Chapter 18 Somatic Recombination and Hypermutation in the Immune System

1. Chapter 18Somatic Recombination and Hypermutation in the Immune System

2. 18.1 The Immune System: Innate and Adaptive Immunity • antigen – A molecule that can bind specifically to an antigen receptor, such as an antibody. • B cell – A lymphocyte that produces antibodies. Development occurs primarily in bone marrow. • B cells emerging from the marrow undergo further differentiation in the bloodstream and peripheral lymphoid organs.

3. 18.1 The Immune System: Innate and Adaptive Immunity • T cells – Lymphocytes of the T (thymic) lineage. T cells differentiate in the thymus from stem cells of bone marrow origin. • They are grouped in several functional types (subsets) according to their phenotype, mainly expression of surface proteins CD4 and CD8. • Different T cell subsets are involved in different cell-mediated immune responses.

4. 18.1 The Immune System: Innate and Adaptive Immunity • adaptive (acquired) immunity – The response mediated by lymphocytes that are activated by their specific interaction with antigen. • The response develops over several days as lymphocytes with antigen-specific receptors are stimulated to proliferate and become effector cells. • It is responsible for immunological memory.

5. 18.1 The Immune System: Innate and Adaptive Immunity • B cell receptor (BCR) – The receptor for antigen expressed on the surface of B lymphocytes. • The BCR has the same structure and specificity of the antibody that will be produced by the same B cell after its activation by antigen.

6. 18.1 The Immune System: Innate and Adaptive Immunity • T cell receptor (TCR) – The antigen receptor on T lymphocytes. • It is clonally expressed and binds to a complex of MHC class I or class II protein and antigen-derived peptide. • Antibody response – An immune response that is mediated primarily by antibodies. It is defined as immunity that can be transferred from one organism to another by serum antibody.

7. 18.1 The Immune System: Innate and Adaptive Immunity • innate immunity – A response triggered by receptors whose specificity is predefined for certain common motifs found in bacteria and other infective agents. • The receptor that triggers the pathway is typically a member of the Toll-like receptor (TLR) family, and the pathway resembles the pathway triggered by Toll receptors during embryonic development. • The pathway culminates in activation of transcription factors that cause genes to be expressed whose products inactivate the infective agent, typically by permeabilizing its membrane.

8. 18.2 The Innate Response Utilizes Conserved Recognition Molecules and Signaling Pathways • Innate immunity is triggered by pattern recognition receptors (PRRs) that recognize highly conserved microbe-associated molecular patterns (MAMPs) found in bacteria, viruses, and other infectious agents. • PAMPs – Pathogen-associated molecular patterns, now referred to as MAMPs, are broadly conserved microbial components, including bacterial flagellin and lipopolysaccharides, that are recognized by PRRs, which critically initiate innate immune responses.

9. 18.2 The Innate Response Utilizes Conserved Recognition Molecules and Signaling Pathways Figure 18.01: Innate immunity; a summary of MAMPs and PRRs.

10. 18.2 The Innate Response Utilizes Conserved Recognition Molecules and Signaling Pathways • Toll-like receptors (TLRs) are important PRRs that directly activate innate immune responses and direct the initial stages of adaptive responses. TLRs are expressed in dendritic cells (DCs), macrophages, neutrophils, B lymphocytes, and some T lymphocytes. • TLR signaling pathways are highly conserved from invertebrates to vertebrates and an analogous pathway is found in plants.

11. 18.2 The Innate Response Utilizes Conserved Recognition Molecules and Signaling Pathways • Toll/interleukin 1/resistance (TIR) domain – The key signaling domain unique to the TLR system. • TIR is located in the cytosolic face of each TLR, and also in TLR adaptors. • Similar to the TLRs, the adaptors are conserved across many species. • These adaptors are MyD88, MyD88-adaptor-like (MAL, also known as TIRAP), TIR-domain-containing adaptor protein inducing IFN (TRIF; also known as TICAM1), TRIF-related adaptor molecule (TRAM; also known as TICAM2), and sterile armadillo-motif-containing protein (SARM).

12. 18.3 Adaptive Immunity • Helper T (Th) cells produce signals required by B cells to enable them to differentiate into antibody-producing cells. • complement – A set of ~20 proteins that function through a cascade of proteolytic actions to lyse infected target cells, or to attract macrophages. • cell-mediated response – The immune response that is mediated primarily by T lymphocytes, and defined based on immunity that cannot be transferred from one organism to another by serum antibody.

13. 18.3 Adaptive Immunity Figure 18.04: Free antibodies bind to antigens to form antigen-antibody complexes.

14. 18.3 Adaptive Immunity • Cytotoxic T cells (CTLs) or killer T cells are responsible for the cell-mediated response in which fragments of foreign antigens are displayed on the surface of a cell. • These fragments are recognized by the TCR expressed on the surface of T cells. Figure 18.05: Cell-mediated immunity.

15. 18.3 Adaptive Immunity • In TCR recognition, the antigen must be presented in conjunction with a major histocompatibility complex (MHC) molecule. • autoimmune disease – A pathological condition in which the immune response is directed to self antigen.

16. 18.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen • clonal selection – The theory proposed that each lymphocyte expresses a single antigen receptor specificity and that only those lymphocytes that bind to a given antigen are stimulated to proliferate and to function in eliminating that antigen. • Thus, the antigen "selects" the lymphocytes to be activated. • Clonal selection was originally a theory, but it is now an established principle in immunology.

17. 18.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen Figure 18.06: The B cell and T cell repertoires include BCRs and TCRs with a variety of specificities.

18. 18.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen • Each B cell expresses a unique BCR and each T cell expresses a unique TCR. • A broad repertoire of BCRs/antibodies and TCRs exists at any time in an organism. • Antigen binding to a BCR or TCR triggers the clonal proliferation of that receptor-bearing B or T cell.

19. 18.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen • hapten – A small molecule that can elicit an immune response only when conjugated with a carrier, such as a large protein. • Once antibodies have been induced by the carrier-conjugated hapten, the hapten will in general bind those antibodies. • epitope – The portion of an antigen that is recognized by the antigen receptor on lymphocytes. • It is also called the antigenic determinant.

20. GENE RECOMBINATION VIDEO http://www.youtube.com/watch?v=AxIMmNByqtM

21. 18.5 Ig Genes are Assembled from Discrete DNA Segments in B Lymphocytes • An antibody (immunoglobulin) consists of a tetramer of two identical light (L) chains and two identical heavy (H) chains. There are two families of L chains (Igλ and Igκ) and a single family of IgH chains. • Each chain has an N-terminal variable (V) region and a C-terminal constant (C) region. The V region recognizes the antigen and the C region mediates the effector response. Figure 18.07: An antibody (immunoglobulin, or Ig) molecule is a heterodimer consisting of two identical heavy chains and two identical light chains.

22. 18.5 Ig Genes are Assembled from Discrete DNA Segments in B Lymphocytes • V and C regions are separately encoded by V(D)J gene segments and C gene segments. • A gene coding for a whole Ig chain is generated by somatic recombination of V(D)J genes (variable, diversity,and joining genes in the H chain; variable and joining genes in the L chain) giving rise to V domains, to be expressed together with a given C gene (C domain).

23. 18.6 L Chains Are Assembled by a Single Recombination Event • A λ chain is assembled through a single recombination event involving a Vλ gene segment and a Jλ-Cλ gene segment. • The Vλ gene segment has a leader exon, an intron, and a Vλ-coding region. The Jλ-Cλ gene segment has a short Jλ-coding exon, an intron, and a Cλ-coding region.

24. 18.6 L Chains Are Assembled by a Single Recombination Event Figure 18.08: The Cl gene segment is preceded by a J segment, so that Vl-Jl recombination generates a productive Vl-JlCl.

25. 18.6 L Chains Are Assembled by a Single Recombination Event • A κ chain is assembled by a single recombination event involving a Vκ gene segment and one of five Jκ segments preceding the Cκ gene. Figure 18.09: The Ck gene segment is preceded by multiple J segments in the germ line.

26. 18.7 H Chains Are Assembled by Two Sequential Recombination Events • The units for H chain recombination are a VH gene, a D segment, and a JH-CH gene segment. • The first recombination joins D to JH-CH. The second recombination joins VH to DJH-CH to yield VH-DJH-CH. • The CH segment consists of four exons.

27. 18.7 H Chains Are Assembled by Two Sequential Recombination Events Figure 18:10: Heavy genes are assembled by sequential recombination events.

28. 18.8 Recombination Generates Extensive Diversity • The human IgH locus can generate in excess of 104 VHDJH sequences. • Imprecision of joining and insertion of unencoded nucleotides further increases VHDJH diversity to 106 sequences. • Recombined VHDJH-CH can be paired with in excess of 104 recombined VκJκ-Cκ or VλJλ-Cλ chains. Figure 18.12: The human and mouse Igk families consist of Vk gene segments and five Jk segments linked to a single Ck gene segment. Figure 18.11: The lambda family consists of Vl gene segments and a small number of Jl-Cl gene segments.

29. 18.8 Recombination Generates Extensive Diversity Figure 18.13: A single gene cluster in humans contains all the information for the IgH chain.

30. 18.9 V(D)J DNA Recombination Uses RSS and Occurs by Deletion or Inversion • The V(D)J recombination machinery uses consensus sequences consisting of a heptamer separated by either 12 or 23 base pairs from a nonamer (recombination signal sequence, RSS). Figure 18.14: RSS sequences are present in inverted orientation at each pair of recombining sites.

31. 18.9 V(D)J DNA Recombination Uses RSS and Occurs by Deletion or Inversion • Recombination occurs by double-strand DNA breaks (DSBs) at the heptamers of two RSSs with different spacers: “12/23 rule.” Figure 18.15: Breakage and recombination at RSSs generate VJC sequences. Adapted from D. B. Roth, Nat. Rev. Immunol. 3 (2003): 656-666.

32. 18.9 V(D)J DNA Recombination Uses RSS and Occurs by Deletion or Inversion • The signal ends of the DNA excised between two DSBs are joined to generate a DNA circle or signal circle. The coding ends are ligated to join VL to JL-CL (L chain), or D to JH-CH and VH to DJH-CH (H chain). If the recombining genes lie in an inverted rather than direct orientation, the intervening DNA is inverted and retained, instead of being excised as a circle.

33. 18.10 Allelic Exclusion Is Triggered by Productive Rearrangements • V(D)J gene rearrangement is productive if it leads to expression of a protein. • A productive V(D)J gene rearrangement prevents any further rearrangement of the same kind from occurring, whereas a nonproductive rearrangement does not. • Allelic exclusion applies separately to L chains (only one VκJκ or VλJλmay be productively rearranged) and to VHDJH chains (one H chain is productively rearranged).

34. 18.10 Allelic Exclusion Is Triggered by Productive Rearrangements Figure 18.16: A successful rearrangement to produce an active light or heavy chain suppresses further rearrangements, resulting in allelic exclusion.

35. 18.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments • The RAG proteins are necessary and sufficient for the cleavage reaction. • RAG1 recognizes the nonamer consensus sequences for recombination. • RAG2 binds to RAG1 and cleaves DNA at the heptamer. • The reaction resembles the topoisomerase-like resolution reaction that occurs in transposition.

36. 18.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments Figure 18.17: Processing of coding ends introduces variability at VkJk, VlJl, or VH-D-JH junctions. Depicted is a Vk-Jk junction.

37. 18.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments • The reaction proceeds through a hairpin intermediate at the coding end; opening of the hairpin is responsible for insertion of extra bases (P nucleotides) in the recombined gene. • Terminal deoxynucleotidyl transferase (TdT) inserts additional unencoded N nucleotides at the V(D)J junctions. • The DSBs at the coding joints are repaired by the same mechanism that has generated the whole V(D)J sequence.

38. 18.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments • severe combined immunodeficiency (SCID) – A syndrome that stems from mutations in different genes that result in B and/or T cell deficiency. • X-linked SCID is due to IL-2R γ chain gene mutations; autosomal recessive SCID can be due to RAG1/RAG2 mutations, Artemis gene mutations, Jak3 gene mutations, ADA gene mutations, IL-7R α-chain mutations, CD3 δ or ε mutations, or CD45 gene mutations.

39. 18.12 B Cell Differentiation: Early IgH Chain Expression Is Modulated by RNA Processing • All B lymphocytes newly emerging from the bone marrow express the membrane-bound monomeric form of IgM (Igμm). • As the B cell matures after exiting the bone marrow, it expresses surface IgD at a high levels. Such IgD consists of Igδm containing the same VHDJH sequence paired with the same recombined κ or λ chain as the IgM expressed by the same cell.

40. 18.12 B Cell Differentiation: Early IgH Chain Expression Is Modulated by RNA Processing • A change in RNA splicing causes μm to be replaced by the secreted form (Igμs) after a mature B cell is activated and begins differentiation to antibody-producing cells in the periphery. Figure 18.19: The 3 end of each CH gene cluster controls the use of splicing junctions so that alternative forms of the heavy gene are expressed.

41. 18.13 Class Switching Is Effected by DNA Recombination (Class Switch DNARecombination, CSR) • Igs comprise five classes according to the type of CH chain. Figure 18.20: Immunoglobulin type and functions are determined by the H chain.

42. 18.13 Class Switching Is Effected by DNA Recombination (Class Switch DNARecombination, CSR) • Class switching is effected by a recombination between S regions that deletes the DNA between the upstream CH region gene cluster and the downstream CH region gene cluster that is the target of recombination. Figure 18.21: Class switching of CH genes.

43. 18.13 Class Switching Is Effected by DNA Recombination (Class Switch DNARecombination, CSR) • CSR relies on a molecular machinery that is different from that of V(D)J recombination and is acting later in B cell differentiation.

44. 18.14 CSR Involves AID and Elements of the NHEJ Pathway • CSR requires activation of intervening promoters (IH promoters) that lie upstream of each of the two S regions involved in the recombination event and germline IH-CH transcription through the respective S regions. • S regions contain highly repetitive 5′-AGCT-3′ motifs. 5′-AGCT-3′ repeats are the main targets of the CSR machinery and DSBs.

45. 18.14 CSR Involves AID and Elements of the NHEJ Pathway Figure 18.22: Class switching passes occurs through sequential and discrete stages.

46. 18.14 CSR Involves AID and Elements of the NHEJ Pathway • activation-induced (cytidine)deaminase (AID) – An enzyme that removes the amino group from the cytidine base in DNA. • AID mediates the first step (deoxycytidine deamination) in the series of events that lead to insertion of DSBs within S regions; the DSBs’ free ends are then religated through an NHEJ-like reaction or A-EJ pathway.

47. 18.15 Somatic Hypermutation (SHM) Generates Additional Diversity • Somatic hypermutation (SHM) introduces somatic mutations in the antigen-binding V(D)J sequence. • Such mutations occur mostly as substitutions of individual bases. Figure 18.24: Somatic mutation occurs in the region surrounding the V segment and extends over the recombined V(D)J segment.

48. 18.15 Somatic Hypermutation (SHM) Generates Additional Diversity • In the IgH chain locus, SHM depends on the iEμ and 3′Eκ that enhance VHDJH-CH transcription. • In the Igκ chain locus, SHM depends on iEκ and 3′Eκ that enhance VκJκ-Cκ transcription. • The λ locus transcription depends on the weaker λ2-4 and λ3-1 enhancers.

49. 18.16 SHM Is Mediated by AID, Ung, Elements of the Mismatch DNA Repair (MMR) Machinery, and Translesion DNA Synthesis (TLS) Polymerases • SHM uses some of the same critical elements of CSR. • Like CSR, SHM requires AID. • Ung intervention influences the pattern of somatic mutations. • Elements of the MMR pathway and of TLS polymerases are involved in SHM and CSR.

50. 18.16 SHM Is Mediated by AID, Ung, Elements of the Mismatch DNA Repair (MMR) Machinery, and Translesion DNA Synthesis (TLS) Polymerases Figure 18.25: Deamination of C by AID gives rise to a U:G mispair.