a critical role in the development of murine collagen-induced arthritis (CIA), a syndrome which shares many features with rheumatoid arthritis. In susceptible mouse strains, immunization with type II collagen (cII) results in chronic inflammation with progressive joint destruction. To examine T cell recognition of cII we have isolated T cell hybridomas specific for a cII peptide fragment, cII260-270 (IAGFKGEQGPK). We assessed the importance of particular amino acid residues to formation of the MHC Class II-peptide complex and interaction of this complex with TCRs. Our results indicate that critical residues are concentrated in the N-terminal half of the peptide, with Lys264 and Glu266 having the greatest influence. Replacement of these residues with alanine resulted in loss of detectable activity. Three other residues, IIe260, Gly262, and Phe263, are also important to T cell stimulation because alanine substitution substantially decreased peptide activity. Truncation analyses supported the conclusion drawn from Ala substitutions that C-terminal residues were dispensable. Other alterations in peptide structure resulted in dramatic increases in stimulatory capacity. For example, replacement of either Gly265 or Gly268 with alanine resulted in a 10-fold increase in potency. The addition of 2-5 residues to the N-terminus of the peptide decreased the dose required for maximal T cell stimulation by <100-fold. The T cell hybridomas exhibited remarkable similarity in their peptide recognition profiles although they express different TCR Vβ elements.

Length and sequence requirements of the cytoplasmic domain of the A beta molecule for class II-mediated B cell signaling.

APC use class II molecules of the MHC to present peptide Ag to Th cells. Interaction of the TCR and CD4 with the class II-peptide complex, together with co-stimulatory signals provided by the APC, activates the T cell. B lymphocytes express class II molecules and can also be induced to express co-stimulatory molecules, allowing them to act as APC to Th cells. In addition to T cell activation, class II binding by T cells has been shown to result in the transmission of signals to B cells. Signal transduction via MHC class II has been well documented in B cells of both mice and humans and is implicated in the processes of cellular adhesion, Ag presentation, and Ag-dependent B cell activation. The regions of the class II MHC molecule which are involved in signal transduction to the B cell are not clearly defined. However, previous studies have suggested that the beta chain of the alpha beta heterodimer has a predominant role in B cell signaling. To examine the role of the cytoplasmic domain of this molecule in class II-mediated signaling to a mouse B cell clone, we have prepared and analyzed a set of subclones expressing sequentially truncated forms of A beta b. Our results demonstrate that only the 8 membrane-proximal amino acids of the cytoplasmic domain are required for signaling. However, specific conserved amino acids within this minimal length are required for successful signal transduction; length alone is not sufficient. Examination of the signaling ability of these truncated beta chains suggests that conserved residues at positions 227 and 228 of the cytoplasmic domain may have particularly important effects on signal transduction. A beta b chains from which the entire cytoplasmic domain have been removed are still capable of transmitting a detectable, although reduced, signal to B cells. Thus, the transmembrane and/or extracellular domains may also be involved in the signaling process.

The influence of LFA 1 on human T cells stimulated by solid-phase immobilized HLA class II-peptide complexes.

Purified HLA class II-peptide complexes immobilized to a solid support induce proliferation of human T-cell clones, indicating that human T-cell clones can proliferate in the absence of secondary signals from accessory cells. We hypothesized that T cells can provide co-stimulatory signals to T cells. LFA-1 molecules play an important role in homotypic interactions of T cells and murine monoclonal antibodies reactive with LFA 1 can inhibit T cell-T cell interactions. LFA-1 reactive monoclonal antibodies inhibited cytolysis of peptide-pulsed T cells by T cells and partially inhibited T-cell proliferation. To study the direct effect of the LFA 1 molecule on T-cell activation, we co-immobilized HLA-class II-peptide complexes with LFA-1 reactive MoAbs. Co-immobilization resulted in an enhanced proliferative response of the T-cell clones. This could indicate that the LFA 1 molecule on T cells is not a passive adhesion molecule, but is capable of transducing a signal that synergizes with the stimulatory signal via the T-cell receptor.

Cloned T cells internalize peptide from bound complexes of peptide and purified class II major histocompatibility complex antigen

Antigen presentation to helper T cells involves the formation of a trimolecular complex consisting of a class II major histocompatibility complex (MHC) antigen combined with an antigenic peptide on the surface of an antigen-presenting cell and a T cell receptor (TCR) on the T cell. The fate of the MHC class II, peptide, or TCR moieties of the ternary complex following antigen presentation is unknown. Using radiolabeled complexes of affinity-purified murine MHC class II molecules and peptides corresponding to T cell epitopes of myelin basic protein (MBP), this report presents evidence that the binding of preformed relevant MHC class II-peptide complexes to cloned T cells in vitro results in internalization of the peptide moiety. Neither the restricting MHC class II molecule nor the TCR moiety of the trimolecular complex was internalized by T cells. The specificity of peptide internalization was demonstrated using complexes of syngeneic MHC class II with an irrelevant MBP peptide analog and by cloned T cells restricted for a different epitope of the same MBP antigen. Furthermore, the peptide translocation mediated by MHC class II and TCR was demonstrated by antibody-blocking experiments using anti-class and anti-TCR monoclonal antibodies. The peptide internalization by T cells was markedly reduced when binding was performed at 4 degrees C as compared with 37 degrees C. In addition, a significant inhibition of peptide translocation was observed in the presence of a metabolic inhibitor (sodium azide) but not in the presence of cytochalasin B. These results together demonstrate that the in vitro interaction of soluble MHC II-peptide complexes with cloned T cells is an active process associated with uptake of the antigenic peptide.

Invariant chain peptides in most HLA-DR molecules of an antigen-processing mutant.

Class II major histocompatibility complexes bind peptides in an endosome-like compartment. When the class II null cell line 721.174 was transfected with class II DR3 genes, DR molecules were produced in normal amounts. However, the DR molecules were abnormally conformed and unstable because deletion of an antigen-processing gene had impaired intracellular formation of most class II-peptide complexes. Yet, 70 percent of the DR molecules still bore peptides, 80 percent of which were 21- to 24-amino acid fragments of the class II-associated invariant chain. These peptides were rare on DR3 from control cells. Thus, a defect in the main antigen-processing pathway revealed a process in which DR molecules bind long peptides derived from proteins present in the same compartment.

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.

Effect of pH on MHC class II-peptide interactions.

The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II-peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II-peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides.

Characterization of the signaling function of MHC class II molecules during antigen presentation by B cells.

In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.

Free ligand-induced dissociation of MHC-antigen complexes.

We have analyzed the stability of Ag-class II complexes on the surface of live APC. It was found that the disappearance of complexes formed by I-Ed and the hen egg lysozyme 107-116 peptide is twice as rapid on the surface of live, as compared to glutaraldehyde-fixed APC. Moreover, the addition of peptides with a high affinity for I-Ed could reduce the half-life of Ag-class II complexes on either live or fixed APC. The same effect was detectable using purified class II molecules adhered on plastic microtiter wells, and even in the case of complexes formed in vitro between radiolabeled Ag and purified class II molecules. This effect of accelerated dissociation appeared to be specific because only I-Ed binding peptides were able to accelerate the dissociation of the hen egg lysozyme 107-116/I-Ed complex either on the surface of cells or in purified form in solution, and high affinity I-Ed binders did not affect the half-life of purified OVA 323-339/I-Ad complexes. These results demonstrate that free ligand can influence the kinetics of dissociation of class II-peptide complexes, and therefore suggest that a mechanism other than the classical first order kinetics may be involved in this process.

Novel pathways of antigen presentation for the maintenance of memory

Follicular dendritic cells (FDC) store native antigen for long periods in lymphoid follicles and so provide a source of continued stimulation for specific B cells. The expression of MHC class II by FDC suggested they might act as antigen-presenting cells for MHC class II-restricted T cells. We show here, however, that the MHC class II molecules found on their surface are not synthesized by the FDC but are picked up from surrounding B cells in germinal centres. Although FDC by themselves cannot present native antigen to T cells, acquired MHC class II-peptide complexes can be recognized by T cells. The true physiological role of FDC seems to be as long-term antigen depots. We demonstrate that antigen localized onto FDC in vivo can be retrieved by antigen-specific B cells, which in turn process and present it to T cells. These presentation pathways are likely to be crucial in both the maintenance of long-term immune responses and the continued survival of memory cells.

Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing.

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.

Invariant chain influences the immunological recognition of MHC class II molecules

Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.

Recognition of the HLA class II-peptide complex by T-cell receptor: reversal of major histocompatibility complex restriction of a T-cell clone by a point mutation in the peptide determinant

Recognition of the HLA DR-peptide complex by an influenza haemagglutinin-specific T-cell clone was examined by assaying a variety of peptide analogues for their ability to be recognized. Consistent with earlier experiments arguing that the peptide blinds the restriction element in a helical conformation, acetylation of the amino terminus and amidation of the carboxy terminus of the natural determinant (residues 307-319) resulted in a peptide that exhibited both greater propensity to form a helix, as judged by circular dichroism, and the ability to stimulate the clone at concentrations approximately two orders of magnitude lower than the native sequence. The peptide was modelled into the potential antigen-combining site of HLA class II based on the ability of analogues containing point mutations to stimulate the T-cell clone. The working model was initially tested by examining the ability of Epstein-Barr-transformed B-cell lines expressing in different DR4 subtypes to present the native haemagglutinin sequence and analogues to the clone. The different alleles could be categorized as high, intermediate, or low responders based on the resulting proliferation. DR4 dw15 was a high-responding allele, dw4, 13, and 14 were intermediate-responding alleles, whereas dw10 was a low responder. Mutation of Gln to Arg at 312 in the haemagglutinin sequence converted the high and intermediate responders to non-responders, while turning the low-responding allele into an intermediate responder. Potential explanations for these effects are discussed in the context of the model of the complex between peptide and the major histocompatibility complex.