Antibody Genetics, W2024, PDF

Learning Objectives 1 Indicate the genetic basis of immunologic specificity, diversity, and self-nonself discrimination. 2 Explain how antibody gene organization contributes to antibody diversity and specificity. 3 Identify the roles of isotypic and allelic exclusion in antibody genetics and a...

Learning Objectives 1 Indicate the genetic basis of immunologic specificity, diversity, and self-nonself discrimination. 2 Explain how antibody gene organization contributes to antibody diversity and specificity. 3 Identify the roles of isotypic and allelic exclusion in antibody genetics and antibody expression. 1 Transcription Review • Alternative splicing* o splicing the primary RNA transcript different ways o creates different mRNA’s &, therefore, different proteins • Example – primary RNA has exons 1,2,3,4 Exon 1 Exon 2 Exon 3 Exon 4 o mRNA #1 – exons 1,2,3 o mRNA #2 – exons 1,2,4 *Alternative splicing is how a single naïve, mature B cell has both IgM & IgD on its surface 2 Alternative splicing DNA 1o RNA mRNA Protein A https://www.genome.gov/Images/EdKit/bio2j_large.gif July 12, 2018 3 Antibody Genetics 4 The conundrum • How can we generate 109 or more different antigen receptors (TCRs & BCRs) when the human has less than an estimated 25,000 genes? o this total variety of antigen receptors is called our antigenic repertoire o each person’s antigenic repertoire develops as a result of a variety of genetic mechanisms so we can recognize the wide variety of pathogens we encounter 5 Antigenic repertoire • A person’s antigenic repertoire of BCRs & TCRs is created through a combination of recombination & mutation events during gene rearrangement • Prior to rearrangement, the genes are in the germline configuration • Recombination only occurs in somatic cells – specifically B & T lymphocytes & is therefore referred to as somatic recombination 6 Gene Organization 7 Organization of L Chain Genes • Each L chain has 2 regions – VL + CL • Variable region of the L chain (VL) is comprised of 2 genes – V & J o Variable (V) gene – Vκ or Vλ o Joining (J) gene - Jκ or Jλ • Constant region of the L chains (CL) is comprised of 1 gene – C o Cκ (kappa L chains) or Cλ (lambda L chains) 8 Organization of L Chain Genes CDR3 CL CDR2 CDR1 VL C gene V gene J gene Vκ Jκ Cκ or Vλ Jλ Cλ CDR is another name for the hypervariable region 9 Janeway’s Immunobiology 2017 V J Organization of H Chain Genes • Variable region of the H chain (VH) is comprised of 3 genes – V, D, J o Variable (V) gene – VH o Diversity (D) gene – DH o Joining (J) gene – JH • Constant region of the H chain (CH) is comprised of 1 gene – C o μ – δ – γ3 – γ1 – ε2 – α1 – γ – γ2 – γ4 – ε – α2 ε2 & γ are non-functional, but are present in the chromosome 10 Organization of H Chain Genes CH1 V gene D gene J gene V D J Janeway, Immunobiology 2017 Hinge CH CDR3 CDR2 CDR1 VH CH2 CH3 CH gene 11 VDJ Recombination • Rearrangement of these genes in somatic cells (somatic recombination) is referred to as V(D)J recombination • These recombination events are regulated by conserved DNA sequences called recombination signal sequences (RSS) o these RSS sequences flank each V, D, & J gene in both the H & L chains & control which genes can/can’t recombine o RSS sequences control TCR genes as well 12 κ L Chain Genes • Vκ genes o approx. 35 of them o each is preceded by its own promoter & leader sequence • Jκ genes o approx. 5 of them • There is 1 Cκ gene 13 κ L Chain Gene Locus Jκ Vκ VL Cκ CL 14 λ L Chain Genes • Vλ genes o approx. 30 of them • Jλ genes o approx. 4 of them • There are 4 Cλ genes o each Cλ gene follows a Jλ gene o a Jλ gene can associate with any downstream Cλ gene ▪ Jλ3 can combine with Cλ3 or Cλ4 o this contributes to λ L chain diversity 15 λ L Chain Gene Locus Jλ1 can combine with Cλ1 or Cλ2 or Cλ3 or Cλ4 Jλ2 can combine with Cλ2 or Cλ3 or Cλ4 Jλ3 can combine with Cλ3 or Cλ4 Jλ Vλ VL Cλ CL 16 H Chain Genes • VH genes o approx. 45 of them • DH genes o approx. 30 of them • JH genes o approx. 6 of them • CH genes o 11 of them – 2 are nonfunctional o μ – δ – γ3 – γ1 – ε2 – α1 – γ – γ2 – γ4 – ε – α2 17 H Chain Gene Locus * * D H JH VH L VH CH1 Hinge Remember only genes 3’ of the current gene can be used for isotype switching in the future CH2 CH CH3 18 Gene Rearrangement Overview 19 Gene Rearrangement • The rearrangement of the V(D)J genes is random • In the L chains - any V gene can be placed next to any J gene • In the H chain - any V gene can be placed next to any D gene, & any D gene can be placed next to any J gene o in the H chain, V genes cannot be juxtaposed to a J gene – this is regulated by the RSS sequences 20 Gene Rearrangement • H chain genes rearrange first • L chain genes will follow, ONLY IF there was a successful rearrangement of H chain genes o κ will attempt to rearrange first – if the κ genes fail to rearrange correctly, the λ genes will attempt rearrangement o λ gene locus is obstructed unless/until needed (if κ genes don’t rearrange correctly) • If either the H chain or L chain genes fail to successfully rearrange, the cell undergoes apoptosis 21 Gene Rearrangement • We have two copies/alleles of each gene – 1 maternal, 1 paternal • Genes from both chromosomes for the H chain (maternal allele & paternal allele) attempt simultaneous rearrangement, but only 1 will be expressed o expression of only 1 of the genes is called allelic exclusion o allelic exclusion prevents both maternal & paternal genes from being expressed in a single cell 22 Gene Rearrangement – Allelic Exclusion • When one allele is successful, the other stops – if the first is not successful, the 2nd continues rearrangement o this ensures that each cell will express only one of the genes/alleles & will have a BCR of one specificity o allelic exclusion applies to both H & L chains ▪ i.e. maternal & paternal alleles will attempt simultaneous rearrangement for H chain, κ chain, & λ chain TCR α chains can, on rare occasion, bypass allelic exclusion 23 Gene Rearrangement – Isotypic Exclusion • κ genes (both maternal & paternal alleles) attempt rearrangement o when one is successful, the other stops, ensuring allelic exclusion in the L chain • If both κ genes are unsuccessful, the λ genes (both maternal & paternal alleles) attempt to rearrange (allelic exclusion) o the inhibition (obstruction) of the λ genes is called isotypic exclusion – meaning each antibody molecule will have κ OR λ chains There is no isotypic exclusion for the H chain genes 24 Isotypic Exclusion* * Allelic exclusion only allows the 2nd allele to be expressed IF the 1st allele is unsuccessful – this occurs for both H & L gene rearrangement * * * * *Both alleles of the κ L chains attempt rearrangement first, preventing the rearrangement of the λ L chain genes = isotypic exclusion 25 Summary of Rearrangement • Both alleles of H chains attempt rearrangement o as soon as one allele is successful, all further rearranging of H chain genes halts (allelic exclusion) ▪ it is possible that the one which finishes rearrangement 1st was unsuccessful in making the rearrangements in frame, so the other will continue until rearrangement is complete o if neither allele makes a successful in frame rearrangement, the cell dies 26 Summary of Rearrangement • Both alleles of the κ chains attempt rearrangement o when one allele is successful, rearranging of κ genes halts (allelic exclusion) o λ gene rearrangement is prevented while κ rearrangement occurs (isotypic exclusion) o if neither κ allele makes a successful rearrangement, the λ genes attempt to rearrange (allelic exclusion will apply) • If neither the κ or λ chain genes make a successful L chain, the cell dies 27 Summary of Rearrangement • In order for a complete Ig to be made/expressed o individual genes of the H chain (V, D, J, C) must rearrange (in frame) to make a mRNA that can be translated into a functional H chain protein o individual genes of the L chain (V, J, C) must rearrange (in frame) to make a mRNA that can be translated into a functional L chain protein 28 H Chain Gene Rearrangement 29 Rearrangement of H Chain • DH – JH juxtaposition • VH – DHJH juxtaposition • Antigen specificity is now set for the H chain • DNA is now permanently altered V1 – V7 V1-V2-V3-V4-V5-V6-V7-V8D3J5--J6--Cμ-Cδ-Cγ3… Immunology A Short Course, 2009 30 Rearrangement of H Chain • Transcription of VHDHJHCμCδ primary RNA transcript (includes introns which can include extra JH genes) = VV 6= 5= 1 –VV 7 V7 P LS V8D3J5----J6----μ--δ Immunology A Short Course, 2009 31 Rearrangement of H Chain • The primary transcript VHDHJHCμCδ undergoes alternative splicing to generate two mature mRNA transcripts for the IgM & IgD H chains* VHDHJHCμ Abbas: Basic Immunology: Functions and Disorders of the Immune System, 2020 VHDHJHCδ *Note: since both the IgM & IgD transcripts (& therefore proteins) have the same VDJ genes, they will have identical antigenic specificity 32 Rearrangement of H Chain = VV 6= 5= 1 –VV 7 V7 Permanently altered from germline DNA as genes have been removed/deleted during rearrangement Alternative splicing removes any extra J genes & introns to make mRNA for IgM & IgD V8D3J5μ Introns J6 & δ removed V8D3J5δ Introns J6 & μ removed Removing the introns during alternative splicing creates 2 mRNA transcripts – 1 for IgM & 1 for IgD 33 Immunology A Short Course, 2009 Rearrangement of H Chain • Once translated into protein, the H chains are transported to the lumen of the ER where they will (later) be joined to the L chains • IgM & IgD* are found on the surface of mature, naïve follicular B-2 B cells *IgM is found on B-1 & B-2 marginal zone B cells, but these lack IgD as they don’t enter the follicles (the function associated with IgD) 34 Isotype Switching H Chain • When isotype switching occurs, the same VHDHJH genes are moved next to a new CH gene (any upstream CH genes are deleted – i.e. permanently removed from the DNA sequence) o each H chain gene (except δ) has a switch region 5’ to the gene to facilitate this process • Is this DNA rearrangement or alternative splicing? 35 Isotype Switching H Chain Rearranged DNA Switch sequences 5’ of the C genes move adjacent to each other & the intervening DNA is removed Newly Rearranged DNA after isotype switching 36 L Chain Gene Rearrangement 37 Rearrangement of κ Chain • Vκ – Jκ juxtaposition • VκJκ – Cκ juxtaposition • Primary transcript (genes + introns) is made (introns can include extra J genes: VκJκ -- Jκ -- Cκ) • Splicing generates the mature mRNA transcript (removes introns – moves VκJκ next to Cκ → VκJκCκ ) • Translation, transport to the ER, & joining of the κ L chain to the μ & δ H chains → expression of full Ig on the surface 38 With any extra Includes extraJJκgenes gene Introns removes & extra Jκextra geneJ genes Splicing have been removed Moved to ER to be linked to H chain 39 Rearrangement of λ Chain* • Vλ – Jλ • VλJλ – Cλ juxtaposition • Primary transcript (genes + introns) is made (introns can include extra J & C genes VλJλ2 --- Cλ2 --- Jλ3 ---Cλ3) • Splicing generates the mature mRNA transcript (VλJλCλ) • Translation, transport to the ER, & joining of the λ L chain to the μ & δ H chains * L chain rearrangement only occurs if both k chain alleles fail to rearrange = isotypic exclusion 40 Learning Objectives 1. Define the regulatory mechanisms of immunoglobulin gene rearrangement. 2. Identify components that facilitate and regulate antibody rearrangement. 3. Recall the types of antibody diversity, how and when they occur, and the enzymes and molecules that facilitate these mechanisms 41 Regulation 42 Regulation of Ig Gene Rearrangement • Recombination signal sequences (RSSs) within the introns only allow o 1 V gene to combine with 1 D gene & 1 D gene to combine with 1 J gene in the H chain ▪ RSSs prevent V genes from combining with J genes or multiple V, D, or J genes from combining o 1 V gene to combine with 1 J gene in the L chain 43 Regulation of Ig Gene Rearrangement • These recombination signal sequences ensure that the distance & rotation of the DNA is correct for splicing & recombination • RSSs flank each V, D, & J gene & must come together in specific ways • RSSs are recognized by the recombination-activating genes (RAG) which facilitate the recombination events 44 Regulation of Ig Gene Rearrangement • Each RSS has 3 components o hepatmer (7 bp) – always closest to the gene o spacer o nonamer (9 bp) – always furthest from the gene RSS RSS nonamer: spacer: heptamer – gene – heptamer: spacer: nonamer 45 Regulation of Ig Gene Rearrangement • There are two types of RSSs which contain “spacers” of different lengths o one-turn RSS has a 12 bp spacer o two-turn RSS has a 23 bp spacer • A 12 bp spacer RSS is always moved next to a 23 bp spacer RSS – called the 12/23 rule or 1 turn/2 turn rule 46 Regulation of Ig Gene Rearrangement one-turn RSS (12 bp spacer) two-turn RSS (23 bp spacer) Abbas: Basic Immunology: Functions and Disorders of the Immune System, 2020 47 Regulation of Ig Gene Rearrangement • H chain VH (23) – (12) DH (12) – (23) JH • κ L chain spacers V κ (12) – (23) J κ • λ L chain spacers V λ (23) – (12) J λ Abbas: Basic Immunology: Functions and Disorders of the Immune System, 2020 48 Regulation of Ig Gene Rearrangement • Heptamers are palindromic & play an important role in recombination & generating additional diversity Immunology, Infection, and Immunity, 2004 49 Immunology, Infection, and Immunity, 2004 Recombination • The DNA is looped, placing the12 bp spacer & 23 bp spacer of the 2 chosen genes next to one another • The palindromic sequences of the heptamers are aligned – one next to the other • DNA of each heptamer is nicked, & the intervening DNA is removed/deleted • Chosen genes are placed adjacent to one another & DNA is repaired 50 1 2 DNA is looped to align the palindromic heptamers DNA is nicked by the endonuclease RAG-1 3 4 Genes are placed adjacent to each other Intervening DNA is removed & joint is repaired Abbas: Basic Immunology: Functions and Disorders of the Immune System, 2020 51 Recombination Immunology, Infection, and Immunity, 2004 RAG-1 RAG-1 52 Immunology, Infection, and Immunity, 2004 Small Group Discussion • What would be the immunological ramifications if a person was born with a mutation in the 12 bp region of a κ L chain gene that made it 5 bp long? Would any antibody be produced? Would the person produce mutated or nonfunctional antibodies? 53 Small Group Discussion • It is highly unlikely that both of the κ alleles (maternal & paternal) would be mutated, thus leaving the other to form normal antibodies • If BOTH κ genes were abnormal, there are still two λ L chain genes to form normal, functional antibodies 54 Generating Diversity 55 Generating Antibody Diversity • There are four ways the immune system can generate diversity within the antibody During o combinatorial diversity o combinatorial association development in the bone marrow Antigen INdependent o junctional diversity o somatic hypermutation Antigen Dependent After activation in the 2o lymphoid organ 56 Combinatorial Diversity • Random joining of VDJ genes of the H chain • Random joining of the VJ genes of the L chains i.e. V(D)J recombination Occurs at the DNA (gene) level 57 Combinatorial Association • Any L chain (κ or λ – with any combination of VJ genes), can associate with any H chain (with any combination of VDJ genes) to form a functional antibody • In two different cells, there could be the same H chain combining with different L chains (or vice versa) Occurs at the protein (chain) level 58 Combinatorial Association V33 J1 V6 Cκ D9 J3 Cell 1: V33,J1, Cκ + V6, D9, J3, Cμ V1 J5 Cλ Cμ Cell 2: V1,J5, Cλ + V6, D9, J3, Cμ 59 Junctional Diversity • Imprecise joining of the VDJ & VJ genes consists of 3 steps o P nucleotide addition o Junctional flexibility o N nucleotide addition Immunology, Infection 2004 , and Immunity, 2004 Immunology, Infection, and Immunity, • Involves the addition or subtraction of nucleotides within the DNA during recombination 60 Junctional Diversity • P nucleotide addition o when the DNA loops around to join the V, D, & J genes (or V & J genes), the DNA is clipped by an endonuclease (RAG-1) o cutting by RAG-1 leaves a hairpin loop which is cleaved open by endonuclease Artemis which leaves an overhang (uneven ends) o DNA polymerase enzyme adds nucleotides to the shorter strand, using the longer strand as a template to even up the two strands o this occurs within the palindromic heptamer sequence of the RSS 61 Rag-1 Artemis DNA Pol P nucleotide addition endonuclease RAG-1 nicks the DNA leaving a hairpin loop which is then cleaved by endonuclease Artemis This process occurs for each of the 2 genes being joined Cutting the hairpin loop leaves an overhang DNA polymerase then adds nucleotides to the shorter strand (using the longer strand as a template) to make the strands the same length Immunology, Infection, and Immunity, 2004 Immunology, Infection, and Immunity, 2004 62 Heptamer Heptamer Rag-1 Artemis Rag-1 DNA Pol DNA Pol Janeway’s Immunobiology, 2008 Heptamers of the RSSs overlap, bringing the D & J genes close together endonuclease RAG-1 cuts both DNA strands within the heptamers, leaving a hairpin at each RSS The hairpins are opened by endonuclease Artemis, leaving uneven DNA strands (i.e. an overhang) – DNA polymerase fills in the 2nd (shorter) strand by each gene 63 Junctional Diversity • Junctional flexibility o when the looped DNA is cut, it is exposed to other enzymes (exonuclease) that can remove nucleotides o exonucleases may remove up to 10 nucleotides – including the recently added P nucleotides & some of the original gene sequence 64 P nucleotide addition Exonuclease Junctional flexibility This process occurs for each of the 2 genes being joined Exonuclease removes nucleotides (some or all of the P nucleotides just added, & possibly some of the nucleotides from the original gene) This process is more common in H chain gene rearrangement Immunology, Infection, and Immunity, 2004 Immunology, Infection, and Immunity, 2004 65 Junctional Diversity • N-nucleotide addition o terminal deoxynucleotidyl transferase (TdT) adds nucleotides o 1-10 non-templated nucleotides can be added to one of the DNA strands o ONLY occurs in H chains • DNA polymerase then fills in the gap on the other (now shorter) DNA strand • Ligase joins the two genes 66 P nucleotide addition Junctional flexibility This process occurs for each of the 2 genes being joined N nucleotide addition TdT adds nucleotides DNA Polymerase fills the gap between the genes Ligase joins the ends 67 Immunology, Infection, and Immunity, 2004 Review of somatic recombination & generating diversity • All of these events occur during B cell development in the bone marrow – PRIOR to (& independent of) antigen exposure o Combinatorial diversity o Combinatorial association o Junctional diversity 68 Review of somatic recombination & generating diversity • Junctional diversity ONLY affects CDR3 • P-nucleotide addition & Junctional flexibility can occur in H & L chains • N-nucleotide addition occurs only in H chains due to the temporal expression of the TdT enzyme • RAG1, RAG2, & TdT are expressed only in lymphoid cells 69 Somatic Hypermutation • The ONLY mechanism of generating diversity that occurs AFTER antigen exposure (i.e. it is antigen DEpendent) • Involves point mutations within the V regions of both the H & L chains o can occur in the HV or FR regions • Does NOT change the number of nucleotides – but can significantly change the character of the protein 70 Somatic Hypermutation • These mutations are the mechanism behind the phenomenon called affinity maturation • Affinity maturation results in the generation of antibodies with increased affinity for their cognate antigen • The reason that this type of immunity is adaptive 71 Somatic Hypermutation Affinity maturation 2 1 3 4 72 Somatic Hypermutation • SHM occurs in the follicles (of spleen or LNs) after B cell activation • Progeny B cells use the same VJ & VDJ genes, but point mutations are introduced during clonal proliferation • Occurs during secondary immune responses (any subsequent challenge by the same antigen) o can also occur at the end of a primary response if it persists long enough 73 Somatic Hypermutation • Activated B cell proliferates, creating many clones of itself, introducing mutations in the V, D, & J genes of the H & L chains as the cells copy their DNA • Antibodies (with mutations) are made & expressed on the surface of the B cell clones • B cells interact with antigen on the FDCs to see if mutations have made their antibodies better, worse, or unchanged • Only B cells with better antibodies receive survival signals from FDCs 74 Somatic Hypermutation 75 Immunology, Infection, and Immunity, 2004 Immunology, Infection, and Immunity, 2004 Somatic Hypermutation B cells proliferating & mutating 6 2 5 1 4 FDCs holding antigen for B cells with mutated abs 3 7 Immunology A Short Course, 2009 76 Small Group Discussions • Why does Junctional diversity ONLY affect CDR3? • Why does somatic mutation affect ALL the CDRs? • Why is it significant that RAG & TdT enzymes are only expressed in B & T lymphocytes? 77