Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Susceptibility to the biological effects of polyaromatic hydrocarbons is influenced by genes of the major histocompatibility complex

Polyaromatic hydrocarbons are ubiquitous environmental chemicals that are important mutagens and carcinogens. The purpose of this study was to determine whether genes within the major histocompatibility complex (MHC) influence their biological activities. Cell-mediated immunity to dimethylbenz(a)anthracene (DMBA) was investigated in congenic strains of mice. On three different backgrounds, H-2k and H-2a haplotype mice developed significantly greater contact-hypersensitivity responses to DMBA than H-2b, H-2d, and H-2s mice. In B10.A(R1) mice, which are Kk and Id, a vigorous contact-hypersensitivity response was present, indicating that the response was governed by class I, rather than class II, MHC genes. C3H/HeN (H-2k) and C3H.SW (H-2s) strains were also compared for the development of skin tumors and the persistence of DMBA–DNA adducts. When subjected to a DMBA initiation, phorbol 12-tetradecanoate 13-acetate (TPA)-promotion skin-tumorigenesis protocol, C3H/HeN mice, (which develop cell-mediated immunity to DMBA) were found to have significantly fewer tumors than C3H.SW mice (a strain that failed to develop a cell-mediated immune response to DMBA). DMBA–DNA adducts were removed more rapidly in C3H/HeN than in C3H.SW mice. The results indicate that genes within the MHC play an important role in several of the biological activities of carcinogenic polyaromatic hydrocarbons. The observations are consistent with the hypothesis that cell-mediated immunity to chemical carcinogens serves to protect individuals by removing mutant cells before they can evolve into clinically apparent neoplasms.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

IMGT/HLA Database—a sequence database for the human major histocompatibility complex

The IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/) specialises in sequences of polymorphic genes of the HLA system, the human major histocompatibility complex (MHC). The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. Many of the genes encode proteins of the immune system and these include the 21 highly polymorphic HLA genes, which influence the outcome of clinical transplantation and confer susceptibility to a wide range of non-infectious diseases. The database contains sequences for all HLA alleles officially recognised by the WHO Nomenclature Committee for Factors of the HLA System and provides users with online tools and facilities for their retrieval and analysis. These include allele reports, alignment tools and detailed descriptions of the source cells. The online IMGT/HLA submission tool allows both new and confirmatory sequences to be submitted directly to the WHO Nomenclature Committee. The latest version (release 1.7.0 July 2000) contains 1220 HLA alleles derived from over 2700 component sequences from the EMBL/GenBank/DDBJ databases. The HLA database provides a model which will be extended to provide specialist databases for polymorphic MHC genes of other species.

The Export of Major Histocompatibility Complex Class I Molecules from the Endoplasmic Reticulum of Rat Brown Adipose Cells Is Acutely Stimulated by Insulin

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase–dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.

Transloading of tumor cells with foreign major histocompatibility complex class I peptide ligand: a novel general strategy for the generation of potent cancer vaccines.

The major hurdle to be cleared in active immunotherapy of cancer is the poor immunogenicity of cancer cells. In previous attempts to overcome this problem, whole tumor cells have been used as vaccines, either admixed with adjuvant(s) or genetically engineered to express nonself proteins or immunomodulatory factors before application. We have developed a novel approach to generate an immunogeneic, highly effective vaccine: major histocompatibility complex (MHC) class I-positive cancer cells are administered together with MHC class I-matched peptide ligands of foreign, nonself origin, generated by a procedure we term transloading. Murine tumor lines of the H2-Kd or the H2-Db haplotype, melanoma M-3 and B16-F10, respectively, as well as colon carcinoma CT-26 (H2-Kd), were transloaded with MHC-matched influenza virus-derived peptides and applied as irradiated vaccines. Mice bearing a deposit of live M-3 melanoma cells were efficiently cured by this treatment. In the CT-26 colon carcinoma and the B16-F10 melanoma, high efficacies were obtained against tumor challenge, suggesting the universal applicability of this new type of vaccine. With foreign peptide ligands adapted to the requirements of a desired MHC class I haplotype, this concept may be used for the treatment of human cancers.

A common major histocompatibility complex class II allele HLA-DQB1* 0301 is present in clinical variants of pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease seen primarily in elderly persons. It is characterized clinically by the development of tense bullae and by the presence of an antibasement membrane antibody. In BP, the antigens involved in the autoimmunity are epidermal basement membrane peptides BPAg1 and BPAg2. We have compared high resolution typing of major histocompatibility complex class II loci (HLA-DRB1, DQB1) in 21 patients with BP, 17 with ocular cicatricial pemphigoid (OCP), and 22 with oral pemphigoid (OP) to a panel of 218 haplotypes of normal individuals. We found that the three diseases (BP, OCP, and OP) have significant association with DQB1*0301 (P = 0.005, P < 0.0001, and P = 0.001, respectively). The frequencies of alleles DQB1*0302, 0303, and 06, which share a specific amino acid sequence from position 71 to 77 (Thr-Arg-Ala-Glu-Leu-Val-Thr) were also increased (P = 0.01). We suggest that an identical major histocompatibility complex class II allele (DQB1*0301) is a common marker for enhanced susceptibility and that the same amino acid residues in positions 71-77 (DQB1*0301, -0302, -0305, -0602, -0603 alleles) are found in patients with BP, OCP and OP. Our findings propose that the autoimmune response in the three different clinical variants of pemphigoid, involves the recognition by T cells of a class II region of DQB1, bound to a peptide from the basement membrane of conjunctiva, oral mucosa, and skin.

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.