Super Antigens

SUPER ANTIGENS SUPER ANTIGENS Definition:    Heterogeneous group of proteins that uses a common extremely efficient mechanism of T-lymphocyte stimulation. It bypasses the antigen processing mechanism. Bind to variable β-domain of T-cell receptor along with MHC II molecule.  Ability to stimulate a large fraction of T-cell from its very small amount (10-9mol/lit) that causes very adverse consequences. Shock Immuno-suppression Auto-immunity Allergy  HISTORY  In 1960 first super antigen isolated by Bergdoll & co-workers as a secreted toxin of Staphylococcus aureus.  In 1981 Schlievert & Bergdoll isolated pyrogenic exotoxin C & staphylococcal enterotoxin F respectively. Finally In 1989 -90 fundamentally a new class of antigen came into existence and Kappler & Marrack termed them SUPERANTIGEN & their properties were described. STRUCTURE  Medium sized 22-29 kDa globular compact protein.  Conserved Two domain architecture (N&C terminal domains and alpha helix in the center of the molecule). N terminal Domain- hydrophobic residues. Determines the lethality of the toxin. C terminal Domain- 4 stranded sheet capped by central helix. Determines the super antigenicity of the toxin. Zinc Ion is required for binding super antigen to MHC II molecule CLASSIFICATION 1. EXOGENOUS 2. ENDOGENOUS 1. T-Cell 2. B-Cell 1. EXOGENOUS: soluble proteins secreted by bacteria & a variety of exotoxins. Eg: staphylococcal enterotoxin, streptococcal pyrogenic exotoxins. 2. ENDOGENOUS: cell membrane proteins encoded by certain viruses that infect mammalian cells. Eg: MMTV, EBV associated super antigen T-Cell Super Antigen: Bind directly to variable Beta-Domain of T-cell receptor & MHC II receptor. B-cell Super antigen: Bind to variable region of heavy chain or light chain of B-cell receptor. Note: Both T & B cells binds outside the conventional antigen binding site. MECHANISM OF ACTION i. ii. iii. Interaction with T-cell receptor Interaction with B-cell receptor Interaction with MHC class II molecule T-CELL RECEPTOR Heterodimer of glycoprotein's chains. Two peptide chains (αβ/ϒδ) Non Covalently associated with CD 3 molecule α-peptide chain has 3 regions: a) V-Variable b) J-junctional c) C-constant Beta peptide chain has one additional region D(Diversity) TH  INTERACTION Note: Biological strength of the super antigen is determined by its affinity for T-cell receptor. Highest affinity elicit strongest response B-CELL RECEPTOR Membrane bound immunoglobulin on the surface Two regions: 1. Constant region 2. Variable region CONSTANT REGION: Remains inserted in the membrane of BCell VARIABLE REGION: act as a Antigen binding site. 3 segments in variable region: V1, V2, V3 B-cell super antigen interacts with variable region of heavy or light chain outside the conventional site. Most commonly to VH3 gene family INTERACTION WITH MHC CLASS II MOLECULE  Super antigens are not MHC restricted.  They show preference for HLA-DQ molecule.  They use different binding sites on MHC II molecule.  They bind as unprocessed protein outside Ag binding groove of class II molecule.  They may attach to polymorphic MHC II beta chain in an interaction mediated by zinc ion.  Without any prior antigen processing super antigen bind to MHC class II molecule on antigen presenting cell This complex is recognized by T-cell receptor or B-cell receptor through variable beta domain region Stimulation of T-cells Massive release of pro inflammatory cytokines Eg: IL-2,TNF alpha, TNF beta, IFN gamma CLASSICAL RESPONSE SUPER ANTIGEN RESPONSE 1 Requires processing by APC Does not require 2 Recognition of antigenic peptide in MHC groove requires Vα, Jα, Vβ, Dβ, Jβ Recognition of Vβ only 3 MHC II restricted Independent of MHC restriction 4 Activation of small fraction Massive T-cell activation (20of T-cell (<0.001) 30% of total T-cells) 5 Highly regulated response Adverse consequences FACTORS AFFECTING SUPERANTIGEN INDUCED RESPONSE  MHC II binding  Concomitant infection  Dose  Type of super antigen  Host immune status CANDIDATES THAT PRODUCE SUPER ANTIGENS PATHOGENS TOXINS STAPHYLOCOCCUS AUREUS • • • STREPTOCOCCUS PYOGENES • • • STAPHYLOCOCCAL ENTEROTOXIN(SE) A-E, G-P TOXIC SHOCK SYNDROME TOXIN(TSST-1) EXFOLIATIVE TOXIN STREPTOCOCCAL PYROGENIC EXOTOXIN(SPE)A, B, C, D, F, G, H, J STREPTOCOCCAL MITOGENIC EXOTOXIN Z (SMEZ) MITOGENIC FACTOR (MF) MYCOPLASMA ARTHRITIDIS • MYCOPLASMA ARTHRITIDIS SUPER ANTIGEN YERSINIA PSEUDOTUBERCULOSIS • YPM (YERSINIA PSEUDOTUBERCULOSIS MITOGEN) MOUSE MAMMARY TUMOUR VIRUS • MMTV SUPER ANTIGEN HUMAN ENDOGENOUS RETROVIRUSES • HIV-HIV-gp120 • EBV-HERVK18env MALASSEZIA FURFUR • FUNGAL SUPER ANTIGEN SUPER ANTIGEN PRODUCED BY STAPHYLOCOCCUS AUREUS 1. STAPHYLOCOCCAL ENTEROTOXIN (SE): Causes emesis & diarrhoea in humans Five Major types: A-E Most common SE-A 2. TSST-1(Toxic Shock Syndrome Toxin-1): Initially designated as SE-F. now it is called TSST1 because of its association with toxic shock syndrome. Members of SE family divided into 5 subgroups according to their sequencing. GROUPING OF MEMBERS OF THE STAPYLOCOCCAL ENTEROTOXIN SUPERFAMILY GROUP I II III IV V MEMBERS PROPERTIES TSST-1 SEB like Central cysteine loop SEA like Central cysteine loop Zinc binding site SPEC like Zinc binding site SPEH like Zinc binding site SETS  STAPHYLOCOCAL ENTEROTOXIN –LIKE TOXIN  CLUSTER OF GENES: that are residents in a pathogenicity.  They have structural homology to SE & SPE in particular to TSST-1.  They form a highly polymorphic family with more than a dozen members.  SET-1  SET-1 bind to IgA inhibit its binding to myeloid cells. also bind complement C5 with nano molar affinity & inhibit complement mediated lysis. STREPTOCOCCUS PYOGENES 1.STREPTOCOCCAL PYOGENIC EXOTOXIN: studied along with SE as they are very much related to them. Both have features of extensive polymorphism, but they don’t have enterotoxin activity like SE. 2.STREPTOCOCCAL MITOGENIC EXOTOXIN (SMEZ): they are powerful stimulators of human Vβ4 & Vβ8 T-cells. Gene encoding SMEZ is chromosomal and almost invariably present in all group A streptococci. MYCOPLASMA ARTHRITIDIS SUPER ANTIGEN (MAS) arthritidis is pathogen for rodents.  Acute inflammation in rats followed by chronic joint disease.  MAS has T-cell stimulatory properties.  It stimulates both CD4 &CD8 T-lymphocytes.  Mycoplasma CAUSES:  Mild toxic shock syndrome  Pronounced T-cell suppression  It has been noted antibodies to MAS are presented in patients with rheumatoid arthritis. YERSINIA PSEUDOTUBERCULOSIS MITOGEN (YPM)  Produced by yersinia pseudotuberculosis.  This protein has all the characteristics of a super Ag in vitro & in-vivo.  Causes acute mesenteric inflammation & lymphadenopathy.  It causes elevation of Vβ3 bearing T-cells.  Also causes Reactive arthritis or Uveitis and Erythema nodosum. MOUSE MAMMARY TUMOR VIRUS (MMTV)  1ST VIRAL SUPER ANTIGEN  Causes mammary carcinoma in mice  It shows high degree of sequence conservation except carboxy terminus which shows extensive polymorphism  This  infect naive B-cell and immature dendritic cells. It stimulates T-cell Vβ receptors which causes proliferation of infected B-cells & increase their number exponentially. HUMAN ENDOGENOUS RETROVIRUS CAN ACT AS SUPER ANTIGENS 1. CMV 2. EBV : transactivates the human endogenous reterovirus HERV-K-18 env gene that encodes a super antigen. 3. RABIES VIRUS 4. HIV ASSOCATION WITH HIV   B-cell superantigen. In patient with AIDS there is imbalance in Vβ region of T-cells which provide indirect evidence for the involvement of superantigen in disease.  It causes deficiency of CD4 & CD8 T-cells.  It also increases viral load in HIV infected patients. FUNGAL SUPER ANTIGEN furfur thought to produce superantigen that causes atopic dermatitis.  Malassezia  It may also related to allergic fungal rhinosinusitis. BIOLOGICAL SIGNIFICANCE OF SUPERANTIGENS VIRULENCE FACTOR Superantigen provides evolutionary advantage to the respective pathogen It releases IL-4 & IL-10 Down regulates production of IFNgamma , MHC class II & co-stimulator molecule on the surface of APC Deletion / anergy of activated T-cells PATHOGENESIS 1. DIRECT EFFECTS 2. INDIRECT EFFECTS 1. DIRECT EFFECTS Nonspecific polyclonal activation of T-cells Massive cytokines release-IFNα, IL-2, IL12, IFNϒ Activate macrophages Over production of pro-inflammatory cytokines like IL-1,16 & TNFα shock Multiple organ failure rashes fever 2. INDIRECT EFFECTS  It causes gastro intestinal toxicity that induces vomiting.  It stimulates recruitment of neutrophils & release of TNFα and other cytokines, that causes inflammation in lungs & intestinal tissues.  It also has synergistic effect with endotoxin which can lead to sepsis.  It also stimulate the release of IFNα which lead to induction of autoimmunity. CONSEQUENCES OF POLYCLONAL T-CELL ACTIVATION Shock like symptom: 1.  Critical mediators are TNF α , IFNϒ which causes capillary leakage leading to hypotension.  It causes toxic shock syndrome leading to:High fever Diffuse erythematous rash Followed by hypotension & Multiple organ failure  Caused by TSST-1 & streptococcus pyrogenic exotoxin. 2.IMMUNOSUPPRESSION      Graded doses of super antigen causes anergy of T-cell & death by apoptosis in T-cells. Low concentration of Sag causes it transiently whereas higher doses cause it permanently. It causes loss of CD2, CD4 & CD8 molecules. Super Ag causes production of IFNϒ which inhibit Ig production and causes humoral immunosuppression. It is also suggested that activated CD8 + T-cells destroy APC or B lymphocytes that have toxin molecules bound to their class II antigens. prolonged exposure to toxin releases IL-4 & IL 10 down regulates production of IFNϒ, MHC class II, co-stimulatory molecule on the surface of APC produces memory cells that are un-responsive to antigen stimulation 3. AUTOIMMUNITY POLYCYCLONAL ACTIVATION OF T-CELL BY SUPER ANTIGEN LEAD TO ACTIVATION OF AUTO REACTIVE B-CELL INDUCTION OF AUTO ANTIBODY PRODUCTION Also molecular mimicry of certain peptides to human protein component may play a role. DISEASES THOUGHT TO BE MEDIATED BY SUPER ANTIGENS  Toxic shock syndrome  Food poisoining  Scalded  Scarlet skin syndrome fever  Kawasaki  Atopic disease dermatitis  Psoriasis  Acute disseminated encephalo-myelitis VIRAL DISEASE  Retroviral disease  Infectious mononucleosis  Burkitt lymphoma  CMV infection  HIV  Rabies AUTOIMMUNE DISEASE:  Rheumatoid arthritis  Chronic arthritis  SLE  Sjogren’s syndrome  Systemic Sclerosis  Crohn’s disease  Arthus Rxn SKIN INFECTION     ATOPIC DERMATITIS KAWASAKI SYNDROME: acute febrile illness with multiple vasculitis primarily affecting infants & young children. PSORIASIS: super antigen causes stimulation of skin keratinocytes, T-cell & endothelial cells leading to psoriasis. Mainly caused by staphylococcal & streptococcal infection SCALDED SKIN SYNDROME: Caused by exfoliative toxin of Staphyloccoccus in which there occur intra dermal separation of layers of skin. DIAGNOSIS Organism producing super Ag are grown till stationary phase is dialyzable in beef heart medium with high aeration at 37 ͦC at Ph 78 Cultures are treated with four volume of cold ethanol to precipitate toxin Toxin is resolublised in pyrogen free water and after that it is subjected to thin layer iso-electric focusing first at Ph 3-10 and then in a narrow range encompassing the isoelectric Ph of toxin Toxin is eluted from the gel & ampholytes are removed by dialysis OTHER METHODS  Polymerase chain reaction that can detect c-DNA of a specific sequences of the toxin.  Immunoassay can be used which can detect minute quantities of toxins.  Antibodies to the toxin may be used but they are very nonspecific as there is lot of variations in toxins.  Western blot analysis can be adopted for detection of Streptococcal super Ag TSST. TREATMENT STRATEGIES  Removal of source of SAg: 1.Drain the abscess 2.Antibiotics :- Clindamycin is effective in treatment of TSS. It down regulates the expression of PBP & streptococcal M protein , inhibiting bacteria cell wall synthesis. 3. IV immunoglobulins :- It is beneficial in the treatment mainly in streptococcal TSS. It reduces cytokine secretion and alter cytokine antagonist production. Also effective in treatment of kawasaki syndrome and in other autoimmune disease. 4. Supportive care especially in case of shock. IMMUNO-MODULATORY DRUGS USEFUL FOR SUPER ANTIGEN ASSOCIATED DISEASE  IV IMMUNOGLOBULIN  CYCLOSPORIN  PENTOXYPHYLLINE  TNF ALPHA INHIBITORS: Adalimumab, infliximab  MONOCLONAL Ab  THALIDOMIDE NOTE: Steroids should not be used in treatment of the super Ag, as super Ag induce the expresion of glucocorticoides receptors, which is an endogenous inhibitors of classic glucocorticoids receptors which leads to super Ag induced glucocorticoids resistance. Also they make peripheral blood mononuclear cells resistant to steroids. BIOLOGICAL SIGNIFICANCE THERAPEUTIC AGENT:  Super antigen causes a massive release of cytokines, so they are suggested as a candidate for anti tumour agent. Super antigen based radioactive yttrium 90 appears to be an option for liver cancer. Genetically engineered super Ag is also used in therapy of cancer.In this super Ag fused with F ab fragments specific for tumour cells which causes activation of T-cells & targeted cells against Ag positive tumour cells. It helps us to discern the mechanism of pathogenesis of a no. of disease linked to microbial infections and also it offers new possibilities for development of novel & effective therapeutic strategies.