Generation of antibody diversity October 18, Ram Savan

Transcription

1 Generation of antibody diversity October 18, 2016 Ram Savan 441 Lecture #10 Slide 1 of 30

2 Three lectures on antigen receptors Part 1 : Structural features of the BCR and TCR Janeway Chapter 4 Part 2 : Assembly of antigen receptor genes Janeway Chapter 5; through section 5-10 Part 3 (Today): Somatic diversification of BCRs: Class switch recombination, hypermutation Janeway Chapter 5; pp (plus a few different slides) 441 Lecture #10 Slide 2 of 30

3 Recap: Antigen receptor genes In developing B and T cells, antigen receptor genes rearrange to generate random specificities. Two types of combinatorial diversity: 1. V(D)J recombination to make variable domains 2. Junctional diversity: random nucleotides added between gene segments This process requires the RAG proteins It occurs during lymphocyte development, before exposure to antigen 441 Lecture #10 Slide 3 of 30

4 Adding up all the numbers V(D)J recombination + Junctional diversity = Astonishing theoretical diversity of lymphocyte antigen receptors!!! For B cells, though, ten trillion is not enough 441 Lecture #10 Slide 4 of 30

5 Several flavors of antibodies The variable region of the heavy chain can be grafted onto one of ~5 different heavy chain constant regions. Light chains remain the same Ig C H differ by: Number of C domains location and number of intrachain disulfide bonds location and number of oligosaccharides length of hinge capacity to form polymers 441 Lecture #10 Slide 5 of 30

6 Ig isotypes differ in abundance, function 441 Lecture #10 Slide 6 of 30

7 Effector functions of Ig C H domains The C H defines the effector function of the Ab molecule. Ig C H regions recruit the help of other cells and molecules to dispose of bound antigens by: Binding to Fc receptors of innate effector cells that mediate phagocytosis (IgG) or secretion of inflammatory molecules (IgE) Initiating the complement cascade to recruit phagocytes to engulf microbes (IgM, IgG) Mediating active transport of Igs to mucous secretions, tears, milk (IgA) Transfer across placenta to protect developing fetus (IgG) 441 Lecture #10 Slide 7 of 30

8 Mature Naïve B cells express both IgM and IgD RNA processing RNA cleavage, polyadenylation, and splicing - control IgM and IgD expression in B cells Naïve B cells express ~ equal amounts of IgM and IgD on their surface Why? There is no good answer for this. No major function for IgD. 441 Lecture #10 Slide 8 of 30

9 Activation of B cells drives antibody secretion B Cell B Cell B Cell MHC Class II B Cell B Cell T Cell T cell help Plasma Cell A plasma cell can secrete ~3,000 Ab molecules per second 441 Lecture #10 Slide 9 of 30

10 Alternate RNA splicing controls membrane bound versus secreted antibody Yellow membrane coding (MC) exons encode a hydrophobic transmembrane domain not present in the Orange secretion coding (SC) exon. Differential inclusion of these exons controls whether Ig is membrane-bound or glycosylated and secreted. 441 Lecture #10 Slide 10 of 30

11 Alternate RNA splicing controls membrane bound versus secreted antibody The switch to secreted antibody involves the regulated use of an internal polyadenylation site that precedes the exons encoding the transmembrane domain Transmembrane domain coding region is excluded from the mature mrna 441 Lecture #10 Slide 11 of 30

12 So far: RNA splicing controls two key events in Ig constant region The differential usage of IgM and IgD coding regions in mature naïve B cells The switch in an activated B cell from membrane bound Ig to secreted antibody molecules These processes occur at the level of RNA, not DNA The rest of the events that diversify Ig genes occur at the level of DNA 441 Lecture #10 Slide 12 of 30

13 Three ways to modify B cell antigen receptor genes These mechanisms occur ONLY in B cells (not T cells) They are activated after a B cell responds to antigen 441 Lecture #10 Slide 13 of 30

14 Three ways to modify B cell antigen receptor genes somatic hypermutation (SHM) results in single base pair mutations in V genes (can change receptor affinity for Ag) gene conversion introduces sequences from V segment pseudogenes (can create additional Ag specificities). Occurs in chickens; nothing further on this class switch recombination (CSR) is replacement of constant region genes encoding one isotype with another (can modify effector function but not Ag specificity) 441 Lecture #10 Slide 14 of 30

15 SHM and CSR occur via similar mechanisms SHM and CSR both require a specific enzyme: Activation-induced cytidine deaminase (AID) AID expression is turned on in antigen-activated B cells (not T cells) AID-deficient people: hyper-igm syndrome Only IgM in serum, no other isotypes AID expression requires a signal from T cells to B cells, and occurs within specialized regions of secondary lymphoid organs called germinal centers more on this later 441 Lecture #10 Slide 15 of 30

16 AID is a DNA deaminase AID initiates a nucleophilic attack on the pyrimidine ring of cytidine 441 Lecture #10 Slide 16 of 30

17 AID is a DNA deaminase Cytidine is deaminated to uridine Uridine is not present in normal DNA It is mutagenic and there are proteins in all of our cells that specifically remove it from DNA 441 Lecture #10 Slide 17 of 30

18 Refresher: DNA base pairs G:U base pair has two misaligned hydrogen bonds; distorts the DNA C U U G Pyrimidine Purine 441 Lecture #10 Slide 18 of 30

19 Targeting AID enzymatic activity AID is recruited to the transcribed regions of the V genes Transcription bubbles produce transient single-stranded DNA structures AID activity is restricted to these single-stranded regions (not double stranded DNA) Multiple fates of the uracil in DNA 441 Lecture #10 Slide 19 of 30

20 AID-induced mutations I: copying the mutated uracil If the uracil is not removed and the cell s DNA undergoes replication, this causes a transition {the exchange of one pyrimidine for another and one purine for another at a former C:G base pair}. This is because DNA polymerases recognize a U as a T DNA replication C U T and so G A T A 441 Lecture #10 Slide 20 of 30

21 AID-induced mutations II: replacing the uracil The base excision repair enzyme uracil DNA glycosylase (UNG) can remove the uracil base, leaving the sugar-phosphate backbone intact Nontemplated (error-prone) replication can place any base at this site This can cause both transitions and transversions A<>G (pu) T<>C (py) nontemplated replication A<>T,C (pu<>py) G<>C,T (pu<>py) X Y 441 Lecture #10 Slide 21 of 30

22 AID-Generated Mutations in SHM Cytidine Transition & transversion mutations The V region mutation rate in SHM is about 1/1000 base pairs per cell division, or ten million times the normal rate. 441 Lecture #10 Slide 22 of 30

23 Outcomes of SHM AID activity is recruited specifically to V region genes during SHM Three outcomes: 1. Mutation disrupts open reading frame: cell dies 2. Mutation reduces affinity of antibody for its antigen: cell dies 3. Mutation increases affinity for antigen: cell is selected to survive and outcompetes the old antigen receptors. More on this later Result: affinity maturation (100- fold or even more over former Ab) 441 Lecture #10 Slide 23 of 30

24 What Do We NOT Know About SHM?? LOTS - there is still no good in vitro model for SHM. One big question is how SHM is targeted mainly to the V gene segments of Ig genes: Ø expression of other targeting proteins? Ø recruitment of error prone repair polymerases? Ø chromatin accessibility? This targeting is not perfect; off-target AID activity can promote B cell leukemia, lymphoma, and myeloma. 441 Lecture #10 Slide 24 of 30

25 Class Switch Recombination: Switching to IgG, IgE, and IgA requires DNA rearrangement RNA splicing DNA rearrangement, after Ag encounter 441 Lecture #10 Slide 25 of 30

26 Initiation of CSR DNA rearrangements also requires AID CSR requires AID, UNG, and apurinic/ apyrimidinic endonuclease (APE-1) to generate clusters of singlestranded nicks in DNA 441 Lecture #10 Slide 26 of 30

27 Switch regions target AID activity for CSR transcription initiated Switch regions are repetitive stretches of DNA in the intron just upstream of the first exon of each C H gene (except Cd) Cytokine signals determine which switch regions are activated at the level of transcription These cytokines come from CD4 T cells, which have been trained by DCs about the nature of the pathogen more on this later 441 Lecture #10 Slide 27 of 30

28 CSR places a new CH gene segment next to the VDJ Coordinated deamination at donor and acceptor switch regions by AID UNG APE1 results in double-stranded DNA breaks (DSBs) Original VDJ These DSBs are repaired by general DNA repair proteins The intervening DNA is excised and is lost forever. Ie: once a cell switches to IgE, it can never become IgM+ or IgD+ or IgG+ (or IgA1+) again Switch regions are intronic, so ALL CSR events are productive (no frameshifts or mutations) 441 Lecture #10 Slide 28 of 30

29 SHM and CSR are mechanistically related Transition & transversion mutations (X) (This figure is from the older version of the text; simpler) 441 Lecture #10 Slide 29 of 30

30 VDJ, CSR, SHM diversify antigen receptor genes (Pre-Ag) (Pre-Ag) (Post-Ag) Irreversible, all productive (Post-Ag) 441 Lecture #10 Slide 30 of 30