BASIC IMMUNOLOGY I. DEFINITIONS •
Acquired (specific / adaptive) Immunity:
A type of host defences that is stimulated by microbes that invade host tissues. In other words, acquired immunity adapts to the presence of microbial invaders. Adjuvants: Substances which enhance the immune response against the immunogens. Affinity: The binding strength of an antigen (having a single antigenic determinant) with a single combining site of an individual antibody. Affinity is the sum of attractive and repulsive forces operating between the antigenic determinant and the antibody combining site. Affinity also refers to the equilibrium constant (K eq eq) that describes the Ag-Ab reaction. K eq eq = [Ag-Ab] / [Ag] X [Ab] Antibody (Ab): A specific protein that is produced in response to an immunogen and reacts with antigens. Antigen (Ag): A substance that reacts with the products of a specific immune response (i.e. antibodies). Agglutination test: A test in which antibodies react with particulate antigens resulting in their agglutination (or clumbing). Note that the agglutination test works only with the particulate antigens. Agglutinin: Antibodies used to agglutinate particulate antigens in the agglutination test. Theoretically, all antibodies can agglutinate antigens, but IgM due to its high valence is good agglutinin. Avidity: A measure of the overall binding strength of an antigen (having many antigenic determinants) with a multivalent antibody. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Complements: 20 different serum proteins that are produced by a variety of cells, including hepatocytes, macrophages and gut epithelial cells. Some complements bind to the immunoglobulins or to cell membranes. Others are proenzymes that when activated cleave one or more complement proteins to yield fragments that activate cells, increase vascular permeability or opsonize bacteria. Complement activation: Alteration of complement proteins, enabling them to interact with another complements. Complement fixation: Utilization of a complement by an antigen-antibody complex. •
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Complement inactivation:
Denaturation (usually by heat) of an early complement component resulting in a loss of its hemolytic activity. Conventional T-dependent antigens: Antigens which monoclonally (or oligoclonally) activate a small fraction of T-cells. Only one in 10 4 - 105 of the T-cell population is able to recognize that type of antigens. Convertase: An altered complement protein , which acts as a proteolytic enzyme for another complement component. •
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Countercurrent Countercurre nt electrophoresis test:
A test in which the antigen and the antibody are placed in wells punched out of an agar gel. The antigen and antibody are then electrophoresed into each other where they form a precipitation line. The test works only if the Ag and Ab have opposite charges. This test is primarily qualitative, although from the thickness of the band you can get some measure of quantity. Cross reactivity (Multispecificity): The ability of an individual antibody combining site to react with more than one antigenic determinant. Cross reactions arise because the cross reacting antigen shares an epitope in common with the immunizing antigen. Domains: Globular folded regions of the immunoglobulin molecule that contain intra-chain disulfide bonds. Domains may be ligh chain domains (VL and CL) or heavy chain domains (VH, CH1-CH3 (or CH4). Direct coombs test: A test used to detect if antibodies are bound to the RBCs surface antigens or not. In this test, the patients RBCs are washed and then incubated with a second antibody (or an antihuman globulin - also called coombs reagent ). If antibodies have been fixed on the RBCs surface, the RBCs in this case will agglutinate when incubated with the antihuman globulin. Epitope (Antigenic determinant): The portion of an antigen that combines with the products of a specific immune response. Fab region: A region that contains the antigen binding sites of the antibody. The Fab region consists of two identical fragments that contain the light chains and the VH and CH1 domains of the heavy chains. Each Fab region is monovalent, whereas the original molecule was divalent. An antibody is able to bind a particular antigenic detrminant, because it has a particular combinations of VH and VL. Thus different combinations of VH and VL result in antibodies that can bind different antigenic determinants. Fc region: A region whose principle function is to mediate the effector functions of immunoglobulins. immunoglobulins. Normally, the ability of an antibody to carry out an effector function requires the prior binding of an antigen. However, there are exceptions for this rule. The Fc region contains the remainder of the two heavy chains (each containing a CH2 and CH3 domains). Haptens: Small molecules which could never induce an immune response when administered by themselves (i.e. non-immunogenic). Free haptens, however, can react with the products of the immune response. This means that haptens have the property of antigenicity, but not immunogenicity. It was found that when a hapten is conjugated (or coupled) with an immunogenic carrier molecule, it can induce an immune response. Hapten-carrier Hapten-carr ier conjugates: Immunogenic molecules, in which haptens have been covalently attached to an immunogenic carrier molecule. Hapten-carrier conjugates are characterized by having native antigenic determinants of the carrier as well as new determinants created by the hapten. Hemagglutination test: erythrocyte antigens resulting in their agglutinaA test in which antibodies react with the erythrocyte tion (or clumping). Hemagglutinin: Antibodies used to agglutinate the erythrocyte antigens in the hemagglutination test. Hinge region: •
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A region of the immunoglobulin molecule, at which the arms of the antibody forms a Yshape, because there is some flexibility in the molecule at this point. Immunoglobulins: Glycoprotein molecules that are produced by the plasma cells in response to an immunogen and function as antibodies. Immunogen: Substance that induces a specific immune response. Incomplete antibodies: Antibodies which bind to the red blood cells, but do not cause their agglutination. Indirect coombs test: A test used to detect circulating antibodies (in the blood serum) against someone else’s RBCs. In this test, the washed RBCs RBCs are incubated with a test serum. If the serum contains anti bodies to antigens on RBCs surface, the antibodies will bind to the surface of the RBCs. In the second step, the RBCs are incubated with an antihuman globulin. If antibodies have been bound to the the RBCs surface in the first step, the RBCs will agglutinate when incubated with the antihuman globulin (also called coombs reagent). Innate immunity: A type of host defences that is usually present in healthy individuals, prepared to block the entry of microbes and to rapidly eliminate microbes that do succeed in entering the host tissues. Passive hemagglutin test: A test in which the antibodies react with the soluble antigens coating the surface of the erythrocyte erythrocyte RBCs resulting in their agglutination. This test is performed just like the agglutination test and used to detect antibodies to soluble antigens (such as viral antigens, polysaccharides, or haptens). Pathogen Associated Molecular Patterns (PAMPS): Broad molecular patterns that are present in many different pathogens and can be recognized by the innate immune system. In other words, PAMPs are the antigenic determinants recognized by the innate immune system. An example of PAMPS is the microbial cell wall components which are recognized by complements. Pattern Recognition Receptors (PRRs): Receptors that have the ability to recognize the PAMPS (or the pathogen associated molecular patterns). A particular PRR can recognize a molecular pattern that may be present on a number of different pathogens and thus enabling the receptor to recognize a wide variety of organisms. PRRs (such as complement) are therefore non-specific in their action. Qualitative agglutination test: Agglutination tests used in an qualitative manner to detect the presence of an antigen or an antibody. For example to determine a person’s blood group, the patient RBCs are mixed with an antibody to a blood group antigen, and a positive test is indicated by the agglutination of these particulate antigen. Radial Immuno-diffusion (Mancini) quantitative precipitation test: A test in which an antibody is incoporated into the agar gel, and different dilutions of the antigens are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed. The diameter of this ring is proportional to the log of the antigen concenteration (since the amount of antibody is constant). Thus, by running different concenterations of a standard antigen, one can generate a standard curve from which we can quantitate the amount of an antigen in an unknown sample. The test is used in the determination of immunoglobulin levels in patient samples. Specificity of antibody: Ability of an individual antibody combining site to react with only1 antigenic determinant. •
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Superantigens:
Antigens which polyclonally activate a large fraction of T-cells (up to 25%). Examples of these antigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal shock toxins, and Streptococcal pyrogenic exotoxins. The diseases associated with the exposure to these superantigens are in part due to hyper-activation of the immune system and subsequent release of biologically active cytokines by the activated T-cells. TH-1 cells: A type of T-helper cells that is produced in the thymus and help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic cells. TH-2 cells: Another type of T-helper cells that is also produced in the thymus, but help the the B cells to differentiate into the plasma cells, which in turn secrete antibodies. Valency of antibody: Number of antigenic determinants that an individual ntibody molecule can bind. The valency of all antibodies is at least two and in some instances more. •
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II. QUESTIONS AND ANSWERS Q1: State the factors affecting Immuogenicity. A1: (I) Contribution of the immunogen: Foreignness - Only foreign molecules are a re immunogenic. • Size - The larger the molecule is, the more immunogenic it is likely to be. • Composition - The more complex, the more immunogenic the substance will be. • Physical form - Particulate antigens are more immunogenic than the soluble ones. • (II) Contibution of the biological system: Genetic factors - Substances may be immunogenic in one species, but not in another. • Age - Usually the very young and very old individuals have a diminished ability to in• duce an ammune response against the immunogen. (II) Method of administration: Antigen dose - There is a dose of antigen above or below which the immune response is • not optimal. Route - Generally the subcutaneous route is better than the intravenous or intra gastric. • Adjuvants - Adjuvants are substances that can enhance the immune response. • Q2: State the factors affecting Antigen-Antibody reactions. A2: Factors affeccting the antigen-antibody reactions include: Affinity: The higher the affinity of the antibody to the antigen, the more stable will be the interaction. Thus, the ease with which one can detect the interaction. Avidity: Reactions between multivalent antigens with multivalent antibodies are also • more stable, and thus easily detected. Ag-Ab ratio: The ratio of the antigen and the antibody influences the detection of the • AgAb complex. This is because the size of the complex formed is related to both the concenteration of the antigen and the antibody. Antigen physical form: Particulate antigens are easily agglutinated by antibodies. • Q3: What are the functions of complements? •
A3: Complements: Complements: (1) Can opsonize bacteria for enhanced enhanced phagocyto phagocytosis, sis, (2) Can activate various cells including PMNs and macrophages, (3) Can participate in regulating antibody responses, (4) Can increase increase vascular vascular permeability, permeability, and (4) Can aid in the clearance clearance of immune com plexes and apoptotic cells. Complements also contribute to inflammation and tissue damage.
Q4: Write about the classical complement pathway? A4: It is dependent upon antibodies to be operational. In this pathway: •
C1 (which is a multi-subunit containing 3 different proteins; C1q, C1r, and C1s) binds to the Fc region of the IgG and IgM antibody molecules that have interacted with the antigen. The binding of C1q to the antibody must cross link at least two antibody molecules and requires calcium and magnesium ions.
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The binding of C1q to the antibody activaties C1r, which in turn activaties C1s. The result is the formation of an activated “C1qrs”. C1qrs is an enzyme which cleaves: C4 into two fragments; C4a and C4b. o o C2 into two fragments; C2a and C2b. The C4b and C2a fragments bind to the membrane to form the C4bC2a complex, whereas the C4a and C2b fragments are released into the surrounding microenvironment.
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The resulting C4bC2a complex is a C3 convertase , which cleaves C3 into two fragments; C3a (which is released released into the microenviro microenvironement nement)) and C3b (which binds binds to the membrmembrane in asscociation with C4b and C2a).
The resulting C4b C2a C3b complex is a C5 convertase. The generation of C5 convertase is the end of the classical pathway. Q5: Write about the lectin pathway? •
A5: The lectin pathway is very similar to the classical pathway, except that it is antibody independent. In this pathway: •
The mannose binding lectin (MBL) – which is similar to C1q – binds to the bacterial (or pathogen) pathogen) surface, resulting resulting in the association association of two serine proteases; MASP-1 (which is similar to C1r) and MASP-2 (which is similar to C1s).
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The formed tri-molecular complex MBL/MASP-1/MASP-2 results in the formation of an activated “MASPs”. MASPs is an enzyme which cleaves: o C4 into two fragments; C4a and C4b. o C2 into two fragments; C2a and C2b. The C4b and C2a fragments bind to the membrane to form the C4bC2a complex, whereas the C4a and C2b fragments are released into the surrounding microenvironment.
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The resulting C4bC2a complex is a C3 convertase , which cleaves C3 into two fragments; C3a (which is released released into the microenviro microenvironement nement)) and C3b (which binds binds to the membrmembrane in asscociation with C4b and C2a).
The resulting C4b C2a C3b complex is a C5 convertase. The generation of C5 convertase is the end of the lectin pathway. Q6: Write about the Membrane Attack Pathway? •
A6: In this pathway, the C5 convertase convertase from the classical classical (C4bC2aC3b), (C4bC2aC3b), lecctin (C4bC2a C3b), or alternative (C3bBbC3b) (C3bBbC3b) pathway cleaves C5 into two fragments; C5a (which remains in the fluid phase) and C5b (which rapidly associates with C6 and C7). At the same time, C8 binds followed by several molecules of C9. The C9 molecules form a pore in the pathogen membrane, through which the cellular contents leak resulting in lysis (which occur due to the membrane physical damage). The complex consisting C5bC6C7C8C9 is known as “Membrane Attack Complex (MAC) ”. Q7: What is the function of the Protein S (vitronectin)? A7: This protein binds to the soluble C5bC6 complex (which have been dissociated from the membrane), and thus preventing the binding of the C5bC6 complex to other bystander cells, which may in turn result in their damage. Q8: Write about the amplification loop of C3b formation in the alternative pathway.
A8: In this pathway, Factor B binds to the C3i (resulted from the spontaneous hydrolysis of C3) and become susceptible susceptible to Factor D. Factor D cleaves Factor B into Bb. The C3iBb complex acts a C3 convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to it and and beco become mess susc suscep epti tibl blee to cleav cleavag agee by Fact Factor or D. the the resul resulti ting ng C3bB C3bBb b comp comple lex x is a C3 convertase that will continue to generate more C3b, and thus amplifying C3b production. Q9: Write about the chemical nature of immunogens. A9: The vast majority of immunogens are proteins (which may be pure proteins or may be glycoproteins or lipoproteins). In general, proteins are usually good immunogens. Immunogens may be also pure polysaccharides or lipopolysaccharides, which are also good immunogens. Nucleic acid, on the other hand are usually poor immunogens. However, they may become immunogenic when they are complexed with proteins. Lipids were found to be non-immunogenic at all. Some glycolipids and phospholipids, however, can stimulate T-cells and produce a cellmediated immune response. Q10: Describe the nature of the t he antigen-antibody reactions. A10: In Ag-Ab reactions, the antigenic determinant of the antigen fits into a cleft formed by the combining site of the antibody. This site is present in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. The Ag-Ab reactions are thus can be viewed as a lock and its key, in which a key (i.e. Ag) fits into a lock (i.e. Ab). Q11: Write about the nature of the bonds involved in the Ag-Ab reactions. A11: The bonds that hold the antigen (Ag) into the antibody combining site are all non covalent in nature. These include: electrostatic bonds, Van der Wals forces, and hydrophobic bonds. Since Ag-Ab reactions occur via non-covalent bonds, they are reversible by their nature. Q12: Write about the general functions of immunoglobulins. A12: The primary function of immunoglobulins is to act as antibodies and bind specifically to one or more few closely related antigens. In fact, each immunoglobulin binds to a specific antigenic determinant. Often the binding of an antibody to an antigen has no direct biological effect. Rather, the significant significant biological biological effects are a consequenc consequencee of secondary secondary “effector functions” functions” of antibodies, such as: Complement fixation that leads to cell lysis as well as the release of other (1) biologically active molecules. Binding of various cell types (such as phagocytic cells, lymphocytes, platelets, (2) mast cells, and basophils) to the immunoglobulins which activate these cells to perform some functions. Q13: Write about the structure of immunoglobulins. A13: Although different immunogens can differ structurally, they all are built from the same basic units, these are: A. Heavy and Light chains: All immunoglobulins have a four chain structure (two identical light chains and two identical heavy chains). B. Disulfide bonds: The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions. There are also intra-chain disulfide bonds within each of the polypeptide chains. C. Variable Variable (V) and and constant constant (C) regio regions. ns. D. Hinge region: At which the molecule forms a Y-shape. E. Domains, which are globular folded regions F. Carbohydrates: These are usually attached to the CH2 domain in most immunoglobulins.
Q14: Write about the cellular barriers of the innate (non-specific) immunity. A14: The following cells are main line of defense in the non-specific immune system. Polymorphonuclear cells (PMNs) and Neutrophiles arrive to the site of infection, where they “phagocytose” invading organisms & kill them. Macrophages also function in phagocytosis
and intracellular killing of microorganisms. Moreover, they act as antigen presenting cells, which are required for the induction of specific immune rsponses. Natural killer (NK) and lymphokine activated killer (LAK) cells, which can non-specifically kill virus infected and tumor cells. Eosinophils have proteins in their granules that are so effective in killing parasites.
3. COMPARISONS 1. Comp Compare are betwee between n innate innate immunity immunity and acquire acquired d immunity. immunity. Innate immunity
Acquired immunity
Specificity
Innate immunity shows no specificity , since its components can recognize broad molecular patterns that can be found in many pathogens, and thus they can recognize and react with a wide variety of different microorganisms
Acquired immunity demonstrate a high degree of specificity . This is because components of the acquired immune system (such as antibodies, and the B and T cells receptors) recognize and react with only particular pathogens
Time
Always present and ready to be mobilized upon infection
Requires some time to react to an invading organism
Memory
Does not have an immunological memory
Demonstrates immunological memory and reacts more rapidly on subsequent exposure to the same organism
2. Compare between the classical, lectin, l ectin, and alternative complement pathways. Classical
Lectin
Alternative
Components
C1, C2, C3 and C4
MBL, MASP-1, MASP-2, C2, C3 and C4
C3, Factor B, Factor D, and properdin
Antibody dependent?
Yes
No
No
Require divalent cations?
Yes
Yes
Yes Yes
No Yes
C5 convertase
C4bC2b C4bC2bC3b
C3bBb C3bBbC3b
Requlatory components
C1-INH, C4-BP, Factor I
FactorH, Factor I, DAF, CR1
Generate prokinin? Generate Anaphylotoxin? C3 Convertase
3. Compare between the activities and controlling factor(s) of the different complement fragments.
C2a / C2b ??
C3a and C4a
C3b C4b
Activity
Effect
Controlling factor(s)
Prokinin (Fluid accumulation)
Edema
C1 – INH *
Basophil and Mast cells degranulation, Enhanced vascular permeability, Smooth muscle contraction
Anaphylaxis
C3a – INA
Opsonin (phagocyte activation)
Fcators I and H Phagocytosis
C4 binding protein and Factor I
C5a
Vascular permeability, Smooth muscle contraction, chemotaxis, Phagocyte activation, Stimulation of respiratory burst and inflammatory cytokines
Anaphylaxis (most potent) and Inflammation
C3a – INA
C5b, C6 and C7
Chemotaxis
Inflammation and Tissue damage
Protein S (vitronectin)
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* The deficiency of the C1-INH inhibitor is usually associated with the development of hereditary angioedema . 4. Comp Compare are between between the the antigenic antigenic determina determinants nts recogniz recognized ed by the B cells cells and those those recognized by the T cells. Deter Determin minant ants s recog recogniz nized ed by B cells cells
Deter Determin minant ants s recog recogniz nized ed by T cells* cells*
Composition
The antigenic determinants recognized by the B cells are created by the 1o sequence of residues in the polymer (linear or sequence determinants) and/or by the 2o, 3o, or 4 o molecule structure (conformational determinants)
Size
The antigenic determinants recognized by the B cells are small and are limited to approximately 4-8 residues (amino acids and/or sugars)
The antigenic determinants recognized by the T cells are small and are limited to approximately 8-15 amino acids
Number
In theory, each 4-8 residues can constitute a separate antigenic determinant. In practice, the no. of antigenic determinants / antigen is much lower than that would theoretically be possible
In theory, each 8-15 residues can constitute a separate antigenic determinant. In practice, the no. of antigenic determinants per antigen is much lower than that would theoretically be possible
The antigenic determinants recognized by the T cells are created by the 1 o sequence of amino acids in proteins.
* Note that : T - cells CAN NOT recognize polysaccharide or nucleic acid antigens. That’s why polysaccharides are T-independent antigens, whereas proteins are T-dependent antigens. Note also that the antigenic determinants recognized by the T-cells are limited to those portions of the antigen that can bind to the MHC molecules. 5. Comp Compare are between between the clinica clinicall implications implications of the differen differentt human immunoglo immunoglobulins bulins.. Increases in:
Decreases in:
IgG
All infections, Hyperimmunization, Liver diseases, and Rheumatoid arthritis
Selective IgG and IgA deficiency, and Chronic lymphoblastic leukemia
IgM
Actinomycosis, Malaria, Infectious mononucleosis, and Trpanosomiasis
Chronic lymphoblastic leukemia
IgA
Live Liverr cirrh cirrhos osis is,, and Rhe Rheum umato atoid id arth arthri riti tiss
Acut Acutee and chro chroni nicc lymph lymphob obla last stic ic leuke leukemi miaa
IgD
Chronic infections and IgD myelomas
???
IgE
Hay fever, Asthma, and IgE myelomas
Congenital agammaglobulinemia
