Antigens and MHC (SEM2-IMSE)

ANTIGENS AND IMMUNOGENS IMMUNOGEN – any agent capable of inducing an immune response ANTIGEN – any agent capable of binding to components of immune response such as lymphocyte and antibodies  Antigen Properties § Immunogenic substance are always antigenic whereas antigens are not necessarily im...

ANTIGENS AND IMMUNOGENS IMMUNOGEN – any agent capable of inducing an immune response ANTIGEN – any agent capable of binding to components of immune response such as lymphocyte and antibodies  Antigen Properties § Immunogenic substance are always antigenic whereas antigens are not necessarily immunogenic. § Immunogens and Antigens are found on: § RBC Surface; WBC Surface; Platelet § They may be introduced into another by: Blood Transfusion Tissue Grafting & Organ Transplant Trans-placental Leakage Ingestion and Injection  HAPTEN AND CARRIER HAPTENS - are called incomplete / partial antigens. Gives the antigen its antibody provoking abilities. CARRIERS - proteins which give the hapten the required sized.  MAIN PARTS OF ANTIGEN CARRIER HAPTEN Protein in nature Nonprotein High Molecular Weight Low Molecular Weight Immunogenic Antigenic Can cause production of Cannot invoke antibody antibody production but can react with its homologous antibody  ANTIGENIC DETERMINANTS §Portion that binds specifically with the binding site of an antibody or to a lymphocyte. §Consist of 4-6 amino acids or 5-7 monosaccharides Aromatic Amino Acids Polysaccharide Hydrophilic Molecules  CLASSIFICATION OF IMMUNOGENS 1. ACCORDING TO DEPENDENCY TO T-CELLS § Thymic Dependent Require help of T-Cells for formation of Antibody. Most Immunogens are thymic independent. § Thymic Independent Antibody production without interacting T Cells. IgM Class.  CLASSIFICATION OF IMMUNOGENS 2. ACCORDING TO THE EPITOPES § Unideterminant, Univalent Antigen 1 Kind and Number of Epitope § Unideterminant, Multivalent Antigen 1 Kind of Epitope ; High number of Epitope § Multideterminant, Univalent Antigen § Multideterminant, Multivalent  FACTORS AFFECTING IMMUNOGENECITY Molecular size Chemical Genetic complexity Immunogenecity composition Foreignness Route, Dosage and timing  FACTORS AFFECTING IMMUNOGENECITY A. FOREIGNNESS The higher the difference = The higher the immune response Antigen must be FOREIGN, ALIEN or NON-SELF to the host : 1. Autologous Antigen: Same Individual ; Autograft 2. Syngeneic Antigens: Between genetically identical individual ; Isograft 3. Allogeneic / Homologous Antigen: Between different individuals with same species ; Homograft / Allograft  FACTORS AFFECTING IMMUNOGENECITY Antigen must be FOREIGN, ALIEN or NON-SELF to the host : 4. Xenogeneic / Heterologous: Between different individuals of different species ; Xenograft 5. Heterogenetic / Heterophilic Antigens: Occur in unrelated animal and plant species _______________  CROSS REACTIVITY AND TOXOID CROSS REACTIVITY - denotes a situation in which two or more compounds that may have various degree of dissimilarity, share antigenic determinants. TOXOID – form of toxin may have one more antigenic determinants in common with the native toxin  TERMS USED TO DESCRIBE ANTIGEN v Autologous (Antigen) – autoantigen; refers to one’s own antigen, which under appropriate circumstances, would stimulate the production of autoantibody v Heterologous (Antigen) – merely an antigen different from that used in the immunization and it may or may not react with the antibody formed, depending upon the chemical similarity to the immunizing antigen v Homologous (Antigen)- refers to the antigen used in the production of antibody v Heterophile (antigen)/ Heterogenetic antigen – antigens that exist in unrelated plants or animals but are either identical or so closely related that antibodies to one will cross-react with antibodies to the other.  TRANSPLANTATION IMMUNOLOGY Most Immunogenic: Bone Marrow Skin Islets Of Langerhans Heart Kidney Liver Bone Least Immunogenic: Cornea  B. Chemical Complexity Pertains to chemical structures of antigen: 1. Protein § Majority of immunogens; Strongest immunogens § High number of epitopes = High number of Antibody 2. Glycoproteins § Immunogens; Best illustrated by RBC blood group Antigens § HLA Antigens B. Chemical Complexity Pertains to chemical structures of antigen: 3. Polysaccharide § Most are HAPTENS § Do not possess sufficient chemical diversity Few polysaccharides which are immunogenic : 1. Pure Polysaccharide substances 2. Lipopolysaccharide Substances B. Chemical Complexity Pertains to chemical structures of antigen: 4. Nucleic Acids Nucleic Acid + Protein = Nucleoprotein = Immunogenic 5. Lipids Lipids + Protein or Polysaccharide = Immunogenic C. Molecular Size Pertains to chemical structures of antigen: 1. Sizes: Non Immunogenic = 10,000 Daltons Excellent Immunogens = >1,000,000 Daltons 2. Soluble antigens is not immunogenic at all times D. Genetic Composition Major Histocompatibility Complex exerts genetic influence of an individual’s ability to respond to an immunogen. E. Route Dosage and Timing Routes: Intravenous (into the vein) Intraperitoneal (into the peritoneal Activity) Intradermal (skin or dermis) Subcutaneous (beneath the skin) Intramuscular (into the muscle) F. Rate of Absorption and Elimination of Antigen  FACTORS AFFECTING IMMUNOGENECITY F. Rate of Absorption and Elimination of Antigen  ADJUVANTS §Substance which when mixed with an immunogen, enhances the immune response against immunogen. §Types of Adjuvants: CFA ( Complete Freund’s Adjuvants ) Toxins and Polysaccharides Alum precipitate***  MITOGENS ü Can be considered mimics of antigens üB Cell Mitogens: o Lipopolysaccharide because the proliferative response o Protein A they initiate in lymphocytes causes o Anti-immunoglobulin üT Cell Mitogens these cells to engage in the same o Concanavalin A (ConA) cellular and chemical activities that o Phytohemagglutinins üCombined B and T cell Mitogens: follow their exposure to antigens o Pokeweed Mitogen o Lectins (substances that bind to specific polysaccharide residues on the cells they agglutinate) Please Study the following in your notes: Kinds of Antigen (based on their reaction with an antibody) Terms used to describe Antigen Mitogens Additional notes about adjuvants MAJOR HISTOCOMPATIBILITY COMPLEX Major Histocompatibility Complex (MHC) § Originally referred to as Human Leukocyte Antigens (HLA). § MHC molecule(determine whether transplanted tissue is histo-compatible and thus accepted or recognized as foreign and rejected). § MHC molecules are actually found on all nucleated cells in the body. Major Histocompatibility Complex (MHC) § Main Function: To bring antigen to the cell surface for recognition by T cells. § Clinically Significance: Transfusion Reactions Graft Rejection Autoimmune Diseases § Genes controlling expression of these molecules are actually a system of genes known as the major histocompatibility complex (MHC). The major histocompatibility complex. Location of the class I, II, and III genes on chromosome 6. Major Histocompatibility Complex (MHC) CLASS I CLASS II CLASS III HLA-A,B,C HLA-DP, DQ, DR C2, C4A, Gene Loci C4B Chain α chain, α chain, Structure β2 chain β chain All Cell Not Nucleated APC & B Cell Distribution expressed Cell Presentation Presentation of code for C2, Function of Antigen to Antigen to CD4+ C4, Factor B, CD8+ T cell T cell TNF Major Histocompatibility Complex (MHC) CLASS I CLASS II CLASS III HLA-A,B,C HLA-DP, DQ, DR C2, C4A, C4B Gene Loci Factor B, TNF Chain α chain, α chain, Structure β2 chain β chain All Cell Not Nucleated APC & B Cell Distribution expressed Cell Presentation Presentation of code for C2,  Class I MHC / Class I Antigens § Molecules are comprised of two polypeptides: POLYMORPHIC HEAVY CHAIN (Alpha Chain) (Encoded within the MHC locus on Chromosome 6) NON-POLYMORPHIC LIGHT CHAIN (B2-Microglobulin) (Encoded within the MHC locus on Chromosome 15) § Each of the three forms of class I molecules, HLA-A, HLA-B, HLA-C are composed on virtually all nucleated cells. STRUCTURE OF CLASS I MHC MOLECULE  CLASS I PEPTIDE INTERACTION  Class II MHC / Class II Antigens § Molecules are comprised of two polypeptide chains designated alpha and beta. § Each of these chains is polymorphic and the genes that encode then are located within the MHC locus. § For the most part, class II MHC molecules HLA- DP, HLA-DQ, HLA-DR are normally expressed on antigen presenting cells. STRUCTURE OF CLASS II MHC MOLECULE  CLASS II PEPTIDE INTERACTION *NON-CLASSICAL/MINOR MHC molecules  Important Aspects of MHC § In general, peptide fragments of proteins in the cytosol associate with class I MHC, those of vesicular proteins associate with class II MHC. § Each is recognize by functionally distinct T cell populations. § The maximum number of class I MHC gene products expressed in an individual is six. That for class II MHC product can exceed six but is also limited.  Important Aspects of MHC § Mature T cells respond to foreign antigens, but not self-protein. The repertoire of antigen recognition is based on selection processes involving MHC molecules that occur mainly in the thymus. This is another level of control of immune responses. § Only a single binding site exists on a class I or class II MHC molecules; all peptides that are capable of binding must bind to the same site. Notes: § Haplotype – total set of MHC alleles inherited from one parent § Genotype – sum of haplotype § Each person inherits 2 copies of chromosome 6 and thus can expressed up to 6 different class I and 6 different class II proteins. Notes: §Histocompatibility Genes. Genes that determines the outcome of the graft. §Histocompatibility Antigens. Protein encoded by those genes.  Importance of HLA typing §Tissue/Organ transplant §Paternity studies §Studies of racial ancestry and migration §For diagnostic and genetic counselling  Association of HLA Alleles and Disease Ankylosing spondylitis HLA B27 Celiac disease HLA DQ2 HLA DQ8 Rheumatoid arthritis HLA DR4 Type 1 diabetes HLA DQ8 HLADQ2 Detection of HLA Class I and II Microlymphocytotoxicity Mixed Cellular Reaction Determines if antigen is Quantify the degree of Class present on the lymphocytes, II between donor and addition of complement will recipient. cause them to take the dye Clinical Histocompatibility Testing HLA Phenotyping MICROLYMPHOCYTOTOXICTY Lymphocytes (of patient or donor) obtained via Ficoll Hypaque solution at 19-22deg C is mixed with known HLA antibodies, once corresponding antigens are present it will undergo complement mediated lysis.  Clinical Histocompatibility Testing HLA Phenotyping T and B lymphocytes are used for HLA class I typing, whereas purified B lymphocytes are used for HLA class II typing because class II antigens are not found on most T cells. After incubation with the antisera, complement is added. Binding of antibody occurs only if the lymphocytes express the HLA antigen targeted by the antisera. The proportion of dead cells is estimated by microscopic examination and scored according to the following scale, established by the American Society for Histocompatibility and Genetics (ASHI): 1 = 0% to 10% cell death; negative 2 = 11% to 20% cell death; doubtful negative 4 = 21% to 50% cell death; weak positive 6 = 51% to 80% cell death; positive 8 = 81% to 100% cell death; strong positive 0 = unreadable Mixed lymphocyte reaction (MLR) INTERPRETATION OF MLC REACTION: THE GREATERTHE AMOUNT OF REACTIONIN THE RECIPIENT.RESPONDER CELLS, THE MORE FOREIGN ARE THE MHC CLASS OF THE DONOR CELLS Mixed Lymphocyte Reaction Lymphocytes from patient are mixed with lymphocytes from donor. If the cells of one individual recognized other’s MHC molecules as foreign, they proliferate and divide. The proliferative response is usually measured with 3H labeled thymidine uptake assay. Mitomycin C is added to prevent the proliferation of donor, so patient will only proliferate. Isolation and Identification of Lymphocytes Isolation Identification Ficoll-hypaque (Density Gradient a. Flow Cytometry Centrifugation) b. Fluorescent-labeled monoclonal antibody 5 layers (top to bottom) 1. Plasma 2. Mononuclear cells 3. Ficoll-Hypaque 4. Granulocytes 5. RBC Ficoll Hypaque Illustration Types of Detection of MHC Types of Detection of MHC Serological “Microlymphocytotoxicity” Cellular “Mixed Cellular Reaction” Molecular