Antibodies Pt. 2 PDF - BMS 545 Immunology

WELCOME! BMS 545 IMMUNOLOGY OCTOBER 21, 2024   CHECK YOUR CANVAS MESSAGES RE: In Class Activity  Office hours:  Tues. 4-5 pm virtual  Thurs 4-5 pm 316J ANNOUNCEMENTS  I will actually be follow...

WELCOME! BMS 545 IMMUNOLOGY OCTOBER 21, 2024   CHECK YOUR CANVAS MESSAGES RE: In Class Activity  Office hours:  Tues. 4-5 pm virtual  Thurs 4-5 pm 316J ANNOUNCEMENTS  I will actually be following the textbook order of the B cell chapters  Chapter 4 is Antibody Development  Chapter 6 is B Cell Development  Chapter 9 is B Cell & Antibody Mediated Immunity WHY SHOULD YOU CARE? *cocktail means it’s a combo of monoclonal antibodies  OBJECTIVES  Identify the five types of immunoglobulins, their structure, function, role, etc.  Compare & contrast the five types of immunoglobulins  Describe how monoclonal antibodies are created (aka slide 7) & describe the main types  Outline the steps of V(D)J recombination  Define & use common terminology (aka vocab words) from this lecture  Describe the importance of alternative mRNA splicing  Describe how junctional diversity is generated during gene rearrangement  Describe how a functional heavy chain is produced  List which immunoglobulins are co-expressed (and why?)  What is the BCR?  Describe how immunoglobulins become transmembrane or secreted  The structural basis of antibody diversity 4-5 A monoclonal antibody is produced by a clone of antibody- producing cells Figure 4.12 Production of a mouse monoclonal antibody 1. Lymphocytes from a mouse immunized with antigen of choice are fused with myeloma cells (cancerous plasma cells) using polyethylene glycol (PEG) 2. Fused cells are grown in presence of a drug that kills myeloma cells but permits growth of hybrid cells  Unfused lymphocytes also die 3. Cultures of hybrid cells (hybridomas) are tested to determine whether they make the desired antibody 4. Cultures that contain a hybridoma making desired antibody are cloned to produce a homogeneous culture of cells making the monoclonal antibody *Myelomas are tumors of plasma cells; those used to make hybridomas were selected not to express antibody heavy & light chains. Thus, hybridomas only express the antibody made by the B-cell partner Figure 4.13 Flow cytometry enables cells to be distinguished by their cell-surface molecules  The structural basis of antibody diversity 4-6 Monoclonal antibodies are used as treatments for a variety of diseases Figure 4.14 Monoclonal antibodies as treatments for disease  Don’t memorize these drug names but appreciate all the cool mAbs we’ve made for such a range of diseases!  You should know the main types (red box)  OVERVIEW  Epitope specificity of immunoglobulins is determined before antigen encounter  # of possible epitope-binding specificities > several genes within the genome  Paradox: How does immune system generate a diverse array of antigen-specific molecules from a limited number of genes?  Generation of immunoglobulin diversity in B cells before encounter with antigen 4-7 The DNA sequence encoding a V region is assembled from two or three gene segments Figure 4.15 The germline organization of the human immunoglobulin heavy-chain and light-chain loci  Top row: λ light-chain locus- has ~30 functional Vλ gene segments & 4 pairs of functional Jλ & a Cλ gene segment  Center row: κ light-chain locus has ~35 functional Vκ gene segments & 5 Jκ gene segments but with ONE Cκ gene segment  Bottom row: heavy-chain locus has ~40 functional VH gene segments, a cluster of ~23 D segments, & 6 JH gene segments * For simplicity, a single CH gene (CH1–9) is shown in this diagram to represent the 9 C genes. The diagram is not to scale. L, leader sequence.  Generation of immunoglobulin diversity in B cells before encounter with antigen 4-8 Random recombination of gene segments creates diversity in the antigen-binding sites of immunoglobulins Figure 4.16 V-region sequences are constructed from gene segments  Left panel: light-chain V-region genes are constructed from TWO segments  A variable (V) gene segment & a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain V-region (VL) exon  After rearrangement, the light-chain gene consists of three exons, which encode the leader (L) peptide, the V region, & the C region & are separated by introns Figure 4.16 V-region sequences are constructed from gene segments  Right panels: heavy-chain V regions are constructed from three gene segments  First the diversity (D) & J gene segments join, then the V gene segment joins to the combined DJ sequence, forming a complete heavy-chain V-region (VH) exon *For simplicity, only the first of the heavy-chain genes, Cμ, is shown here.  Each immunoglobulin domain is encoded by a separate exon, & two additional membrane-coding exons (MC; light blue) specify hydrophobic sequence that will anchor heavy chain to B-cell membrane. Figure 4.17 The numbers of functional gene segments available to construct the variable and constant regions of human immunoglobulin heavy chains and light chains V(D)J RECOMBINATION  https://digital.wwnorton.com/immunesystem5 Figure 4.18 Each V, D, or J gene segment is flanked by recombination signal sequences (RSSs) Recombination signal sequences (RSSs)- conserved sequences of noncoding DNA that are recognized by RAG1/RAG2 enzyme complex during V(D)J recombination in immature B cells  There are two types of RSS 1. A nonamer (9 bp, shown in purple) & a heptamer (7 bp, shown in orange) separated by a spacer of 12 bp (white) 2. The same 9- and 7-bp sequences separated by a 23-bp spacer (white) Figure 4.19 Rearrangement of V gene segments is required to make immunoglobulin genes functional  The V(D)J recombinase is a Y- shaped structure that consists of 2 RAG-1 & 2 RAG-2 subunits  Each RAG-1 subunit has binding sites for nonamer (N) & heptamer (H) nucleotide motifs that flank V & J gene segments  In V gene segment, nonamer & heptamer are separated by a 12-bp spacer, & in J gene segment they are separated by a 23-bp spacer. 1. 1 RAG-1 subunit binds to nonamer & heptamer of V gene segment, while the other binds to nonamer & heptamer of J gene segment. 2. This creates a scaffold that brings together V & J gene segments that will be cut & then spliced together.  Generation of immunoglobulin diversity in B cells before encounter with antigen 4-9 Recombination enzymes produce additional diversity in the antigen-binding site Figure 4.20 The generation of junctional diversity during gene rearrangement (Part 1)  D to J rearrangement 1. The RSSs are brought together & RAG complex cleaves (arrows) between the heptamer sequences & the gene segments (top panel) 1. This leads to excision of DNA that separates D & J segments 2. Ends of the two strands of DNA double helix in D & J segments are joined to form structures known as ‘hairpins’ 3. Further cleavage (arrows) on one DNA strand of D & J segments opens hairpins & generates short single-stranded sequences at the ends of D & J segments 1. The extra nucleotides are known as P nucleotides because they make a palindromic sequence in the final double-stranded DNA 4. Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the ends of the single strands 1. These nucleotides, which are not encoded in the germline, are known as N nucleotides Figure 4.20 The generation of junctional diversity during gene rearrangement (Part 2) 5. The single strands pair, &through the action of exonuclease, DNA polymerase, & DNA ligase, the double-stranded DNA molecule is repaired to give the coding joint  Generation of immunoglobulin diversity in B cells before encounter with antigen 4-10 In naive B cells alternative mRNA splicing produces IgM and IgD of the same antigen specificity Figure 4.21 Rearrangement of V, D, and J segments produces a functional heavy-chain gene  The assembled VDJ sequence lies some distance from the cluster of C genes  Only functional C genes are shown here  The four different γ genes specify four different subtypes of the γ heavy chain, whereas the two α genes specify two subtypes of the α heavy chain *For simplicity, individual exons in the C genes are not shown **diagram is not to scale Figure 4.22 Coexpression of IgD and IgM is regulated by RNA processing In mature B cells, transcription initiated at VH promoter extends through both Cμ and Cδ genes For simplicity we have not shown all the individual C-gene exons but only those relevant to the production of IgM and IgD The long primary transcript is then processed by cleavage, polyadenylation, & splicing Figure 4.22 Coexpression of IgD and IgM is regulated by RNA processing Cleavage & polyadenylation at μ site (pAμm; the ‘m’ denotes that this site produces membrane-bound IgM) & splicing between Cμ exons yields an mRNA encoding the μ heavy chain (left panel) Cleavage & polyadenylation at δ site (pAδm) & a different pattern of splicing that removes Cμ exons yields mRNA encoding the δ heavy chain (right panel) *AAA designates the poly(A) tail. MC, exons that encode the transmembrane region of the heavy chain.  Generation of immunoglobulin diversity in B cells before encounter with antigen 4-11 Immunoglobulin is first made in a membrane-bound form that is present on the B-cell surface Figure 4.23 Membrane-bound immunoglobulins are associated with two other proteins, Igα and Igβ  Igα & Igβ form a disulfide-linked complex that interacts with the immunoglobulin molecule  Have long cytoplasmic tails that interact with intracellular signaling proteins, & both Igα & Igβ have binding sites for immunoglobulin C region on their extracellular portions.  Complex of immunoglobulin with Igα & Igβ serves as the functional B-cell receptor *Immunoglobulin shown here is IgM, but all isotypes can serve as B-cell receptors *Igα & Igβ are also called CD79a & CD79b Diversification of antibodies after B cells encounter antigen 4-12 Secreted antibodies are produced by an alternative pattern of heavy-chain RNA processing Figure 4.24 The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by alternative RNA processing  Each heavy-chain C gene has two exons (membrane-coding, MC; light blue) & a secretion-coding (SC) sequence (orange) encoding the carboxy terminus of the secreted form  Membrane-coding (MC)- encodes the transmembrane region & cytoplasmic tail of surface form of isotype  Secretion-coding (SC) sequence- encodes the carboxy terminus of the secreted form  Whether a heavy-chain RNA will result in a secreted OR transmembrane immunoglobulin occurs during processing of the initial transcript & depends on alternative RNA processing Each heavy-chain C gene has two potential polyadenylation sites (pAμs & pAμm) *AAA designates the poly(A) tail Figure 4.24 The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by alternative RNA processing Generation of transmembrane form of heavy chain: AAA designates  Transcript is cleaved & polyadenylated at second the poly(A) tail polyadenylation site (pAμm)  Splicing between a site located between fourth Cμ exon & SC sequence & a second site at 5ʹ end of MC exons removes SC sequence & joins MC exons to the fourth Cμ exon. Each heavy-chain C gene has two potential polyadenylation sites (pAμs & pAμm) Figure 4.24 The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by alternative RNA processing Generation of secreted form of heavy chain: AAA designates the poly(A) tail  The primary transcript is cleaved & polyadenylated at the first polyadenylation site (pAμs), eliminating the MC exons & giving rise to the secreted form of the heavy chain Each heavy-chain C gene has two potential polyadenylation sites (pAμs & pAμm) ALSO A SCIENTIST  Interestingly, I could not find much on Z. L Awdeh… it’s possible it’s Dr. Zuheir Audeh who passed away in 2022, but not much of a record is present besides some patents and PubMed hits.  Check out our girl, Brigitte Askonas though!  What is she most known for?  And who is the first author?  What did Awdeh & Askonas discover?