3 B Lymphocyte Biology.pdf

The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi Learning Objectives 1. Compare and contrast the order of events that generates an immunoglobulin heavy chain compared to a light chain. 2. Explain the importance of RAG-1 and RAG-2 proteins in gene segment rearrangement. 3. Differentiat...

The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi Learning Objectives 1. Compare and contrast the order of events that generates an immunoglobulin heavy chain compared to a light chain. 2. Explain the importance of RAG-1 and RAG-2 proteins in gene segment rearrangement. 3. Differentiate between ‘junctional diversity’ and ‘combinatorial diversity’ and explain the roles of terminal deoxynucleotidyl transferase (TdT) and exonuclease. 4. Describe somatic hypermutation and isotype switching, and explain how the processes are accomplished. 5. Discuss the disorder(s) that may arise in an individual that does not possess a functional RAG-1, RAG-2, AID, CD40 or CD40L. 6. Discuss the B cell developmental events that occur before contact with antigen and those that occur after contact with antigen. 1. Germline Diversity V, D and J gene segments are present in multiple copies. This generates what is known as germline diversity. The heavy chain genes are made of V, D and J segments while the light chain genes are made of only V and J segments. 2. Combinatorial Diversity 1 The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi The random recombination of the various VJ and VDJ gene segments results in combinatorial diversity. This recombination exercise is controlled by the enzymes RAG-1 and RAG-2 that occur at the ends of VDJ genes and function to separate, shuffle, and rejoin the VDJ genes. These enzymes are products of recombination- activating genes, RAGs. RAG-1 and RAG-2 appear to work synergistically and their absence results in a failure to generate immunoglobulins, BCRs or TCRs. The number of gene segments in each locus is shown above and it is easy to see how millions and possibly billions of possible combinations may be achieved. 3. Junctional Diversity The formation of the junction between gene segments involves DNA cleavage and the addition or deletion of nucleotides to create a joint. This random addition by the enzyme terminal deoxynuceotidyl transferase TdT, or random deletion by exonuclease, results in a further level of variability or junctional diversity. An estimate of the total theoretical diversity is given in the table below. For example, using an imaginary binding site 1) …GCG.CGA.AAU.A…. translates to →…Ala.Arg.Asn…. but the deletion of A from CGA changes the sequence to 2) …GCG.CGA.AUA….. that translates to →…Ala.Arg.Ile……. and, more usually, the addition of G after CGA changes the sequence to 3) …GCG.CGA.GAA.UA….. that translates to →…Ala.Arg.Glu… 2 The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi 4. Combination Multiplicity Since any heavy chain may associate with any light chain and both chains contribute to the binding specificity, the random assortment of the hundreds of different heavy and light chains creates further variability. It is important to understand that the processes described above occur in the developing B cell in the bone marrow. The cell eventually becomes mature but naïve. That is, the processes occur before the cell encounters antigen. 5. Somatic Hypermutation and Affinity Maturation After the B cell encounters its specific antigen in the peripheral lymphoid tissue, it must undergo a number of further changes to fine tune its response to the antigen and produce antibody with the best fit, the highest affinity and the most appropriate biological functions. First, the B cell must produce a different type of IgM, changing from a cell bound form (BCR) to a soluble, secreted form. Thus the first antibody produced by the B cell progeny, the plasma cell, is IgM. IgM is also called ‘early’ antibody for the same reason. The activated B cell may now be directed to improve the quality of antibody produced through point mutations in the V domain. This process is called somatic hypermutation and it requires signals from T cells and from IL-4, among others. Antigen that is handled independently of T cells, the so-called T-independent antigen, does not require somatic hypermutation in the responding B cell. 3 The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi The process of somatic hypermutation is mediated by the enzyme activation-induced cytidine deaminase (AID) and the synthesis of this enzyme is controlled by IL-4 and CD40 activation signals. AID creates point mutations by converting cytosine to uracil in the antibody variable domain and creating a mismatch that the B cells attempt to repair. Like many mutations, some turn out to be beneficial to the cell and others prove not to be. If the resulting variable domain possesses increased binding affinity, the B cell will be positively selected and the clone will proliferate. Hence, somatic hypermutation generates further antibody diversity and results in affinity maturation, a vastly increased bonding strength between antibody and antigen. This is evidenced by a great increase in the hyper-variability of the CDR domains. 6. Isotype switch The enzyme AID is also involved in the process of isotype switch or class switch, in which the immunoglobulin class is switched from say, IgM, to IgG. 4 The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi Using the illustration above, a switch from IgM (Cµ) to IgG3 (Cγ3) would involve the deletion (looping out) of the entire sequence between the switch for IgM (Sµ) and the switch for IgG3 (Sγ3). To switch from IgM to IgA, for example, the entire sequence from Sµ to Sα would be looped out. An isotype switch involves a change of the C domain only (see below). The variable domain, and the antibody specificity, remains the same. same specificity 5 The Generation of Diversity- Core Concepts Dr Raymond F Adebiyi AID Deficiency (Hyper-IgM Syndrome) In the absence of AID, somatic hypermutation and isotype switching do not occur. The B cell is able to secrete IgM only and this leads to very high levels of serum IgM and low levels of other isotypes. Somatic hypermutation, affinity maturation and isotype switching events occur only with cooperation between B cells and T cells. With T-independent antigens, such as pure polysaccharides or carbohydrates, B cells respond in the absence of T cell help and only low affinity IgM antibody is produced. The activation of a B cell following interaction with a T-dependent antigen requires the delivery of 3 signals. 1. The first signal is derived from the presentation of peptide with MHC, pMHC, to the T cell receptor. 2. The second signal comes from activated CD40 upon binding with CD40L of the T cell. 3. The third signal is delivered from cytokine binding to its receptor on the B cell. Upon receiving all 3 signals, the B cell is able to produce antibody of the appropriate specificity, affinity and class. Highly Recommended VDJ Gene Recombination YouTube video by Daniel Levy (2013) https://www.youtube.com/watch?v=QTOBSFJWogE 6