Antibodies Pt. 3 PDF - BMS 545 Immunology - October 23, 2024

WELCOME! BMS 545 IMMUNOLOGY OCTOBER 23, 2024   Office hours:  Tues 4-5 pm virtual  Thurs 4-5 pm 316J ANNOUNCEMENTS  B cell textbook chapters:  Chapter 4: Antibody Development  Chapter 6: B Cell Developme...

WELCOME! BMS 545 IMMUNOLOGY OCTOBER 23, 2024   Office hours:  Tues 4-5 pm virtual  Thurs 4-5 pm 316J ANNOUNCEMENTS  B cell textbook chapters:  Chapter 4: Antibody Development  Chapter 6: B Cell Development  Chapter 9: B Cell & Antibody Mediated Immunity WHY SHOULD YOU CARE?  OBJECTIVES  Define & describe how isotype switching and somatic hypermutation occur & key players involved in each  What are the consequences of isotype switching and somatic hypermutation?  Identify the five types of immunoglobulins, their structure, function, role, & compare & contrast  Define & use the definitions from today’s class as you talk about BCR development  List the chromosomes where you can find the light and heavy chains  Illustrate the flexibility of IgG (aka how does it move & why?)  Provide examples of the immunoglobulin types (and subtypes) & their effector functions (slide 27)  Compare & contrast the 4 subtypes of IgG  List the changes in immunoglobulin genes and whether they are reversible or not Diversification of antibodies after B cells encounter antigen 4-13 Rearranged V-region sequences are further diversified by somatic hypermutation Somatic hypermutation- mutation that occurs at high frequency in the rearranged variable-region DNA segments of immunoglobulin genes in activated B cells, resulting in the production of variant antibodies, some of which have higher affinity for the antigen  SOMATIC HYPERMUTATION  Upon subsequent epitope exposure, B cells may also accumulate small point mutations in DNA encoding their VL or VH regions during the rapid proliferation that follows restimulation  Provides additional variation to “fine-tune” antibody responses to antigens that are frequently/chronically present  Some mutations may increase binding affinity of antibody for its epitope & increased affinity causes those cells to proliferate more rapidly after binding to antigen  Interaction of antibody with a specific epitope become tighter & more effective over time = affinity maturation  *Somatic hypermutation occurs only in B cells, not T cells AFFINITY MATURATION Figure 4.25 Somatic hypermutation is targeted to the rearranged gene segments that encode immunoglobulin V regions  The frequency of mutations at positions in & around the rearranged VJ sequence— which encodes the V region— of an expressed light-chain gene is shown here Figure 4.26 Somatic hypermutation enables selection of B cells making higher-affinity antibodies  B cells were collected 1 & 2 weeks after immunization with same epitope & used to make hybridomas secreting monoclonal antibodies  Amino acid sequences of heavy & light chains of epitope-specific monoclonal antibodies were determined  Each thick horizontal line represents one variant, & red bars represent amino acid positions that differ from prototypic sequence  1week after primary immunization, most B cells make IgM, which shows some new sequence variation in V region  This variation is confined to the CDRs, which form the antigen-binding site  2 weeks after immunization, both IgG- & IgM-producing B cells are present, & their antibodies show increased variation & higher affinity that involves all six CDRs of antigen-binding site Diversification of antibodies after B cells encounter antigen 4-14 Isotype switching produces immunoglobulin with a different constant region but identical antigen specificity Isotype switching- process by which a B cell changes the class of immunoglobulin it makes while preserving antigenic specificity of the immunoglobulin. Involves a somatic recombination process that attaches a different heavy-chain constant region gene to the existing variable region exon (aka “Class switching”) Figure 4.28 Isotype switching involves recombination between specific switch regions (Part 1)  Repetitive DNA sequences are found to the 5ʹ side of each of the heavy-chain C genes, with exception of the δ gene  Immunoglobulin isotype switching occurs by recombination between these switch regions (S), with deletion of intervening DNA  Switch regions are targeted by AID, which leads to nicks being Activation-induced cytidine deaminase (AID)- enzyme that deaminates DNA made in both strands of the at cytosine residues converting them to uracil The activity of AID & consequent repair of damaged DNA are basis of somatic DNA hypermutation & isotype switching in activated B cells Figure 4.28 Isotype switching involves recombination between specific switch regions (Part 2) Continued down from previous slide  These nicks facilitate recombination between the switch regions, which leads to excision of intervening DNA as a nonfunctional circle of DNA & brings the rearranged VDJ sequence into juxtaposition with a different C gene  The first switch a clone of B cells makes is from the μ isotype to another isotype  A switch from μ to the γ1 isotype is shown here  Further switching to other isotypes can take place subsequently ISOTYPE SWITCHING  https://digital.wwnorton.com/immunesystem5 ISOTYPE SWITCH IN MEMORY B CELLS  Antibodies produced by B cells with prolonged or repeated exposure to same epitope may undergo an isotype switch induced by T cell cytokines  Availability of multiple isotypes having the same specificity allows humoral response to initiate various mechanisms (e.g., complement activation) to be directed against the same epitope  Serial reactivation of memory B cells allows the isotype switch to occur during each restimulation  IgM is predominant isotype seen in primary responses, while secondary responses are mostly IgG, with IgA & IgE  IgM → IgG → IgA → IgE Diversification of antibodies after B cells encounter antigen 4-15 Antibodies with different constant regions have different effector functions  GENE CLUSTERS ENCODING BCRS  Gene clusters encoding κ light chains = chromosome 2  Gene clusters encoding λ light chains = chromosome 22  Heavy chain gene cluster = chromosome 14  Potential antigen-binding combinations > 26 MILLION  REFRESHER FROM EARLIER- IMMUNOGLOBULIN BASIC STRUCTURE  Light chains, termed κ (kappa) or λ (lambda) are on chromosomes 2 & 22, respectively  Five types of heavy chains all encoded on chromosome 14  Mu (μ)  Delta (δ)  Gamma (γ)  Epsilon (ε)  Alpha (α)  Isotypes- genetically different forms of light chains (κ & λ) & of heavy chains (μ, δ, γ, ε, & α)  Immunoglobulin class or subclass is determined by the heavy chain isotype Figure 4.5 The structures of the human immunoglobulin classes  Note the differences in length of heavy-chain C regions, locations of the disulfide bonds linking the chains, & presence of a hinge region in IgG, IgA, & IgD, but NOT in IgM & IgE.  Heavy-chain isotype in each antibody is indicated by the Greek letter (where we get the name)  The isotypes also differ in distribution of N-linked carbohydrate groups (turquoise)  All these immunoglobulins occur as monomers in their membrane-bound form  In their soluble, secreted form, IgD, IgE, & IgG are monomers  IgA forms monomers & dimers, & IgM forms pentamers Figure 4.29 The physical properties of the human immunoglobulin isotypes  Molecular mass given for IgM is the pentamer (the form present in serum)  Molecular mass given for IgA is the monomer  Large amounts of IgA are also produced in the form of dimers, which are secreted at mucosal surfaces (i.e. in breast milk) Figure 4.30 Each human immunoglobulin isotype has specialized functions correlated with distinctive properties  Major effector functions of each isotype (+++) are shaded in dark red; lesser functions (++) are shown in dark pink, and minor functions (+) in pale pink  Opsonization refers to the ability of the antibody itself to facilitate phagocytosis  Antibodies that activate complement system indirectly cause opsonization via complement -Properties of IgA1& IgA2 are similar & combined under “IgA” *IgG2 acts as an opsonin in the presence of one genetic variant of its phagocyte Fc receptor, which is found in ~50% of people of European origin. Figure 4.27 IgM is secreted as a pentamer of immunoglobulin monomers  Schematic diagrams of IgM monomer & pentamer  IgM pentamer is held together by a polypeptide called the J chain, for joining chain (not to be confused with a junctional (J) segment)  Monomers are cross-linked by disulfide bonds to each other & to J chain  The lack of a hinge region in IgM monomer makes molecule less flexible than others (e.g., IgG) but this is compensated for by pentamer having five times as many antigen-binding sites as IgG  With pathogen with multiple identical epitopes on surface, IgM can attach with several binding sites simultaneously Figure 4.31 IgA molecules can form dimers  In mucosal lymphoid tissue, IgA is synthesized as a dimer in association with the same J chain that is present in pentameric IgM  In dimeric IgA, the monomers have disulfide bonds to the J chain but not to each other Figure 4.32 IgG is a highly flexible molecule  Most flexible part of IgG molecule = hinge  Allows Fab arms to wave & rotate & accommodate antibody to orientation of epitopes on pathogen surfaces  “Elbow” within the Fab adds more flexibility that allows variable domains to bend with respect to constant domains  Similarly, “wagging” of Fc tail allows IgG molecules that have bound to antigen to accommodate to binding of C1q & other effector molecules *Shown is a molecule of the IgG1 subclass Diversification of antibodies after B cells encounter antigen 4-16 The four subclasses of IgG have different and complementary functions Figure 4.33 Different hinge structures distinguish the four subclasses of IgG  Relative lengths of hinge & # of disulfide bonds in hinge that cross-link the two heavy chains are shown  Not shown are other differences in amino acid sequence, particularly glycine & proline residues, that influence hinge flexibility Figure 4.34 The four subclasses of IgG have different and complementary functions Figure 4.35 IgG4 is present in the circulation in a functionally monovalent form  Like other IgG subclasses, IgG4 is synthesized in a form that has two heavy chains, two light chains, & two identical antigen-binding sites  Unlike other IgGs, molecules of IgG4 can interact in the circulation & exchange one heavy chain & its associated light chain  Because of this property, most IgG4 molecules in circulation have TWO different binding sites for antigen  Thus, they only interact with a pathogen or a protein antigen through one binding site Figure 4.36 Gene rearrangement and the synthesis of cell-surface IgM in B cells Summary slide of heavy & light chain rearrangement & development  Before immunoglobulin light-chain (center) & heavy-chain (right) genes can be expressed, rearrangements of gene segments are needed to produce exons encoding V regions  Once this has been achieved, genes are transcribed to give primary transcripts containing both exons & introns  The latter are spliced out to produce mRNAs that are translated to give κ or λ light chains & μ heavy chains that assemble inside the cell & are expressed as membrane- bound IgM at cell surface  The main stages in the biosynthesis of the heavy & light chains are shown in the panel on the left. Figure 4.37 Changes in the immunoglobulin genes that occur during a B cell’s lifetime Summary slide of things that can happen to immunoglobulin genes and consequences (basic summary of last 3 classes) ALSO A SCIENTIST  Winifred Ashby, PhD (1879-1975)  First researcher to establish the lifespan of red blood cells in humans was longer than 2-3 weeks  Using the principle of agglutination, Ashby was able to determine the lifespan of transfused blood cells was 30+ days – Further research determined the lifespan we now known as 110 days  This process, which she used for her PhD dissertation, was coined the Ashby Method, and was used for years in the treatment of chronic anemia and revolutionized blood transfusions during WWII  Her and her family emigrated from England to Chicago when she was 14, earned her BS at University of Chicago, MS at Washington University of St. Louis, and completed her doctorate in immunology and pathology at the Mayo Foundation