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Immunogenetics. Introduction . How can the body identify and eliminate foreign invaders. especially since microorganisms are constantly evolving ways to avoid detection?

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introduction
Introduction
  • How can the body identify and eliminate foreign invaders. especially since microorganisms are constantly evolving ways to avoid detection?
  • The trick is to be able to distinguish between “self” and “non-self”: to recognize molecules in the body that don’t belong there.
  • Foreign molecules (often on the surface of foreign organisms) raise an immune response in the body.
  • The primary defense is a set of antibody molecules (also called immunoglobulins, Ig). The human body produces over 1,000,000 different antibodies for this purpose.
  • Antibody molecules bind to antigens, which are molecules that are non-self. Each antibody is specific for a particular antigen.
  • Some antibodies are present in the blood and other body spaces as soluble molecules. The antibodies cause antigens to clump up and form precipitates that scavenger cells pick up and digest.
  • Some soluble antibodies mark foreign cells for attack by the complement system, a series of proteins that punches holes in the cell membranes.
  • Other antibodies are on the surface of immune system cells (lymphocytes), which are stimulated to engulf and digest foreign cells by phagocytosis.
complement
Complement
  • The complement system is a way of rupturing the membrane of invading cells.
  • Antigens on the surface of the invader bind to soluble antibodies.
  • Then a series of other proteins (already circulating in the blood) is activated: the complement cascade.
  • These proteins bind to the complex in a specific sequence, creating a large hole, which usually kills the cell.

This is a compliment.

the immune system
The Immune System
  • The lymphocytes, or white blood cells, mostly travel through the body in the lymph vessels, a separate circulatory system that is connected to the blood system. The cells collect in lymph nodes, where large numbers of lymphocytes can attack foreign invaders.
  • There are two main branches of immune system: T cells and B cells. Both originate in the bone marrow, from the same stem cells as the red blood cells.
    • T cells then move to the thymus gland to mature.
    • B cells were originally named for their site of maturation in birds: the Bursa of Fabricius. This organ doesn’t exist in humans. B cells probably mature in the bone marrow.
  • B cells secrete soluble antibodies: humoral immunity.
  • T cells interact directly with their targets: cellular immunity
  • In the early 1900’s, there are a major debate as to whether the immune response was humoral or cellular. Turned out they were both right.
antibody molecules
Antibody Molecules
  • Basic structure: 2 heavy chains plus 2 light chains, joined together by disulfide bridges between cysteine amino acids.
  • The molecule has a "Y" shape, with the two ends of the fork being composed of both heavy and light chain regions.
  • These ends are the regions that bind the antigens (Ag). Each Ab molecule has two identical Ag binding regions, and thus the Ab molecules can bind together large groups of Ag's. This makes an insoluble complex that is easy for other cells in the immune system to find and eat.
more antibody molecules
More Antibody Molecules
  • Each light (L) chain has 2 domains, a variable (V) region and a constant (C) region. There are only a small number of C regions in each person, but there are very many different V regions. Note that the V and C regions are together on the same polypeptide chain!
  • Each heavy (H) chain has 4 domains, a V domain followed by 3 C domains. The C domains determine the class (IgG, IgM, etc) of the antibody.
  • Ig's come in 5 classes: IgM (early response), IgG (main blood Ig), IgA (in body secretions), IgE (allergic response), and IgD (mostly a cell surface molecule in the early response).
    • IgM comes in both membrane-bound and soluble forms. The soluble form is 5 Ig’s connected together in a star shape.
    • IgA is two Ig’s connected together tail to tail.
  • In many cases, the constant class-specific regions of the H chains bind to receptors on the surface of specific cells. For instance, IgA binds to secretory cells so it gets secreted into tears, mucus, etc. Also, IgE binds to mast cells that trigger histamine release and other rapid responses to invasion.
generation of antibody diversity
Generation of Antibody Diversity
  • There isn't room in the genome for 1,000,000 different antibodies.
  • Coding is done in pieces, with a unique DNA splicing mechanism used to assemble the L and H chains. Each immune system cell splices the DNA differently.
  • Thus, the DNA in the region of the H and L chain genes in B and T cells is not identical to the DNA in other body cells.
light chain splicing
Light Chain Splicing
  • There are two different L chain genes, called kappa and lambda. Immunoglobulins use one or the other.
  • At the kappa L chain gene, there is a single region of DNA that codes for the C domain. Upstream from the C domain is a group of about 250 V domains and another group of 5 J (for "joining") regions.
    • Each V region has a 5’UTR segment, a separate exon, attached to it: the leader.
  • During the development of the B cell, a randomly chosen V domain joins with a randomly chosen J domain to form a VJ domain. This occurs by splicing out the DNA between them.
    • Note that this is a very different mechanism from intron spicing that occurs in the RNA of most genes!
  • Once the VJ domain is formed, it can be transcribed into RNA along with the C domain and any DNA that lies between the VJ and C. All the intervening RNA is spliced out as an intron, so the final messenger RNA has VJC all together; this is then translated into a L chain protein.
  • Lambda light chain genes are slightly different: fewer V regions, and four different C regions each of which has its own J. Lambda chains use only a single DNA splice, to join a randomly selected V region to one of the 4 J-C regions.
heavy chains
Heavy Chains
  • The process of producing a heavy chain is very similar to L chains, except that there is an additional group of 5 D regions between the V regions and the J regions.
  • Two DNA splices occur for H chains: first a random J joins with a random D to form a DJ region, then a random V region is added to form a VDJ domain.
  • Just as in the L chain, this VDJ is transcribed along with the C region, and any intervening RNA is spliced out as an intron. The final product has VDJC all joined together and gets translated into an H chain protein.
  • All of the C regions are at the heavy chain locus. Initially, the CM region is transcribed. CD is also transcribed and expressed using an alternative RNA splicing mechanism.
summary of splices
Summary of Splices
  • Light chain
    • V-JC joining by DNA splice (lambda)
    • V-J joining by DNA splice (kappa)
    • VJ-C intron removal (RNA splice)
  • Heavy chain
  • D-J joining by DNA splice
  • V-DJ joining by DNA splice
  • VDJ-C intron removal by RNA splice
  • IgM membrane bound 3rd C domain to soluble 3rd C domain by RNA splice
  • Class switching by DNA splice
additional diversity mechanisms
Additional Diversity Mechanisms
  • In addition to the DNA splicing, other variants in the antibody molecules are generated by two mechanisms:
  • First, the DNA splices do not occur at a precise point: they can vary by several bases, which can lead to the addition or deletion of 1 or 2 amino acids at each splice point.
    • These variations can lead to different specificities of the antibodies.
    • The enzymes that do the DNA splicing (RAG1 and RAG2) produce double stranded breaks in the DNA, which is repaired imprecisely.
somatic hypermutation
Somatic Hypermutation
  • Second, there is a “somatic hypermutation” mechanism by which random base change mutations occur in the V regions in B cells.
    • This mechanism doesn't work in other cells and doesn't affect other genes: only a region of about 1.5 kb is affected.
    • It only occurs as the B cell is maturing: after it has been stimulated to divide by an antigen, somatic hypermutation occurs to modify the antigen binding region.
    • Those cells that bind the antigen most tightly survive and divide more than the others. This process is called “affinity maturation”.
    • It is triggered by the enzyme “activation-induced cytidine deaminase” (AID), which deaminates cytidine to uracil. This base mismatch can be incorrectly repaired by several different mechanisms to generate mutations.
allelic exclusion
Allelic Exclusion
  • These diversity mechanisms often generate non-functional Ig genes: genes that contain stop codons or don't stay in the proper reading frame. The developing B cells use a mechanism called "allelic exclusion", in which each B cell makes only 1 active L chain and 1 active H chain. The cell tries each copy of the L genes and each copy of the H genes in turn:
    • If an active chain is made, no further DNA splicing occurs.
    • However, if a non-functional Ig is made, the cell then tries the next L or H gene.
    • This process continues until an active product is made from both H and L, or until all genes have been tried (in which case the cell dies).
class switching
Class Switching
  • Heavy chains fall into 5 classes, based on their C regions.
  • Each H gene has C regions for all 5 classes arranged on the chromosome, with the IgM C region nearest to the V regions.
  • There are several different C regions for some of the classes.
  • IgM is the initial Ab made by each B cell.
  • However, after a while the B cell switches to a different class.
  • This is done using a third DNA splice, in which the DNA between the VDJ and the constant region for the new class is spliced out.
alternative splicing in early activation
Alternative Splicing in Early Activation
  • Before it is activated, the B cell carries IgM on its surface. This happens because the third Constant region domain in the IgM molecule contains a stretch of hydrophobic amino acids that anchor it to the membrane.
  • However, after the B cell is activated, it starts to secrete IgM molecules with the same V region on their H chain but with a different third C domain.
  • This happens because there are 2 third C domains for IgM; which one is used depends on alternate RNA splicing: before B cell activation, the membrane-bound domain is used, and after activation this domain is spliced out as an intron and the soluble domain is used instead.
t cell receptors
T Cell Receptors
  • The TCR protein has 2 subunits and one antigen binding site.
  • The alpha subunit has V and J segments (similar to Ig light chains)
  • The beta subunit has V, D and J regions, like the Ig heavy chain.
  • Both segments undergo DNA splicing rearrangements like the Ig genes. The joining is not precise and short additions or deletions of bases can occur, as in the Ig genes. However, affinity maturation and somatic hypermutation do not occur.
  • The TCR protein is membrane bound. It is only found on T cells.
mhc proteins
MHC Proteins
  • Class 1 MHC molecules are found on the surface of all nucleated cells. They are involved in cellular immunity.
  • Class 2 MHC molecules are only found on the surface of cells that display antigens: macrophages and B cells. They are involved in humoral immunity.
  • Structure: Both class 1 and class 2 molecules have 4 domains, but they are divided differently. MHC1 has 3 domains on the alpha subunit and one domain on the beta subunit. MHC2 has two domains on both alpha and beta subunits.
  • The peptide-binding groove is between the alpha-1 and alpha-2 domains on MHC1, and between alpha-1 and beta-1 in MHC2. Most of the differences between the many different MHC alleles lie in this region, which allows binding of different peptides.
  • The MHC proteins are encoded by a series of genes at the MHC locus on chromosome 6. MHC genes are well known to be very polymorphic. This polymorphism is the major cause of tissue graft rejection: the immune system almost never recognizes MHC genes from another individual as self.
    • There are three main MHC class 1 genes: A, B, and C, which encode the alpha subunit. The beta subunit (beta2 microglobulin), is not very polymorphic, and it is encoded elsewhere. Also, there are about 25 “non-classical” class 1b genes and pseudogenes clustered nearby. The class 1b genes are mostly monomorphic and their function is not well understood.
    • The class 2 genes are encoded by 6 regions: DM, DN, DO, DP, DQ, and DR. Each region has at least one alpha and one beta gene. There are also some other genes and pseudogenes in the region.
activation of b cells
Activation of B Cells
  • In the embryo, each B cell undergoes the DNA splicing necessary to produce a single type of antibody. This antibody is an IgM bound to the surface of the cell. No antibody is secreted at this time.
  • Foreign invaders are swallowed (phagocytosis) by macrophages, which are lymphocytes that non-selectively eat particles found in the body.
  • The macrophage partially digests the invader, converting it into peptides and other small molecules.
  • The macrophage then combines the foreign peptides with an MHC class 2 molecule that gets “displayed” on the surface of the macrophage cell.
  • Helper T cells that have a T cell receptor proteins that match (bind to) the displayed antigen get activated.
    • Helper T cells are the central regulatory element in humoral immunity. They are also the primary target of the AIDS virus.
more b cell activation
More B Cell Activation
  • The activated helper T cells then divide (proliferate). They also activate B cells.
  • The inactive B cells start the activation process by binding to an antigen with their IgM molecules, engulfing it and processing it. The antigen peptides are displayed on MHC 2 molecules on the cell surface, just like the macrophages do.
  • Activated helper T cells interact with B cells that have the same antigen displayed on their surface: the TCR on the helper T cell binds to the antigen displayed on MHC2 on the surface of the B cell.
    • the CD4 protein on the surface of the helper T cell is also necessary for this interaction.
  • The helper T cells secrete cytokines, which stimulate the B cells to proliferate and differentiate into plasma cells, which secrete antibodies.
    • this is clonal selection: only those B cells with usable antibodies are selected to proliferate.
  • Some descendants of the activated B cell become plasma cells, while others become memory cells. Memory cells stay present in the blood for long periods of time. The next time their specific antigen appears, the memory cells quickly start proliferating and differentiating into plasma cells. This provides a rapid response to a second appearance of the antigen.
cellular immunity
Cellular Immunity
  • Cytotoxic (or killer) T cells are the main actor in cellular immunity. Cytotoxic cells (also called CD8 cells) kill cells that are infected with viruses or otherwise contain foreign proteins.
  • Cytotoxic T cells are activated in the same way that helper T cells are.
    • Macrophages eat things in the blood, digest them to peptide fragments, and display the fragments on their surfaces bound to MHC class 1 molecules (as opposed to class 2 for helper T cells).
    • Inactive cytotoxic T cells have a specific T cell receptor protein on their surface. If their TCR binds to the peptide displayed on a macrophage, the cytotoxic T cell is activated.
    • the CD8 preotein on the T cell surface facilitates this interaction.
  • All cells digest proteins in their lysosomes. Some of the resulting peptide fragments are displayed in MHC 1 proteins on the surface of the cell. Thus, every cell displays a summary of the proteins inside it on its surface. Mostly these are acceptable proteins, but if the cell has been infected by a virus, foreign viral protein fragments will be displayed.
  • If the TCR on an activated cytotoxic T cell binds to a peptide fragment displayed on the surface of a cell, the T cell kills it by secreting perforin proteins that punch holes in the cell’s membrane.
self vs non self
Self vs. Non-self
  • If every cellular protein is displayed on the cell’s surface, and macrophages eat everything non-selectively, why don’t we have an immune response against normal cellular proteins?
  • The phenomenon of not attacking “self” proteins is called “tolerance”, and it is the result of clonal deletion.
  • Around the time of birth, all the V-D-J joining involved in making T cell receptors and IgM molecules has already occurred. Any T cell (helper or cytotoxic) that binds to an antigen present in the body at that time is killed (apoptosis). Thus, “self” is defined as any molecule circulating in the blood around the time of birth.
    • Clonal deletion occurs in the thymus.
    • since B cells only become activated by helper T cells, clonal deletion of T cells ensures that B cells that might produce antibodies against self molecules never get activated.
  • Autoimmune diseases are the result of self proteins being incorrectly recognized as non-self.
immunological memory
Immunological Memory
  • The first time a new antigen is seen, the response is slow and relatively weak.
  • However, a second appearance of that antigen gives a rapid, strong reaction. This is the result of memory cells formed during the first exposure. The memory cells have already proliferated and they quickly start to secrete antibodies.
    • Note that the secondary response is not general: it is only to the specific antigen that caused the primary response.
  • This is the basis of vaccinations: introducing a small amount of killed or weakened virus into the body, sometimes several times. The body develops a weak immune response, but builds up memory cells. When the pathogen tries to infect the body, a strong immune response is generated.
  • Cellular immunity has a similar system of memory cells.
rh incompatibility
Rh Incompatibility
  • The Rhesus factor is an antigen on the surface of red blood cells. It was originally found in rhesus monkeys. About 85% of people have the Rh antigen (Rh+) and 15% lack it (Rh-). The Rh+ allele is dominant, so heterozygotes are Rh+.
  • Because the + allele is dominant, it is possible for an Rh- mother to have an Rh+ baby.
  • In general, the fetus’s blood is separated from the mother’s: there is a barrier against blood mixing at the placenta.
  • However, during childbirth some of the fetus’s blood often enters the mother’s body. This causes her immune system to develop antibodies against the Rh factor.
  • Since antibodies do cross the placental barrier, a subsequent Rh+ fetus will have its blood cells attacked by the mother’s anti-Rh antibodies. This leads to “hemolytic disease of the newborn”, which used to be a major cause of stillbirth.
  • However, these days Rh- mothers are given anti-Rh antiserum shortly after they give birth to an Rh+ baby. This antiserum removes any Rh+ blood cells from the mother’s blood, and so she does not develop an immune response against Rh.
severe combined immunodeficiency scid
Severe Combined Immunodeficiency (SCID)
  • This disease involves a completely non-functional immune system due to a lack of T cells.
  • The “boy in the bubble”. Normally SCID is lethal within the first year of life. One child was immediately put into a sterile environment and kept there until age 12. At this point he was given an experimental treatment (at his own request). Unfortunately it failed and he died.
  • The disease is caused by several different mutant genes, including those involved with DNA splicing, interleukins (which signal between macrophages, T cells, and B cells), the CD4 and CD8 surface proteins, and others.
  • Basic treatment: transplant of bone marrow (which contains blood stem cells) from someone who has similar tissue antigens.
  • One form, X-linked SCID, is due to the lack of an inerleukin receptor. Attempts to cure it by transfecting a normal version of the gene into blood stem cells has been fairly successful. However, 2 patients developed leukemia, presumably due to the insertion of the transfected gene into an oncogene; gene therapy trial have been suspended.
  • Another form of SCID is caused by the absence of adenosine deaminase. This results in the accumulation of dATP; lymphoid stem cells are very sensitive to this and die.
autoimmune diseases
Autoimmune Diseases
  • A wide spectrum of diseases: rheumatoid arthritis, Crohn’s disease, type 1 diabetes, lupus, and many others.
  • Normally, immune system cells that react with self antigens are deleted early in life.
  • One cause is sequestered antigens: body proteins that did not reach thymus during the critical period of clonal deletion around birth. Eye lens crystallin is not generally exposed to the blood, but eye injuries sometimes allow contact and the development of anti-crystallin antibodies. This can result in blindness.
  • Another possible cause is clones of self-reactive immune cells that escape clonal deletion due to chance events.
  • A third possible cause is cross reactive antigens. A pathogen carries an antigen that is similar to a normal body protein. As the body mounts an immune response against the pathogen, the degree of similarity causes cross reaction with the body proteins. An example is scarlet fever, which is caused by a Streptococcus infection. Antibodies produced against these bacteria often cross react with a protein found on heart valve cells. The result is heart damage.
  • No good treatment exists. The main treatments attempt to relieve the symptoms.
hypersensitive states
Hypersensitive States
  • The body over-reacts to the presence of an antigen.
  • There are several types.
    • Anaphylactic reaction is a very rapid response that comes from mast cells releasing large amounts of histamine as a result of binding the antigen to IgE molecules. Histamine causes the blood vessels to leak, causing allergy symptoms such as runny nose and weepy eyes, as well as bronchoconstriction (as in asthma).
    • Some external antigens, especially certain small molecules, accumulate of the surface of cells. The body’s immune response can kill the cells.
    • accumulation of antibody-antigen complexes in body tissues can lead to inflammation and tissue damage.
    • Contact dermatitis, such as is found in poison ivy rashes, is due to T cell memory cells,