ABSTRACT
This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first basic protocol describes electroelution of the fragment of interest from standard agarose gels using buffer-filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA-45 paper is also described and provides relatively high yields for fragments smaller than 2000 bp. Protocols are also provided for separating fragments larger than 1000 bp using low gelling/melting agarose gels and purified by phenol extraction, b-agarase digestion of the gel, or via glass beads extraction. Sieving agarose gels can also be used to resolve very small DNA fragments. Removing linkers from a fragment using a column rather than a gel is included, followed by a method for estimating DNA concentrations in solution.
Subject(s)
DNA/analysis , DNA/isolation & purification , Animals , Chromatography, Liquid/methods , DNA Restriction Enzymes/chemistry , Electrophoresis, Agar Gel/methods , Glycoside Hydrolases/chemistry , HumansABSTRACT
Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA. The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A(+)) mRNAs from total RNA.
Subject(s)
Eukaryotic Cells/chemistry , RNA/isolation & purification , Animals , Cell Nucleus/chemistry , Cells, Cultured/chemistry , Centrifugation/methods , Centrifugation, Density Gradient , Cytoplasm/chemistry , Detergents , Guanidines , Organ Culture Techniques , Phenol , RNA, Messenger/isolation & purification , ThiocyanatesABSTRACT
Dynamic interactions between major histocompatibility complex (MHC) class I heavy chains and beta2-microglobulin (beta2m) play a critical role in their stability on the cell surface, and their ability to present peptide antigens to CD8+ T-cells. A cursory review of protein sequence homologies and three-dimensional crystal structures of MHC complexes might indicate very similar modes of interaction between the heavy and light chains. In this report, a panel of human beta2m mutants was screened to probe the interactions of beta2m with the murine MHC molecules H-2Kb, -Db, -Kd, -Ld, and -Dd. Binding experiments coupled with analyses of existing three-dimensional crystal structures demonstrate allelic differences in their interaction with beta2m. A comprehensive analysis of the existing murine MHC structures indicates a conformational flexibility on the part of murine beta2m that is not present in beta2m of the human structures. This flexibility is in a region directly interacting with the heavy chain and may relate to its lower affinity for murine heavy chains relative to human beta2m. This defined panel of beta2m mutants of differing affinity may also be useful for subsequent studies of thymic selection, T-cell recognition, and more refined algorithms for protein structure prediction.
Subject(s)
H-2 Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Membrane Proteins/metabolism , Point Mutation , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolism , Animals , Computer Simulation , Crystallography , Databases, Factual , Mice , Models, Molecular , Protein BindingABSTRACT
The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous beta2-microglobulin (beta2m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and beta2m, we engineered variants of human beta2m (hbeta2m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/beta2m interface and displace an ordered water molecule. Compared with hbeta2m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of hbeta2m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50-80-fold. Therefore, the inclusion of hbeta2m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.
Subject(s)
Antigen Presentation , HLA-A Antigens/metabolism , Peptides/metabolism , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolism , Drug Design , Genetic Variation , Humans , Mutagenesis, Site-Directed , Protein Binding , Protein Engineering , T-Lymphocytes, Cytotoxic , Vaccines/immunologyABSTRACT
The monoclonal antibody W6/32 is one of the most commonly used pan-HLA-ABC antibodies in studying human MHC I structure and function. We have discovered that the reactivity of W6/32 is absolutely sensitive to the amino terminus of human beta2-microglobulin (hbeta2m). Bacterially expressed recombinant forms of hbeta2m that have been extensively used in structural and biochemical studies of MHC I molecules often have an additional methionine at their amino terminus. Cell surface MHC I molecules reconstituted with allele-specific peptides and recombinant hbeta2m are reactive with various HLA-specific mAbs, but not W6/32. In contrast, cell surface HLA molecules reconstituted with peptide and native hbeta2m, which has no amino terminal methionine, are recognized by W6/32 as well as other HLA-specific mAbs. Thus, the specificity of W6/32 includes the amino terminus of hbeta2m.
Subject(s)
Epitope Mapping , Epitopes, B-Lymphocyte/immunology , HLA-A2 Antigen/immunology , beta 2-Microglobulin/immunology , Animals , Antibodies, Monoclonal/immunology , HLA-A2 Antigen/genetics , Humans , MiceABSTRACT
The mouse NK inhibitory Ly-49A receptor specifically interacts with a peptide-induced conformational determinant on its MHC class I ligand, H-2Dd. In addition, it binds the polysaccharide fucoidan, consistent with its C-type lectin homology and the hypothesis that Ly-49A interacts with carbohydrates on Dd. Herein, however, we demonstrate that Ly-49A recognizes Dd mutants lacking N-glycosylation. Fucoidan competes for binding with anti-Ly-49A antibodies that inhibit Ly-49A-Dd interaction, and blocks apparent Ly-49A binding to unglycosylated Dd. We confirm that Ly-49A recognizes the alpha1 and amino-terminal alpha2 domains of Dd by analysis of recombinant H-2Kd-H-2Dd molecules. These studies indicate that Ly-49A recognizes carbohydrate-independent epitope(s) on Dd and suggest that Ly-49A has two distinct ligands, carbohydrate and MHC class I.
Subject(s)
Antigens, Ly , Carrier Proteins/immunology , Glycoproteins/immunology , H-2 Antigens/immunology , Killer Cells, Natural/immunology , Lectins/immunology , Membrane Proteins/immunology , Receptors, Immunologic/immunology , Animals , Binding, Competitive , Carrier Proteins/metabolism , Cytotoxicity Tests, Immunologic , Epitopes , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , H-2 Antigens/chemistry , H-2 Antigens/genetics , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Killer Cells, Lymphokine-Activated/immunology , Lectins/metabolism , Lectins, C-Type , Ligands , Membrane Proteins/metabolism , Mice , Models, Molecular , Mutation , Polysaccharides/metabolism , Protein Binding , Receptors, Immunologic/metabolism , Receptors, NK Cell Lectin-LikeABSTRACT
Human beta 2m (h beta 2m) binds to murine MHC I molecules with higher affinity than does murine beta2m and therefore can be used as a model system to define and dissect the interactions between beta2m and MHC I heavy chains that promote the stability of the complex. In the present study we compare three-dimensional crystal structures of human and murine MHC I molecules and use functional studies of chimeric human:murine beta 2m variants to define a region of beta2m that is involved in the higher affinity of h beta 2m for murine MHC I heavy chains. Further examination of the three-dimensional structure in this region revealed conformational differences between human and murine beta2m that affect the ability of an aspartic acid residue at position 53 (D53) conserved in both beta 2ms to form an ionic bond with arginine residues at positions 35 and 48 of the heavy chain. Mutation of residue D53 to either asparagine (D53N) or valine (D53V) largely abrogated the stabilizing effects of h beta 2m on murine MHC I expression in a predictable manner. Based on this observation a variant of h beta 2m was engineered to create an ionic bond between the heavy chain and beta 2m. This variant stabilizes cell surface H-2Dd heavy chains to a greater extent than wild-type h beta 2m. Studying these interactions in light of the growing database of MHC I crystal structures should allow the rational design of higher affinity h beta 2m variants for use in novel peptide-based vaccines capable of inducing cell-mediated immune responses to viruses and tumors.
Subject(s)
Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Protein Engineering , beta 2-Microglobulin/chemistry , beta 2-Microglobulin/metabolism , Animals , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/drug effects , Histocompatibility Antigens Class I/genetics , Humans , L Cells , Mice , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Point Mutation , Protein Biosynthesis , Protein Conformation , Protein Engineering/methods , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , beta 2-Microglobulin/genetics , beta 2-Microglobulin/pharmacologyABSTRACT
Fetal calf serum is a well known source of bovine beta2-microglobulin (beta2m) which can exchange with endogenous beta2m from, as well as promote peptide binding to, class I major histocompatibility (MHC I) molecules on cells cultured in vitro. Recombinant bovine beta2m was expressed and purified for direct functional comparison to human and murine beta2m for interactions with murine MHC I molecules H-2Kb, Db, Kd, Ld, and Dd. Bovine and human beta2m were equivalent in stabilizing MHC I heavy chains and facilitating peptide loading, suggesting similar affinities for murine MHC I heavy chains. The activity of murine beta2m was significantly weaker, consistent with previous work that demonstrated the lower affinity of murine human beta2m for murine heavy chains compared to human beta2m. Analysis of bovine beta2m in fetal calf serum revealed ten-fold higher concentrations than in adult bovine serum, levels shown to significantly affect MHC I stability and peptide loading. The ramifications for the study of MHC I molecules from cells in culture and the evolutionary implications of the higher affinity interactions of human and bovine beta2m are discussed.
Subject(s)
beta 2-Microglobulin/physiology , Amino Acid Sequence , Animals , Cattle , Cell Line , Fetal Blood/chemistry , Histocompatibility Antigens Class I/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Mice , Molecular Sequence Data , Peptides/drug effects , Peptides/metabolism , Protein Binding/drug effects , Recombinant Proteins/pharmacology , beta 2-Microglobulin/analysis , beta 2-Microglobulin/pharmacologyABSTRACT
CD69 is expressed on the surface of all hematopoietically derived leukocytes and is suggested to function as a multipurpose cell-surface trigger molecule important in the development and activation of many different cell types. Human CD69 contains only a single consensus sequence for N-linked oligosaccharide addition within its extracellular domain (Asn-Val-Thr), yet exists as two distinct glycoforms that are assembled together into disulfide-linked homodimers and heterodimers. The molecular basis for human CD69 heterogeneity has remained elusive. In the current report we show that human CD69 glycoforms are generated before the egress of CD69 proteins from the endoplasmic reticulum to the Golgi and are synthesized under conditions where Golgi processing is inhibited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation site in the Golgi complex. Importantly, these data demonstrate that contrary to current belief, not one but two sites for N-glycan addition exist within the human CD69 extracellular domain and identify the second, "cryptic" CD69 N-glycan attachment site as the atypical Cys-containing glycosylation motif, Asn-Ala-Cys. The results in this study provide a molecular basis for human CD69 heterogeneity and show that multiple dimeric forms of human CD69 result from the variable addition of N-glycans to atypical and typical glycosylation motifs within the CD69 extracellular domain.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Asparagine/metabolism , Consensus Sequence , Glycoproteins/metabolism , Protein Processing, Post-Translational , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/chemistry , Antigens, Differentiation, T-Lymphocyte/genetics , Cell-Free System , Consensus Sequence/genetics , DNA Mutational Analysis , Dimerization , Disulfides/metabolism , Endoplasmic Reticulum/metabolism , Glycoproteins/chemistry , Glycoproteins/genetics , Glycosylation , Golgi Apparatus/metabolism , Humans , Lectins, C-Type , Models, Molecular , Mutagenesis, Site-Directed , Polysaccharides/metabolism , Protein Biosynthesis , T-Lymphocytes/metabolism , Transcription, GeneticABSTRACT
To identify residues that control the interactions between MHC-heavy chains and (beta 2m) sequence comparisons were made between murine class I MHC molecules with high (H-2Dd, H-2Kb) and low (H-2Ld, H-2Db) affinities for beta 2m. A single residue at position 9 was evaluated for its contribution to the stability of the complex. Mutagenesis of the glutamic acid at position 9 of H-2Ld to valine, as is found in H-2Dd and H-2Kb, resulted in both qualitative and quantitative effects on inter-chain interactions, intracellular transport, peptide binding, and peptide presentation. In in vitro translation and assembly studies, the E9V mutation resulted in a more stable association of beta 2m with the heavy chain after immunoprecipitation with the alpha 2 domain-specific Ab 30-5-7 in the presence of an H-2Ld-restricted peptide. E9V variant expressed in transfected L cells had similar surface expression compared with H-2Ld despite exhibiting a slower rate of maturation. However, cells expressing E9V were unable to present peptide Ag to a specific T cell hybridoma. H-2LdE9V in E-3 cells, which are defective in TAP-dependent peptide transport, was expressed at higher levels than H-2Ld and was stabilized more efficiently by the addition of exogenous human beta 2m. Thus, amino acid position 9 not only plays an important role in the interaction of the MHC-1 molecule with the beta 2m, it also qualitatively and quantitatively influences peptide binding and Ag presentation.
Subject(s)
Histocompatibility Antigens Class I/genetics , Viral Proteins/immunology , beta 2-Microglobulin/immunology , Amino Acid Sequence , Animals , Antigen Presentation/genetics , Base Sequence , Cell Line , DNA, Complementary , Gene Transfer Techniques , Histocompatibility Antigens Class I/immunology , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymorphism, Genetic , Receptor Aggregation , Sequence AlignmentABSTRACT
As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2Dd class I MHC molecule (SC beta Ddm) which has beta 2-microglobulin (beta 2m) covalently linked to the amino terminus of a full-length H-2Dd heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2Dd-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SC beta Ddm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27 degrees or 37 degrees C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SC beta Ddm can be stably expressed in beta 2m-negative cells, showing that the single-chain molecule uses its own beta 2m domain to achieve correct folding and surface expression. Furthermore, the beta 2m domain of SC beta Ddm, unlike transfected free beta 2m, does not rescue surface expression of endogenous class I MHC in the beta 2m-negative cells. This strict cis activity of the beta 2m domain of SC beta Ddm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.
Subject(s)
Histocompatibility Antigens Class I/biosynthesis , Recombinant Proteins/biosynthesis , beta 2-Microglobulin/biosynthesis , Animals , Antigen Presentation , Base Sequence , Cells, Cultured , Histocompatibility Antigens Class I/genetics , Humans , Molecular Sequence Data , Precipitin Tests , Temperature , Transfection , beta 2-Microglobulin/geneticsABSTRACT
Although reported examples of endogenous antigen (Ag) presentation by major histocompatibility complex (MHC) class II molecules have increased, the mechanisms governing this process remain poorly defined. In this communication, we describe an experimental system designed to examine the mechanisms governing class II presentation of internal Ag. Our target peptide is processed from a transmembrane protein constitutively expressed by a variety of nucleated cells (MHC class I, H-2Ld), is naturally displayed by MHC class II molecules in vivo, and is recognized by a class II-restricted, CD4+ T cell hybridoma. Our results indicate that presentation of the Ld target Ag is independent of its plasma membrane expression, may not involve endosomal proteolysis, and thus may be distinct from the classically defined class II presentation pathway. In addition, the observations that Ld presentation does not require a functional TAP-1 complex, is not blocked by invariant chain, and cannot utilize cytoplasmic forms of H-2Ld, suggest that a classical class I pathway is not involved in this presentation event. Finally, our data suggest that different cofactors participate in MHC class II presentation of exogenous and endogenous Ag, and that disparate Ag presenting cells, such as B, T, and pancreatic islet cells, may differentially express these two class II pathways of Ag presentation.
Subject(s)
Antigen-Presenting Cells/physiology , Antigens, Differentiation, B-Lymphocyte , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Animals , Clone Cells , H-2 Antigens/analysis , Histocompatibility Antigens Class II/physiology , Hybridomas , Islets of Langerhans/immunology , Mice , Mice, Inbred BALB C , T-Lymphocytes/immunologyABSTRACT
Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines.
Subject(s)
H-2 Antigens/immunology , beta 2-Microglobulin/immunology , Animals , Antibodies, Monoclonal , Base Sequence , Cell Division , Cell Line , DNA/genetics , DNA/isolation & purification , Enzyme-Linked Immunosorbent Assay , Genes, MHC Class I , H-2 Antigens/genetics , Liver/immunology , Macromolecular Substances , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oligodeoxyribonucleotides , Oligonucleotides, Antisense , Polymerase Chain Reaction , Protein Folding , Recombinant Proteins/immunology , Restriction Mapping , Transfection , beta 2-Microglobulin/geneticsABSTRACT
We have examined the post-translational processing, intrachain disulfide bond formation, folding, and assembly of MHC class I H chains with beta 2-microglobulin after coupled in vitro translation of homogeneous mRNA and transport of nascent chains into canine microsomal vesicles. The formation of native alpha 3 domain conformation was dependent on conditions that optimized intrachain disulfide bond formation, and efficient folding of the alpha 1 alpha 2 domain required exposure to antigenic peptide. beta 2-microglobulin and peptide acted synergistically in forming native alpha 1 alpha 2 domain structure, and a small proportion of molecules with native alpha 1 alpha 2, but non-native alpha 3 structure were detected, indicating that alpha 3 domain folding is not an absolute prerequisite for the formation of native alpha 1 alpha 2 domain structure.
Subject(s)
Gene Products, gag/pharmacology , H-2 Antigens/biosynthesis , Immediate-Early Proteins , Protein Processing, Post-Translational , Viral Proteins/pharmacology , beta 2-Microglobulin/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Drug Synergism , Electrophoresis, Polyacrylamide Gel , Glutathione/pharmacology , Histocompatibility Antigen H-2D , In Vitro Techniques , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microsomes/physiology , Molecular Conformation , Molecular Sequence Data , Protein Biosynthesis/drug effects , Transcription, Genetic , gag Gene Products, Human Immunodeficiency VirusABSTRACT
The molecular basis of target cell recognition by CD3- natural killer (NK) cells is poorly understood, despite the ability of NK cells to lyse specific tumour cells. In general, target cell major histocompatibility complex (MHC) class I antigen expression correlates with resistance to NK cell-mediated lysis, possibly because NK cell-surface molecules engage MHC class I antigens and consequently deliver inhibitory signals. Natural killer cell allospecificity involves the MHC class I peptide-binding cleft, and further understanding of this allospecificity should provide insight into the molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecule is expressed by 20% of CD3- NK cells in C57BL/6 mice (H-2b). Here we show that C57BL/6-derived, interleukin-2-activated NK cells expressing Ly-49 do not lyse target cells displaying H-2d or H-2k despite efficient spontaneous lysis by Ly-49- effector cells. This preferential resistance correlates with expression of target cell MHC class I antigens. Transfection and expression of H-2Dd, but not H-2Kd or H-2Ld, renders a susceptible target (H-2b) resistant to Ly-49+ effector cells. The transfected resistance is abrogated by monoclonal antibodies directed against Ly-49 or the alpha 1/alpha 2 domains of H-2Dd, suggesting that Ly-49 specifically interacts with the peptide-binding domains of the MHC class I alloantigen, H-2Dd. Inasmuch as Ly-49+ effector cells cannot be stimulated to lyse H-2Dd targets, our results indicate that NK cells may possess inhibitory receptors that specifically recognize MHC class I antigens.
Subject(s)
Cytotoxicity, Immunologic , H-2 Antigens/immunology , Immunity, Cellular , Killer Cells, Lymphokine-Activated/immunology , Killer Cells, Natural/immunology , Animals , Antigens, Ly/analysis , Cells, Cultured , H-2 Antigens/genetics , In Vitro Techniques , Lymphocyte Activation , Lymphocyte Subsets/immunology , Mice , TransfectionABSTRACT
We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.
Subject(s)
H-2 Antigens/metabolism , Peptides/chemistry , beta 2-Microglobulin/physiology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Biological Transport , Cell Compartmentation , Cell Membrane/metabolism , Cells, Cultured , H-2 Antigens/ultrastructure , In Vitro Techniques , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Mice , Molecular Sequence Data , Peptides/metabolism , Protein ConformationABSTRACT
The influence of CD8 on the fine specificity of MHC class I-restricted T cell allorecognition was evaluated by comparing the reactivity of CD8- and CD8-transfected forms of an allospecific, H-2Kb-restricted T hybridoma. The CD8- T hybridoma responded to cells expressing H-2Kb, H-2Kbm6, and the individual H-2Kb----bm10 back mutations 165V----M, 173K----E, and 174N----L. Under the same conditions the CD8- T hybridoma responded poorly or not at all to cells expressing H-2Kbm10, H-2Kbm8, the individual H-2Kb----bm10 back mutants 163T----A and 167W----S, and the individual H-2Kb----bm8 back mutations 22Y----F and 24E----S. In contrast, T hybridoma cells expressing high levels of CD8 reacted strongly with antigen presenting cells (APC) expressing H-2Kb and H-2Kbm6 molecules, as well as APC expressing H-2Kbm10 (weakly), H-2Kbm8, and all five individual H-2Kb----bm10 and the two H-2Kb----bm8 back mutants 22Y----F and 24E----S. The mutations which distinguish the T cell recognition of both H-2Kbm10 and H-2Kbm8 from H-2Kb are predicted to control the interaction of these class I molecules with antigenic peptides in the binding site, implying an important role for peptide antigen in T cell allorecognition. Nonetheless, CD8 expression by the H-2Kb-restricted T cells conferred novel or enhanced alloreactivity with cells expressing H-2Kbm10, H-2Kbm8, and each of the individual H-2Kb----bm10 and H-2Kb----bm8 back mutants. These findings reflect an important role for CD8 in influencing the fine specificity of MHC class I recognition by T cells and may indicate a limited structural role for peptide antigen in defining the ligand recognized by these alloreactive T cells.
Subject(s)
CD8 Antigens , H-2 Antigens , T-Lymphocytes/immunology , Animals , Base Sequence , DNA/genetics , H-2 Antigens/genetics , Hybridomas/immunology , Lymphocyte Activation , Mice , Molecular Sequence Data , Mutation , TransfectionSubject(s)
Antigens, Ly , H-2 Antigens , Killer Cells, Natural/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/genetics , Antigens, Ly/genetics , Antilymphocyte Serum/pharmacology , Cell Line , Cytotoxicity, Immunologic/genetics , H-2 Antigens/genetics , Histocompatibility Antigen H-2D , Mice , Multigene Family , TransfectionABSTRACT
It has long been recognized that the absence of expression of products of the major histocompatibility complex (MHC) during early development might allow the fetus to escape recognition by maternal lymphocytes. In addition to the MHC class I heavy chain and beta 2-microglobulin, antigenic peptide is an essential structural component of the class I molecule. Indeed, there is evidence that MHC-linked genes encoding peptide transporter molecules and possibly components of a proteolytic complex are necessary for MHC class I assembly and stability at the cell surface. Here we demonstrate that embryonic cells in general show a defect in MHC class I assembly. Surface expression was rescued in the presence of an appropriate antigenic peptide, or by treatment with interferon. Consistent with this, HAM1 messenger RNA was not constitutively expressed, but was inducible by interferon, and during differentiation in vitro. Thus, tolerance of the fetal allograft may in part be controlled at the level of peptide-dependent MHC class I assembly.
Subject(s)
Embryo, Mammalian/immunology , Gene Expression Regulation/drug effects , H-2 Antigens/biosynthesis , Interferons/pharmacology , Peptides/pharmacology , Animals , Blotting, Northern , Cell Line , Cell Membrane/immunology , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Embryo, Mammalian/metabolism , Gene Products, gag/pharmacology , Mice , RNA/biosynthesis , Transfection , beta 2-Microglobulin/pharmacology , gag Gene Products, Human Immunodeficiency VirusABSTRACT
To study levels of neutrophil chemotactic activity in human gastric secretions, these secretions were collected via the endoscope during elective esophagogastroduodenoscopy. Fresh samples were then prepared for the neutrophil chemotactic assay with the use of human peripheral neutrophils in modified Boyden chambers. Mean maximum chemotactic activity was 45.8% +/- 11.2% in patients with gastric inflammation compared with only 10.1% +/- 4.2% in patients with normal results of endoscopic examination. The chemotactic factors responsible for this chemotactic activity may play a role in the recruitment of neutrophils to areas of gastric mucosal injury.