Module 3 and 4

define affinity

measure of the strength with which one molecule binds to another at a single binding site.

Explain IgG

most abundant isotope in serum predominant isotope in secondary response 4 sub types

when IgG is cleaved with a protease that targets the hinge region, what is generated?

two Fab fragments and one Fc fragment

what is a dimer?

two identical molecules linked together

describe how antibodies can neutralize viral and bacterial infections and toxins? what is the role of Fab?

-Fab binds virus preventing attachment to receptor -prevents bacterial colonization: fab binds pathogen preventing attachment. -fab blocks toxins: fab binds toxin preventing attachement receptor. -blocks the pathogen from entering host cell. prevents the initiation of infection by blocking the antigen from binding to host cell. -can create agglutination-clumping together

Explain IgA

-secreted as dimer with 4 Ag binding sites -predominant isotope in secretions: breast milk and mucous -resistant to proteases -passive immunity in breast milk

Explain IgM

-secreted as pentamer with 10 Ag binding sites -predominant isotope in primary response -first Ab formed in primary response

What is an epitope?

-the part of an antigen molecule to which an antibody attaches itself -usually carbs or proteins bc surface of molecules of pathogens are commonly glycoproteins, polysachs, glycolipids, and peptidoglycan. -interacts with the CDR/FV

What is junctional diversity and when does it occur?

P+N additions -resolution of hair-pin loops: P additions -random nucleotide addition via TdT enzyme: N additions -· Nucleotides can be added to the DNA during VDJ recombination when the chromosomes have been nicked by the RAG recombinase enzymes. · Specifically an enzyme called TdT, terminal deoxynucleotidyl transferase, add random nucleotides to exposed chromosome ends N additions. · The hair pins can be opened up and refilled by DNA repair enzymes to create P additions.

explain how rearrangement and modifications of TCR genes in T cells are both similar and different to that of Ig genes in B cells

T cells have more diversity than the B cells.

which domains, in an antibody, form an antigen binding site?

VH and Vl domains, variable domain V of light and heavy chains together

what is carboxyl?

acid -COOH, present in most organic acids

what is affinity maturation?

as the adaptive immune response to infection proceeds, antibodies of progressively higher affinity for the infecting pathogen are produced. its improved selection for binding to a pathogen.

what does opsonization do?

coats a pathogen for phagocytosis

how do the four classes of IgG differ?

differ in the hinge regions

Why is CDR so important?

differences in the loop between different antibodies create both the specificity of antigen binding sites and their diversity. creates the specificity

What is a non-covalent bond?

does not involve the sharing of electrons, but involves more dispersed variations of electromagnetic interactions between molecules or within a molecule

what happens when antibodies bind to their specific antigens?

effector function of antibodies: -neutralization -opsonization -complement activation-classical pathway -ADCC

what environment do antibodies function in and why?

extracellular environments since they can withstand the harsh conditions because of their immunoglobulin domain. how they fold up is very stable.

Define humoral immunity, plasma, and serum

humors=body fluids plasma=clotting factors, proteins, 55% of blood serum=plasma-clotting factors

what is allelic exclusion and why do we have it?

in developing B cell the process of immunoglobulin gene rearrangement is tightly controlled so that only on heavy chain and one light chain are finally expressed this ensures that each B cell produces IgM and IgD of a single antigen specificity

What kind of epitopes can a protein-based antigen have?

linear epitopes and discontinuous epitopes

what are monoclonal antibodies?

-Antibodies produced from a single group of genetically identical B-cells -used for diagnostic test and therapeutic agents.

what is a catalytic antibody and what are they used for?

-antibodies that catalyze chemical reactions involving the antigen they bind. -possible use for these catalytic antibodies would be to convert a toxic chemical within the body into an unharmful product. -have been made to bind to cocaine or methamphetamines and convert the highly addictive drugs to derivatives that have no psychostimulating activity.

which isotopes are expressed on naive B cells and what does it mean to be a naive B cell. How are they made?

-circulating B cells that have yet to encounter antigen are naive B cells. -they express both IgM and IgD on their surfaces alternative splicing of RNA transcript -same VDJ thus same Ag specificity -· IgM is the first antibody expressed because the constant region for IgM, Cmu, is located directly downstream from the rearranged VDJ. Both isotypes will include the same rearranged VDJ segments. · The BCR besides the antibody IgM and IgD it will also have Ig alpha and Ig beta · The B cell's receptor includes a transmembrane domain that anchors it into the plasma membrane · The secreted receptor antibodies through alternative splicing at the RNA level this enables the immunoglobulin protein to pass through the cellular secretory pathway and to be secreted from the cell in the form of soluble antibody proteins

What is VDJ recombination/ somatic recombination?

-during the development of B cells, the arrays of V, D, and J segments are cut and re-spliced by DNA recombination. -Somatic recombination it brings together a single gene segment of each type to form a DNA sequence encoding the V region. -For light chains there is single recombination between VL and JL segments. -For heavy chains two recombinations are needed, the first to join D+JH segments, and the second to join DJ segments to a VH segment. -In each case, the particular V,D, and J gene segments that are joined together are selected at random. Bc of the multiple gene segments of each type, numerous different combinations of V,D, J gene segments are possible. this generates many different V region sequences in the B cell population -joining of V and C gene regions occurs at the level of RNA not the DNA level -mRNA is when you have VJ or VDJ combined and fused together. not in the DNA level. -· VDJ is recombination is mediated by the RAG1/RAG2 recombinase enzymes that are expressed in developing B cells.

What are CDR's?

-hypervariable regions HV or complementarity determining regions CDR 1, 2, 3 these regions are in the V domain (variable domain in the heavy and light chains) within this variable domain there are differences in aa sequence and are concentrated within this HV. there are 3 HV/ CDR regions located on the three loops farthest from the constant region. -contributes much of the antigen specificity of the antigen binding site. most variability in aa sequence -they provide a binding surface that is complementary to that of the antigen -interacts with the epitope. binds the antigen

what is somatic hypermutation dependent upon and what does it do and why is it important?

-on the enzyme activation induced cytidine deaminase AID, which is made only by proliferating B cells. -AID converts cytosine in single stranded DNA to uracil, a normal component of RNA but not DNA. AID acts during transcription when the two DNA strands of the immunoglobulin gene become temp separated, other enzymes repair and modification, then convert the uracil to any one of the four bases of normal DNA causing a mutation. -some of these mutant immunoglobulins have substitutions in the antigen binding site that increase its affinity for the antigen. -B cells bearing the mutant high affinity immunoglobulin receptors compete most effectively for binding to antigen and are preferentially selected to mature into antibody secreting plasma cells. They don't have a random distribution of aa substitutions. the changes are concentrated at positions in the heavy and light chain CDR loops that form the antigen binding site and directly contact antigen. -the AID does point mutations in the variable regions. not the constant regions. · The variable region of the rearranged immunoglobulin DNA is targeted for excessive point mutations that can alter the affinity of the antibody for its antigen. · Affinity maturation: when there is a higher affinity for an antigen and that is chose for proliferation and differentiation

what is somatic hypermutation and affinity maturation and how is this adding another diversification to the V region

-once a B cell has been activated by antigen, further diversification of the whole of the V domain coding sequence occurs through the process of somatic hypermutation. -this introduces point mutations (single nucleotide substitutions) almost randomly and at a high rate throughout the arranged V regions of of heavy and light chain genes. -of the variable regions-point mutations-have different Ab affinities. -some of these mutant immunoglobulins have substitutions in the antigen binding site that increase its affinity for the antigen.

what is clonal selection/expansion

-the T or B cell receptor encounters/ recognizes that pathogen and then cells within the area rapidly increase and change into B or T cell with the same receptor binding site for that specific pathogen. The number of the B or T cells increase and attack the pathogen. -when the specific T or B cells is activated by the antigen, they start replicating itself with the same receptor. -simplified: it selects an a B or T cell then clones itself

explain the diversity of the light and heavy chains DNA sequence. Explain the germline organization of the human immunoglobulin heavy and light chains

-the V regions are encoded by two VL or 3 VH gene segments and rearranged to produce an exon that can be transcribed. -for the light chain V region there are V variable and J joining gene segments. -the heavy chain locus includes diversity D gene segments that lie between V and J segments. -V region of light chain: combination of one V and one J and the C regions is encoded by a single C gene segment. -V region of light chain gene has only one: kappa or lambda: VJ -V region of heavy chain: VDJ -diversity of CDR 1+2 is determined by differences in the sequences of heavy chain V gene segments; CDR3 is determined by differences in the D gene segments and the junctions they make with the V+J gene segments. ----The C region of a heavy chain is encoded by one of the C genes.

What is an isotope?

-the class of an immunoglobulin that is IgM, IgG, IgD or IgE each of which has a distinct heavy chain constant region encoded by a different constant region gene. -the heavy chain constant region determines the effector properties of each antibody class. -differ in the constant region -the stem contains binding sites for cells and effector molecules that enable bound antigen to be cleared from the body

Explain RAG 1+2 and junctional diversity

-the set of enzymes needed to recombine V,D,J segments are called V(D)J recombinase. the two component proteins are made only in lymphocytes and are specified by the recombination activating genes RAG 1+2. The RAG 1+2 proteins interact with each other and with other proteins known as the high mobility group of proteins a RAG complex. 1. RAG complex binds to one of the RSSs flanking the sequences to be joined and then recruits the other RSS to the complex. 2. the endonuclease functions of RAG cleve the DNA to generate a clean break at the ends of the two heptameter sequences. The RSSs are aligned and held in place by the RAG complex called non homologous end joining. this brings together the ends of the two gene segments to for a coding joint in the chromosome while joining the ends of the removed DNA in a signal joint. the recombination process initiated by the RAG complex in a developing B cell introduces changes in nucleotide sequence and adds nucleotides at random at the coding joints, thus introducing additional diversity into the variable regions. 3. the initial cleavage of the DNA within the RAG complex generates single stranded structures known as DNA hairpins at the ends of the cleaved gene segments. DNA repair enzymes then open the hairpins. the knick that opens the hairpin can occur at any of serval positions, which is one source of diversity. it generates a single stranded end in which bases that were complementary in the two DNA strands are now on the same strand. this creates a new nucleotide sequence that could be a palindrome in double stranded DNA is a sequence of base pairs that is identical when read from either end. this addition of nucleotides is called P nucleotides. the ends of the opened hair pins can then be variable modified by exonuclease that remove gremlin encoded nucleotides and by the enzyme terminal deoxynucleotidyl transferase TdT, which randomly adds nucleotides generating further diversity. these new nucleotides are called N nucleotides bc they are not encoded in gremlin DNA. once the single stranded tails of the two gene segments are able to pair, the single strand gaps are filled in with complementary nucleotides to complete the coding joint. junctional diversity: the contribution of P and N nucleotides to the coding joints and it increases diversity resulting in CDR3s

what depends on the type of antigen being bound by an antibody?

-the shape of the antigen-binding site: antigens that are small molecules can be bound within deep pockets; linear epitopes from proteins or carbs can be bound within clefts or grooves; and the binding of conformational epitopes of folded proteins takes place over an extended surface area.

Explain why we have diversity of immunoglobulin V regions

1. VDJ recombination/ somatic recombination; light and heavy chains. 2. RAG 1+2, junctional diversity, N+P nucleotide additions 3. somatic hypermutation

Describe the overall organization of immunoglobulin genes in germ-line configuration

3 chromosome locations; the heavy chain locus on chromosome 14, the kappa light chain locus on chromosome 2, the lambda light chain locus on chromosome 22. -leader peptide L, variable region V, joining gene segments J, diversity gene segment D. and C constant region.

explain how antibodies activate complement by the classical pathway; what are the roles of Fab, C1q, Fc?

C1q binds Fc activating complement cascade. IgG molecules bind to antigens on bacterial surfaces, C1q binds to two or more IgG molecules and initiates complement activation. -IgM molecules bind to soluble multivalent antigen, C1q binds to soluble immune complex and initiates complement activation --recruiting the effector cells and molecules to kill the pathogen. -->activation of complement coats the pathogen with C3b, facilitating their uptake and destruction by phagocytes. But IgM is bulky so it goes through isotype switching. -when an antibody more likely IgM or IgG, bind to pathogen surface. - Each IgG molecule has a single binding site for C1q in its Fc region (the C1q binding to Fc activates complement cascade). Need C1q to cross link tow or more IgG molecules bound to antigen on the pathogens surface, and antigen bound IgG molecules must be sufficiently close for C1q to span them. Complement activation by IgG depends more on the amount and density of antibodies bound to the pathogen surface than complement activation by IgM. After the complexes of antigen and IgG have activated C1, the classical pathway proceeds. Once the pathogen is coated with C3b it can be efficiently phagocytksed by a neutrophil or macrophage using its CR1 complement receptors. -->· The antibody Fab first binds to the surface of an antigen or multivalent antigen. C1q complement component then binds to the Fc region of the bound antibodies leading to activation of complement cascade.

identify effector functions associated with each various isotypes

Fab: neutralization: IgM, IgG, IgA Fc: opsonization: IgG, IgA Fc: sensitization of killing by NK cells;ADCC : IgG Fc: sensitization of mast cells: IgG, IgE Fc: activation of complement system: IgM, IgG, IgA

describe antibody mediated opsonization and ADCC; what are roles of Fab, Fc, FcR?

Fc/FcR antibody binds to antigen on the surface of target cell, Fc receptors on NK cells recognize bound antibody, cross-linking of Fc receptors signals the NK cell to kill the target cell, target cell dies by apoptosis. --recruiting the effector cells and molecules to kill the pathogen. -intracellular infections like viruses or if the pathogen is too large to phagocytize the pathogen. The antibody binds to these antigen and the Fc region of the antibody is recognized by leukocytes like NK cells and eosinophils. these cells have specific Fc receptors on their surface, so they bind the antibodies by the Fc receptors. then degranulation; the cytotoxic granules are released. this disrupts the cell membrane of the pathogen and lysis of the cell occurs. -->· Interaction with Fc receptors with Fc domains

For the effector function for antibody what does opsonization function work?

Fc/FcR antibody binds to bacterium, antibody coated bacterium binds to Fc receptors on the cell surface, macrophage membrane surrounds bacterium, macrophage membrane fuse, creating a membrane-bounded vesicle, the phagosome, lysosomes fuse with phagosome creating the phagolysosome. --recruiting the effector cells and molecules to kill the pathogen. -the antibody coats the pathogen letting the macrophage know its a pathogen and to kill it. -The Fc receptors on the macrophage bind to the Fc region of the antibody and the antigen undergoes phagocytosis. -->Phagocytes including neutrophils and macrophages have Fc receptors for IgG. When a phagocyte binds to Fc portions of antibodies that have coated the surface of a pathogen, the uptake of the pathogen is enhanced.

understand the changes that occur in Ig genes during the life span of a B cell.

In the bone marrow you have naive B cells: there is VDJ recombination, junctional diversity, transcription happens, expression of IgM, IgD. in the secondary lyphoid tissue the B cell is activated by a T helper cell or a dendritic cell: somatic hypermutation, isotype switching, somatic hypermutation and affinity maturation

define immunoglobulin

Ig the general name for antibodies and B cell antigen receptors

what is immunoglobulin domain?

Ig domain protein structure module with 100 aa that fold into a sandwich of 2 beta sheets held together by a disulfide bond.

distinguish between the 5 antibody isotypes

IgG= most abundant in serum; 4 sub types, major part of secondary response IgM=first Ab formed in primary response; pentamer IgA=present in external secretions, dimer IgE=sensitizes mast cells and basophils leading to symptoms of allergy; helminth protection IgD=antibody activity rarely demonstrated; expressed on naive B cell surface

describe in detail, the processing and presentation pathways for exogenous protein antigens

Professor: · The endocytosed material is degraded following fusion with a lysosome, forming a phagolysosome. Meanwhile, MHC II molecules are synthesized by the cellular machinery through the standard route used for expression of cell surface proteins, which includes passage through the ER and golgi apparatus. While in the ER MHC II proteins are complexed with a protein called the invariant chain, which serves to block the peptide antigen binding site from interacting with other proteins present in the ER. The invariant chain provides a mechanism for preventing MHC I class appropriate peptides from binding to MHC II. Vesicles containing the MHC class II and invariant chain are transported through the golgi apparatus, and the invariant chain is cleaved, leaving behind a fragment called CLIP that remains bound to the peptide binding site. Eventually, the vesicle will fuse with the phagolysosome. Here the CLIP is exchanged for a peptide derived from the exogenous antigen. Now the whole complex is able to move to the cell surface, and display the MHC class II bound to peptide to CD 4 Th cells. MHC II will not go to the cell surface without a peptide loaded in the antigen binding site, ad in the absence of infections, MHC II are usually loaded with self derived peptides. · Have rough ER in an APC cell, lysosomes (containing acids), golgi · Pathogen that was phagocytized by the APC like a macrophage, the macrophage will destroy the bacteria by allowing the fusion of both the lysosome and the phagosome and this will form the phagolysosome which has an acidic environment and bc of the acidic environment this will cause the content/ the pathogen to break down leaving pathogen peptides/antigens. · Now the ribosomes are synthesizing the MHC class II molecules. The MHC is made up of alpha 1,2 domains and beta 1,2. As soon as the MHC is synthesized it has another protein interacting with it. This prevents ER proteins to bind to the groove of the MHC> . The complex exits the ER into the golgi and forms a new endosome which is acidic. This environment degrades the protein attached to the MHC leaving a fragment called CLIP on the binding site of the MHC class II, thus allowing no peptides or antigens to bind to the binding site of the MHC class II. Next another protein called h a DM will interact with MHC class 2 releasing CLIP from the binding groove. The phagolysosomes containing antigen peptides will fuse with the MCH class 2 endosome this allows the exogenous antigen bind onto MHC class 2. Now the whole complex is able to move to the cell surface and display the MHC class 2 bound to peptide to CD 4 Th cells. · The CD 4 receptor on the Th cell will check the MHC molecule to make sure it's an MHC class 2 molecule. It will bind to the alpa portion of the MHC class 2. Then the TCR will check that it can bind to the antigen presented. Then a single transduction occurs which will activate the CD4 Th cell. Now the Th can do things like activate a B cell.

understand the process of isotype switching, somatic hypermutation and affinity maturation.

The two antibodies that are expressed on a naive B cell is IgM and IgD when the B cell is matured. · the 'activated' B cell undergoes two other important changes; somatic hypermutation and isotype switching. -AID will introduce mutations on the variable region of the DNA, this hypermutation will change the antibody increasing its affinity or decreasing its affinity for an antigen, the B cells proliferate into many different mature B cells with increased affinity or decreased affinity. If a B cell had a disadvantage mutation and can't make contact with dendritic cell or T helper cell (with the antigen present to B cell) will go through apoptosis. IF the B cell has an improved affinity due to the mutation, it recognizes the antigen on the T helper cell and or the dendritic cell. the body would want to produce more of this cell. This cell will go through isotype switching and differentiation where it will become either a plasma cell or memory cell with a different antibody class. -When the B cell is 'activated' by a T helper cell or dendritic cell, the B cell will go through a single point mutation by AID in the variable region, causes a point mutation in the gene of a mature B cell. The B cell will proliferate into many different B cells and these B cells will have an increased or decreased affinity to the antigen that was presented on the T helper cell. · During isotype switching, there is another DNA rearrangement that takes place, but this time between the constant regions resulting in exchange of this CMu coding for one of the constant regions for a different isotype. This recombination also has a hairpin loop, · An enzyme called activation induced cytidine deaminase AID is involved in both somatic hypermutation and isotype switching In switching, the AID targets so called switch sites that are located adjacent to the different isotype constant region coding sequences. -somatic hypermutation targets the rearrangement gene segments encoding the variable regions. in the isotype switching it targets the constant region. Somatic hypermutation and affinity maturation: · The variable region of the rearranged immunoglobulin DNA is targeted for excessive point mutations that can alter the affinity of the antibody for its antigen. · Affinity maturation: when there is a higher affinity for an antigen and that is chosen for proliferation and differentiation

Explain IgD

antibody activity rarely demonstrated; expressed on naive B cell surface

Explain how P and N additions contribute to junctional diversity and relationship to hyper variable regions.

To increase the specificity/diversity of the light chain new nucleotides are randomly added during somatic recombination. After the RAG complex cleaves off the RSS from V and J, TdT protein comes in and adds new nucleotides. When the RAG complex cleaves RSS other proteins come and bind the segments to create a hairpin loop, this is one are that new nucleotides can be added in called P additions. Then TdT comes in and adds new nucleotides randomly called N additions and then DNA ligase and other proteins will ligate the ends tougher. The P+N additions is when cytosine is replaced with uracil and then uracil is removed and replaced by a new nucleotide. this creates junctional diversity because of the new nucleotides that are randomly added in during the end of the chromosome and when the hairpin loop opens up. This creates diversity in the hyper variable region. there are 10^7 total potential diversity in humans.

understand how antibody can sensitize mast cells and the consequences of this sensitization; what are the roles of Fab, Fc, FcR?

Type 1 hypersensitivity-allergy Fc/FcR resting mast cell has preformed granules containing histamine and other inflammatory mediators. -multivalent antigen cross links IgE antibody bound at the mast cell surface causing release of granule contents. -the Fc region of IgE is bound by an FcR which is carried by mast cells, basophils, and activated eosinophils. FcR binds only to IgE antibodies, and the binding is so strong that IgE cannot dissociate one it has bound. IgE doesn't function by binding to antigens as a soluble antibody but is used as a cell surface receptor for antigen. The IgE that are secreted by plasma cells become quickly attached to the surfaces of mast cells waiting to bind pathogens and their antigens. When a pathogen binds to IgE on a mast cell and cross links tow or more FcR molecules in the mast cell surface it activates the cell to secrete active mediators that act on smooth muscle to cause violent reactions like sneezing to forcibly eject pathogens. the cytoplasm of the rising mast cell is filled with large granules containing histamine and other molecules that contribute to inflammation. mast cells become activated to release their granules when antigen binds to the IgE (Fc region). molecules bound to FcR on the mast cell surface. to activate the cell, the antigen must cross link at lease two IgE molecules (multivalent antigen) and their associated receptors. cross linking of FcR generates the signal that initiates the release of the mast cell granules. - causes inflammation: inflammatory mediators cause increase permeability of local blood vessels, enabling other cells/molecules of immune system to move out of the bloodstream and into tissues.

what is a plasma cell?

a fully differentiated B cell that produces a single type of antibody.

what is a monomer?

a molecule that can be bonded to other identical molecules to form a polymer

define antigen

a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies

What is a multivalent antigen?

an antigen that contains more than one epitope or more than one copy of the same epitope

what is a protease?

an enzyme which breaks down proteins and peptides.

what bonds do antigens bind to antibodies and how does this correlate to antigen and antibody binding?

based solely on noncovalent forces. several different antibodies may recognize the same epitope, but the small differences in the shapes and chemical properties of the binding sites give these antibodies different binding strengths or affinities for the epitope. creates -different affinities for an antigen.

how do do we get different chains of the heavy chain locus?

both u and s chains from the same heavy chain locus is accomplished by differential splicing of the same primary RNA transcript a process that involves no rearrangement of genomic DNA

describe the general procedures for creating chimeric, humanized, and fully humanized MAbs

chimeric: 70% humanized. · Created using mouse monoclonal hybridomas. · The mRNA from the hybridomas is removed and modified by recombinant DNA tech to replace most of the antibody protein coding info with the human equivalent. For the chimeric antibodies, the entire mouse v regions are maintained humanized: 90% humanized. · For humanized antibodies, only the murine hypervariable regions, CDR 1,2,3 are maintained and all other sequences are swapped for the human sequences. · Recall that the hyper variable regions are where the specific interactions with the epitope occur. fully humanized transgenic mice: One strategy for creating fully humanized monoclonal antibodies require the creation of knock out transgenic mice in which the murine immunoglobulin locus was removed and then replaced with the human non rearranged immunoglobulin genes. Can reaarange and assemble the human antibody genes and express these antibodies as normal fully humanized phage: · Another technique for full humnanized monoclonal antibodies doesn't use mice at all. A DNA library of human immunoglobulin heavy and light chains variable regions is transferred into bacteriophage where the Fab fragments are expressed on the surface of the phage coats.

what is Fc?

constant region when an antibody (after it becomes a plasma cell) binds to an epitope or antigen and tags the antigen for a phagocyte cell to come and engulf it. the constant region tells the phagocyte cell to phagocytosis the cell its attached to.

explain what occurs during VDJ recombination and the role of RAG 1+2

during VDJ recombination: somatic recombination: -have the germline DNA: have different chromosomes for the heavy and light chains. There are many V, D, J segments. The DNA has introns and exons. The first is making the heavy chain. the process that occurs is DJ recombination is where J will bind to the D region to bring C to close proximity. Next is VDJ recombination which brings the constant region in close proximity in the RNA. Then there is splicing to remove the introns in the RNA to become mRNA to make protein. This also happens for the light chain gene but it doesn't have the D diversity segment. There are two classes of the light chain; lambda and kappa light chains. RAG: for example if you have the kappa light chain gene you have the variable and joining segments. These many V and J segments have motifs called the RSSs. they can be a 23 bp spacer or 12bp spacer. The J usually have the 23 bp and the V segments have the 12bp spacers. The RSSs have a special rule called 12/23 rule, only a 12/23 can combine together. In order for recombination to occur, there are two ways for recombination to occur. One is for the gene to create a hairpin loop where a V and J are parallel to each other. now the V and J can go through recombination, then protein will cut off the base pair sequences to be left with VJ segments bound together. To increase the specificity/diversity of the light chain new nucleotides are added. -in recombination RAG 1+2 proteins will bind to the RSSs to a 12bp spacer and a 23 bp spacer. this will cause the RAG 1 and RAG 2 to bind together bc they have an affinity for each other. when they bind together they will form a hairpin loop with V and J parallel to each other. The RAG 1+2 will cleave off RSSs. Other proteins will bind to the segments creating a hairpin loop where RAG has broken off the RSS. after creating the loop. Then TdT comes in and adds new nucleotides randomly. Then DNA ligase and another protein will ligate the ends together which will create a new and unique repaired VJ recombined segment.

what is FcR?

fc receptor cell surface receptor for the Fc portion of immunoglobulin isotopes. there are different Fc receptors for the different isotopes and subtypes. the FcR binds to the Fc part of the antibody.

what is isotope switching?

first start with IgM, secondary response IgG. involves AID · In the lymphoid tissues the 'activated' B cell undergoes two other important changes; somatic hypermutation and isotype switching. · During isotype switching, there is another DNA rearrangemnnt that takes place, but this time between the constant regions resulting in exchange o fthis CMu coding for one of the constant regions for a different isotype. · An enzyme called activation induced cytidine deaminase AID is invllved in both somatic hypermutation and isotype switching · In switching, the AID targets so called switch sites that are located adjacent to the different isotype constant region coding sequences.

why is IgA considered passive immunity in breast milk?

no memory B cells passed down from mother through breast milk

when does isotope switching occur?

only occurs during an active immune response, when the B cell is activated, and the patterns of isotope switching are regulated by cytokines secreted by antigen activated T cells.

the antigen binding site of an immunoglobulin is formed from?

paired V regions of a single heavy chain and a single light chain

Distinguish between polyclonal vs. monoclonal antibodies.

polyclonal: pathogens with multiple antibodies, activation of multiple clones. mixture of heterogeneous which are usually produced by different B cell clones. Recognize and bind to many different epitopes of a single antigen. monoclonal: MAb, antibody by cloning a specific B cell. only binds to the same epitope.

Explain RSSs

recombination of V,J,D gene segments is directed by recombination signal sequences RSSs. There are 2 types of RSS; RSS with 12bp spacer and RSSs with 23 bp spacer. provides a recognition sites for the enzymes that cut and rejoin the DNA, RSSs ensure that the gene segments are joined in the correct order. 12/23 rule, recombination in the heavy chain DNA can't join a VH directly to a JH without DH bc VH and JH are flanked by the same type of RSS.

Explain IgE

sensitizes mast cells and basophils leading to symptoms of allergy; helminth protection parasites

what is a covalent bond?

sharing of electrons

what is the difference between membrane bound and secreted immunoglobulin?

the difference lies at the carboxyl terminus of the heavy chain; here membrane associated immunoglobulin has a hydrophobic anchor sequence that is inserted into the membrane, whereas antibody has a hydrophilic sequence. due to differential RNA splicing and processing of the same primary RNA transcript. this makes sense because antibodies that are membrane bound need to be hydrophobic and the free antibodies hydrophilic because they are not bound to a any membrane.

why is immunoglobulin domain so important?

the structure of the immunoglobulin domain provides the capacity for localized variability within a structurally constant framework. (how it folds up)

what determines the affinity of an antigen binding to an antibody?

the sum strength of the non covalent bonds that determine the affinity that an antibody possesses for a particular antigen

for each of the experimental techniques for antibody testing, explain what kinds of experimental information can be obtained

western blot: the western blot procedure is an essential tool for protein analysis of complex systems, and the identification of potential mechanisms underlying aberrant tissue function or disease. ELISA: · Specifically detecting antigens or antibodies · Widely used in research labs around the world and used in clinical labs for diagnosing diseases. immunoaffinity chromatography: · Purification of a particular macromolecule. · This technique is commonly used in research for large scale purifications and in the biotech industry it is used for purifying biological molecules that may be used as therapeutic agents. immunofluorescence microscopy: detection of cellular structures; · This technique has been useful in the field of cellular biology for identifying and studying the distribution and functions of cellular structures. FACS: · Useful in identification of different types of leukocytes based on differential expression of CD molecules. analysis of Ag expression in cellular populations.

describe the following experimental techniques: western blotting, Elisa, Immunoaffinity chromatography, immunofluorescence microscopy, and FACS

western blot: · Used for qualitative, semi quantitative id of a particular protein from within a mixture of proteins. · First, separation by electrophoresis through a polyacrylamide denaturing gel, SDS-PAGE. · Second, proteins are then transferred in place on a filter paper that is exposed to the specific antibody that is allowed to bind specifically to the protein of interest. · The excess antibody is washed off and the bound antibody is revealed by mixing with a second antibody that is specific for the Fc region of the primary antibody. · The second antibody has been conjugated to an enzyme such as alkaline phosphatase or beta galactosidase that can react with the substrate to form a color and thus reveal the location of the protein antigen. Elisa: · Specifically detecting antigens or antibodies · Technique involves of 96 well microtiter plates. Each well is coded with a protein sample and then a primary antibody is added to each well. · If the specific antigen is present in the well the antibody will bind. Excess antibody is removed and then a secondary antibody specific for the primary antibody The secondary antibody has been conjugated to an enzyme that will create a color when the substrate is added. In this case the amount of color can be measured by using a spectrophotometer Immunoaffinity chromatography:· Purification of a particular macromolecule · The antibody specific for the substance of interest is conjugated through chromatography beads that are loaded into a column. · A mixture of macromolecules such as mixtures derived from cellular lysates is passed over the column. The molecule of interest will bind to the antibody while all other substances pass through. · The bound molecule can subsequently be eluted from the column by altering the salt or pH of the washing buffer that is passed over the column. immunofluorescence microscopy: Use of antibodies that are conjugated to a fluorescent molecule that gives off a distinct color that can be detected in a fluorescent microscope. --florescence labeled Ab, detection of cellular structures FACS: · Involves the use of antibodies that are conjugated to a fluorescent molecule. · Enables the cells to be physically separated and counted from those who are not labeled with antibody. · A mixture of cells is allowed to combine with the labeled antibody and then passed through a flow cytometer nozzle that allows the passage of only one cell at a time. · A laser activates the fluorescent antibody and that is then detected and recorded on a computer.--analysis of Ag expression in cellular populations. semi quantitive, separation of different cellular populations.

when you talk about a B cell differentiating what does this mean?

when a B cell goes through clonal selection it differentiates into the B cells with that antigen receptor (that are effector cells) and also into memory B cells. the effector cells actually do something they turn into plasma cells- antibody factories- they start producing the antibodies that can uniquely bind to the pathogen

describe the general procedure for creating a mouse derived monoclonal antibodies.

· 1970 nobel prize: hybridomas that are created by physicaly fusing normal B cells with cancerous B cells called myelomas. · Cyeloma cells can grow indefinitely in culture so this can be used to enable B cells to be grown in the lab. · To make a monoclonal antibody: 1. Immunization of mice with the antigen of interest. Once the mice have mounted an immune response B cells are removed and fused with myeloma cells. The hybridomas must then be analyzed by flow cytometry and ELISA in an effort to id the hybridomas that are secreting antibodies that actually bind to the antigen.

Distinguish between the alpha/beta and gamma/delta T cells

· Similar structures · Completely different gene sets · Gamm delta (yS) o Only ~1-5% of total o Not MHC restricted · Alpha beta (aB) o Major class o MHC restricted · TCRs are heterodimers composed of one alpha chain and one beta chain or one gamma chain and one delta chain. · Most T cells express alpha beta type T cell receptors · Gamma delta plan a more limited functional role and have different activation requirements. · Gamma delta T cells: they don't require presentation by classical MHC molecules and antigen presenting cells.

Explain the general function of the CD3 complex

· TCR polypeptides have short cytoplasmic tails and are always accompanied by a group of proteins called the CD3 complex that mediates signal transduction events when the T cell is stimulated as a result of antigen recognition.

compare and contrast the structures of immunoglobulin and TCR proteins.

· The TCR is similar to a single Fab of an immunoglobulin. · Each TCR chain has an amino terminal variable region and carboxyl terminal constant region. · Antibodies are capable of binding to free and accessible antigen, the TCR can only bind to antigen formally presented on a surface of an antigen presenting cell. APC: macrophages, dendritic cells, B cells. · Antibodies have two identical binding sites, TCRs have only one. · TCRs are restricted to recognition of protein fragments, peptide antigens that are presented in an MHC molecule on the surface of an APC.

describe in detail, the processing and presentation pathways for endogenous pathway

· Any endogenous cell that can process endogenous antigens o Have a nucleated cell: rough ER, golgi, proteasome, ribosome o A pathogen enters the cell- the pathogen takes over cell machinery, infects the cell makes its own protein in host cell. Endogenous proteins of the pathogen. o The protein products enter the proteasome, a complex that will degrade these cytosolic proteins and they will come out as small fragments ~15 aa in length. o Then these small fragments can enter the ER through the TAP 1+2 transporter and when these 15 aa fragments these antigen peptides enter the rough ER they can get further broken down to 8-10 aa by specific enzymes within the ER o The ribosome are machines that make proteins so its making proteins an MHC I protein however its incomplete because its only composed of alpha 1,2,3 domains, this incomplete MHC 1 is bound to a protein called calnexin now calnexins function is as a chaperone bring the MHC 1 through the ER to prepare it. o Following this, another protein which is a macroglobulin beta 2m will actually to this incomplete MHC I which will essentially complete it so now we have a complete MHC I made up of alpha 1, 2, 3 and beta 2 domains. At the same time there will be other proteins attaching on this MHC I. these proteins will cover the binding site of the MHC I which will prevent peptides/antigens to interact with the binding site of MHC I. These proteins will also act as a chaperone it will bring the MHC I molecule towards the TAP transporter while the MHC I will interact with another protein called tapassin, when it interacts with tapassin the other proteins previously will disassociate which will allow these small pathogen peptide fragments to bind to the binding site of the MHC I. the MHC I binding site/groove is very unique and only fits to a specific antigen and this is bc it contains a variable region, alpha 1, 2 is unique bc it's a variable region and binds to specific antigen. o Then the MHC I with the antigen peptide can leave the ER in an endosome and go through the golgi leave the golgi in the endosome the MHC I together with the peptide will be presented on the cell surface. Now it will wait for interaction with a CD 8 Tc cell. o The T cell has a CD 8 receptor and it will interact with the MHC I molecule. The infected cell will present the antigen peptide to a CD 8 Tc cell then the CD 8 receptor will check if it's a MHC class I being expressed. Then the Tc cell has the TCR role is to check the antigen bcs the TCR only fits to a specific antigen. If all this is ok, then the Tc can release chemicals cytokines that will the infected cell. Professor: · For normal physiology: cellular proteins are regularly broken down and degraded by a barrel shaped protein complex proteolytic enzymes called proteasome, peptide fragments generated by passage through a proteasome are actively transported by a complex of transport proteins Tap 1 and Tap 2 into the ER. Once in the ER the peptide can be loaded onto MHC I molecules that are being assembled and stabilized by a committee of proteins. The peptide loaded class I molecules are released in vesicles, which are eventually transported to the cell surface for presentation. The MHC I will not exit the ER without a peptide. This process occurs continuously, even in the absence of an intracellular infection, most MHC class I molecules display normal self peptides.

describe the germ line organization of alpha and beta T cells.

· Beta chain has the VDJ: similar to heavy chains · The alpha regions is similar to light chains · T cell development occurs in the thymus ·

draw the interaction between TCR/co-receptors with AG/MHC indicating the regions where TCR, CD4 and CD8 bind to either MHC class II or class I molecules

· CD4 and 8 are co receptors because they function alongside the TRC in bind to MHC molecules on the surface of the antigen presenting cell · CD 8 co receptor binds to the alpha 3 domain of MHC class 1, which is not involved in antigen binding and that the CD 4 co receptor binds to the beta 2 of the MHC II which is also not involved in antigen binding. · The TCR binds to both the antigen and the MHC thus must be able to recognize a specific antigen within the context of the appropriate MHC molecule Most nucleated cells of the body express MHC I molecules. MHC II expression only occurs on professional antigen presenting cells like dendritic cells, macrophages, B cells

understand the binding of peptide antigens to MHC molecules

· CD4 and 8 are co receptors because they function alongside the TRC in bind to MHC molecules on the surface of the antigen presenting cell · CD 8 co receptor binds to the alpha 3 domain of MHC class 1, which is not involved in antigen binding and that the CD 4 co receptor binds to the beta 2 of the MHC II which is also not involved in antigen binding. · The TCR binds to both the antigen and the MHC thus must be able to recognize a specific antigen within the context of the appropriate MHC molecule Most nucleated cells of the body express MHC I molecules. MHC II expression only occurs on professional antigen presenting cells like dendritic cells, macrophages, B cells

describe the general functional differences of CD8 T cells and CD4 T cells

· CTL (Tc)= CD8 o Binds to pathogen inside the cell and then presenting it out. o MHC is on infected cell o CD8 receptor—checks if MHC (on infected cell) expresses MHC I, bc CD8 only interacts with MHC I. o The TCR—checks for specific antigen o If everything matches the cytotoxic T cell releases cytokines—kill infected cell: releases molecules like perforins—creates holes in membrane in infected cell. o The cytotoxic T cell has many CD8 receptors and TCR receptors on its surface—same class of receptors. Very specific. o Once Tc binds to MHC I its going to replicate into memory cells and effector cells. Effector=actually does something. o Infected cells can act as their own antigen presenting cell. · Th: MHC II—CD4 o Only by antigen presenting cell—APC § Dendritic cells, B cells and macrophages o MHC II will have an antigen presented to a naïve CD4 cell (Th cell) o If CD4 receptor checks MHC II and TCR checks antigen and everything is good—signal transduction cascade—activation of naïve CD4 Th cell to become a T helper cell which promotes immune response by activating B cells or other cells. o Presenting an antigen that initially found outside of the cells, engulfing and then taken out as MHC/antigen receptor. o Professional antigen presenting cell

summarize the physiological significance of the endocytic and cytosolic processing pathways in respect to the functional roles of CTLS and Th cells.

· Compartmentalization of two distinct pathways; induce appropriate effector functions to antigens of different origins · Endogenous Ags presented to CTLs: o Elimination of infected cells or diseased self o 'red flag' indicates state of inner health or disease · Exogenous Ags presented to Th cells: o Infectious agents in body's tissue o Stimulate B cells to make Abs o Activate phagocytes

describe the structures and expression profiles of MHC class I and MHC class II molecules

· In humans there are 3 kinds of MHC class 1 molecules that present antigen to CD 8 T cells: HLA-A, HLA-B and HLA-C o These class 1 proteins are co dominantly expressed which means that any given MHC class I expressing cell will display all 3 types on its surface. · 3 kinds of MHC II that function in antigen presentation to CD 4 T cells: HLA-DQ, HLA-DP, HLA-DR. are co dominantly expressed by are usually expressed only the surface of professional antigen presenting cells. · Thus human APCs express all six types of HLA surface proteins;

explain the significance of MHC polymorphism to pathogen recognition and transplantation

· Polymprphism: this means that there is a great genetic variability in the MHC genes from person to person. · Somewhat like a genetic fingerprint that distinguishes one person from another. · The polymorphisms are particularly concentrated in the regions of peptide bonding · Provides advantages to both the individual and to the population at large, problematic for transplantation. · For individual having a variance (alleles) increases the odds of presenting relevant pathogen derived peptides for T cell activation. · Good for members of a particular population as diversity of MHC presentation at the individual level decreases the odds of pathogen survival and transmission to others within the same geographical area.

explain the limitation s of suing murine MAbs as therapeutic agents

· The hope was for monoclonal antibodies to be the answer that could selectively be used for treating cancer and other cell based diseases. While these mouse derived antibodies have been valuable tools for experimental biology, they fell short in their utility as therapeutic agents. Since the human immune system reacts against these mouse antibodies by creating human anti-mouse antibodies HAMA, the strategy didn't initially work. · Now there are humanized monoclonal antibodies that are clinically approved for treating a variety of human diseases including breast cancer, rheumatoid arthritis,