Generation of naturally processed peptide/MHC class I complexes is independent of the stability of endogenously synthesized precursors

Proteolysis of endogenously synthesized cellular proteins is essential for constitutive display of processed peptide/MHC class I complexes on the APC surface for stimulating CD8+ T cells. However, the extent to which normal protein turnover serves as the source of processed peptides is not clear. To address this question, we used pairs of novel N-end rule substrates that varied in their intracellular stability and served as precursors for generating peptide/MHC class I (OVA257-264/Kb or influenza nucleoprotein 366-374/Db) complexes. Surprisingly, although each of three precursor pairs tested varied profoundly in their intracellular stability, they were indistinguishable in either T cell stimulation assays, or in the amounts of naturally processed peptides in the APC extracts. Our findings demonstrate that the proteolytic turnover of endogenously synthesized proteins is not directly proportional to the generation of processed antigenic peptide/MHC class I complexes.     

Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.     

Alterations in TCR-MHC contacts subsequent to cross-recognition of class I MHC and singly substituted peptide variants

Vesicular stomatitis virus (VSV) elicits H-2Kb-restricted CTLs specific for the immunodominant VSV octapeptide RGYVYQGL. To study the structural features important for interaction between the TCR beta-chain and the peptide/MHC complex, we immunized TCR alpha-chain transgenic mice with the VSV peptide and raised a panel of anti-VSV CTL clones with identical TCR alpha-chains. Consistent with our previous analysis of uncloned populations of primary CTLs, the anti-VSV CTL clones were all Vbeta13+ and expressed TCR beta-chains with highly homologous complementarity-determining region 3 (CDR3) loops. Although the clones expressed similar TCRs, they differed in their ability to cross-react with VSV peptide variants singly substituted at TCR contact positions 4 and 6. These findings allowed us to identify short stretches of amino acids in the C-terminal region of the CDR3beta loop that, when altered, modify the cross-reaction capability of the TCR to position 4 and position 6 variant peptides. To further probe the structural correlates of biologic cross-reactivity, we used cross-reactive CTL clones and cell lines expressing point mutations in H-2Kb to investigate the effect of single amino acid changes in the peptide on the pattern of recognition of the TCR for the peptide/MHC complex. Single conservative substitutions in the peptide were sufficient to alter the recognition contacts between a cross-reactive TCR and the MHC molecule, supporting the idea that the TCR can make overall structural adjustments in MHC contacts to accommodate single amino acid changes in the peptide.     

Application of an artificial neural network to predict specific class I MHC binding peptide sequences

We report a 22-year-old man who successfully underwent coronary stent implantation after an acute myocardial infarction. Lupus anticoagulant and antibodies against cardiolipin and prothrombin were detected despite the absence of any underlying disease. Therefore, long-term oral anticoagulation was instituted and appeared to be effective. To our knowledge this is the first report on coronary stenting in primary antiphospholipid syndrome.     

Knowledge-based structure prediction of MHC class I bound peptides: a study of 23 complexes

BACKGROUND: The binding of T-cell antigenic peptides to MHC molecules is a prerequisite for their immunogenicity. The ability to identify binding peptides based on the protein sequence is of great importance to the rational design of peptide vaccines. As the requirements for peptide binding cannot be fully explained by the peptide sequence per se, structural considerations should be taken into account and are expected to improve predictive algorithms. The first step in such an algorithm requires accurate and fast modeling of the peptide structure in the MHC-binding groove. RESULTS: We have used 23 solved peptide-MHC class I complexes as a source of structural information in the development of a modeling algorithm. The peptide backbones and MHC structures were used as the templates for prediction. Sidechain conformations were built based on a rotamer library, using the 'dead end elimination' approach. A simple energy function selects the favorable combination of rotamers for a given sequence. It further selects the correct backbone structure from a limited library. The influence of different parameters on the prediction quality was assessed. With a specific rotamer library that incorporates information from the peptide sidechains in the solved complexes, the algorithm correctly identifies 85% (92%) of all (buried) sidechains and selects the correct backbones. Under cross-validation, 70% (78%) of all (buried) residues are correctly predicted and most of all backbones. The interaction between peptide sidechains has a negligible effect on the prediction quality. CONCLUSIONS: The structure of the peptide sidechains follows from the interactions with the MHC and the peptide backbone, as the prediction is hardly influenced by sidechain interactions. The proposed methodology was able to select the correct backbone from a limited set. The impairment in performance under cross-validation suggests that, currently, the specific rotamer library is not satisfactorily representative. The predictions might improve with an increase in the data.     

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 +/- 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these alphabeta Ii complexes are converted into alphabeta-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the alphabeta Ii complexes. More than 80% of alphabeta dimers originating from internalized alphabeta Ii complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface alphbeta Ii into SDS-stable and -unstable alphabeta dimers. Altogether, these data suggest that newly liberated class II alphabeta heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.     

Differential contact of disparate class I/peptide complexes as the basis for epitope cross-recognition by a single T cell receptor

In an effort to better understand functional recognition of structurally dissimilar ligands by a single TCR, a model system for studying cross-recognition of disparate peptide/class I complexes was developed using the murine (H-2b) CTL clone AHIII12.2, which is reactive to a human self-peptide (p1049) bound to an HLA-A2.1 molecule. We identified a second complex comprised of a synthetic peptide, designated p1058, bound to H-2Db that is recognized by clone AHIII12.2. In cytolysis assays, dose-response profiles for peptides p1049 and p1058 pulsed onto the appropriate target cells were comparable, suggesting that p1049/A2.1 and p1058/Db form functionally equivalent epitopes. To probe the interaction between each complex and the TCR of AHIII12.2, singly substituted analogues of each peptide were tested for their activity in lysis assays. Differences were observed between the two epitopes with respect to permissible residue substitutions at each peptide position from P3 to P8; marked differences were evident at P3 and at P8. The results obtained suggest that this TCR forms critical contacts with atoms at peptide positions P3 and P5 of p1049/A2.1 and at P5 and P8 of p1058/Db, and that TCR cross-recognition of these ligands is a function of both shared and complex-specific contacts made with each epitope. These findings further highlight the versatile reactivity that may be shown by a single TCR and suggest a basis for the recognition of peptide ligands sharing only a limited set of structural features.     

Induction of tolerance in experimental autoimmune myasthenia gravis with solubilized MHC class II:acetylcholine receptor peptide complexes

Stimulation of T lymphocytes through the T cell receptor in the absence of costimulatory signal(s) induces a state of unresponsiveness to subsequent antigen presentation. We have employed solubilized complexes consisting of rat class II MHC molecules containing an immunodominant peptide of the acetylcholine receptor (AChR alpha 100-116) to induce unresponsiveness in the autoreactive T lymphocytes mediating an animal model of myasthenia gravis. In vitro incubation of rat T cell lines specific for peptide AChR alpha 100-116 with solubilized complexes of MHC II and AChR alpha 100-116 (MHC II:AChR alpha 100-116) rendered the T cells unresponsive to subsequent stimulation by antigen presenting cells and the peptide. T cell lines with a broader specificity to the entire AChR protein pentamer had an 81% reduction in proliferation to AChR following a preincubation with solubilized MHC II:AChR alpha 100-116. Treatment with the solubilized MHC II:AChR alpha 100-116 induced phosphatidylinositol 4,5-bisphosphate hydrolysis, an early signalling event associated with binding to the TCR. Rats primed with AChR and injected intravenously with MHC II:AChR alpha 100-116 had reduced in vitro T cell proliferation to the AChR alpha 100-116 peptide and to whole AChR. Solubilized MHC II:AChR alpha 100-116 injected i.v. into rats exhibiting serological clinical symptoms of experimental autoimmune myasthenia gravis (EAMG) prevented death in 67% of the treated animals, compared to a 0-20% survival rate in all other control groups. These results demonstrate that solubilized MHC II complexed with an immunodominant autoantigenic peptide is tolerogenic and improves the survival rate of rats with EAMG, suggesting the basis for an antigen-specific therapy in autoimmune diseases such as MG.     

The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed B loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class II evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci.     

Immunodominance of a single CTL epitope in a primate species with limited MHC class I polymorphism

MHC class I molecules play a crucial role in immunity to viral infections by presenting viral peptides to cytotoxic T lymphocytes. One of the hallmarks of MHC class I genes in outbred populations is their extraordinary polymorphism, yet the significance of this diversity is poorly understood. Certain species with reduced MHC class I diversity, such as the cotton-top tamarin (Saguinus oedipus), are more susceptible to fatal viral infections. To explore the relationship between this primate's limited MHC class I diversity and its susceptibility to viruses, we infected five cotton-top tamarins with influenza virus. Every tamarin recognized the same immunodominant CTL epitope of the influenza nucleoprotein. Surprisingly, this nucleoprotein peptide was bound by Saoe-G*08, an MHC class I molecule expressed by every cotton-top tamarin. Two tamarins also made a subdominant response to an epitope of the matrix (M1) protein. This peptide appeared to be bound by another common MHC class I molecule. With the exception of an additional subdominant response to the polymerase (PB2) protein in one individual, no other influenza-specific CTL responses were detected. In populations or species with limited MHC class I polymorphism like the cotton-top tamarin, a dependence on shared MHC class I molecules may enhance susceptibility to viral infection, since viruses that evade MHC class I-restricted recognition in one individual will likely evade recognition in the majority of individuals.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas

A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen-specific stimulation of class I-restricted T cell hybridomas, as assessed by IL-2 release, does not depend on CD8.     

Immunosuppressive peptides corresponding to MHC class I sequences

The induction of tolerance is a long-standing goal in transplantation. Intact MHC molecules, or fragments of them, are being used to render T cells unresponsive both in vitro and in vivo. Elucidation of the mechanisms underlying the effects of these treatments should aid the design of novel therapies for transplantation.     

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.     

In vitro induction of naive cytotoxic T lymphocytes with complexes of peptide and recombinant MHC class I molecules coated onto beads: role of TCR/ligand density

We previously reported that complexes of peptide with soluble single-chain recombinant MHC (SC-MHC) class I molecules are able to induce cytotoxic T lymphocytes (CTL) in vitro in a murine system with an efficiency comparable to that observed with peptide-pulsed dendritic cells as antigen-presenting cells. In this report, we have assessed the capacity of preformed peptide/SC-Kd complexes in monomeric or dimeric form as well as of peptide/SC-Kd-loaded beads to generate in vitro specific CTL responses from naive DBA/2 spleen cells. Peptide/SC-Kd-coated beads were consistently more efficient. We evaluated the role of costimulatory molecules, using monoclonal antibodies anti-CD80 or anti-CD86. In addition, the capacity of peptide/SC-Kd-coated beads to generate a CTL response from purified naive CD8+ T cells was ascertained. Taken together, the results indicate that, under our conditions, CTL priming does not require the participation of co-stimulatory molecules and is the consequence of a direct interaction between the cognate TCR on peptide-specific CTL precursors and the peptide/SC-Kd-loaded beads. Titration of the amount of preformed complexes of SC-Kd and peptide 170-179 of HLA-CW3 that need to be coated onto the beads to prime CTL precursors shows an activation threshold which can be calculated to be between 25000 and 50000 complexes. In effect, in cultures stimulated with specific peptide CW3/SC-Kd complexes representing less than 50% occupancy of the total (10(5)) complexes on the beads, no peptide-specific cytolytic activity was observed. These results suggest that the efficiency of the primary CTL induction depends on the density of specific peptide/SC-Kd complexes present on the beads.     

Role of MHC class I molecules in autoimmune disease

The MHC class I molecules play a pivotal role in triggering cellular immune responses, binding and presenting intracellularly derived peptide antigens. Studies of MHC class I expression revealed a complex regulatory mechanism that integrates tissue-specific and hormonal modulation. Dynamic regulation occurs in the thyroid, in response to hormonal repression by TSH and stimulation by thyroid hormone. This dynamic cycle provides the basis for proposing the model that such regulation is important to maintain tolerance to self-antigens in tissues synthesizing large amounts of secretory proteins. Failure to appropriately regulate class I levels is predicted to result in autoimmunity. In support of this model, we found that class I-deficient mice are resistant to the experimentally induced autoimmune diseases, SLE, and blepharitis. Furthermore, pharmacological treatment with an agent that reduces class I expression also reduces the incidence and severity of both experimental and spontaneous autoimmune SLE.     

Interpreting MHC class I expression and class I/class II reciprocity in the CNS: reconciling divergent findings

MHC-restricted T cells are thought to contribute to clinical demyelination in MS and other circumstances. The step-by-step mechanisms involved and ways of controlling them are still being defined. Identification of the MHC+ cells in the CNS in situ has been controversial. This chapter reviews MHC expression in neural tissue, including normal, pathological, experimental, and developing tissue in situ and isolated cells in vitro. A basic pattern is defined, in which MHC expression is limited to nonneural cells and strongest class I and II expression are on different cell types. Variations from the basic pattern are reviewed. Ways of reconciling divergent findings are discussed, including the use of "mock tissue" to help choose between technical and biological bases for divergent findings, the potential contribution of internal antigen to the in situ staining patterns, and the possibility that class I upregulation is actively suppressed in situ. Functional implications of the observed patterns of MHC expression and ways of confirming the function of each MHC+ cell type in situ are described. It is suggested that modulating MHC expression in different cell types at different times or in different directions might be desirable.     

NK cells can recognize different forms of class I MHC

NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.     

Antigenic peptide binding by class I and class II histocompatibility proteins

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)