What is the 12/23 rule?
Combinatorial Diversity
• Sequencing of junctions of V, D and J in B cells did not match predicted sequences – imprecise joining leads to junctional diversity
• V, D and J segments flanked by recombination signal sequences (RSS) consisting of a conserved heptamer and a conserved nonamer, separated by 12 or 23 bases.
• This is the 12/23 rule
• Means you always get V joining to J in light chains, and V – D – J in heavy chains → cant get V to J because this would be 23/23 and breaking the rule
• The only time rule breaking has been seen is in the CDR3 → additional diversity is seen from generation of a CDR3 by joining of a D to a D – found in 5% of antibodies.
What are the molecular mechanics of recombination?
- Alignment of RSS nonamers following the 12/23 rule so a V-J and V-D-J
- Cleavage od DNA to form hairpins and generation of P nucleotides
− When the nonamers align, the DNA strand with the heptamers is cleaved by RAG1/2
− The intervening DNA is excised and the ends of the V and D genes now contain parts of the heptamer sequence
− The two strands of DNA at the ends of the V and D genes join to form a hairpin
− The hairpins are cleaved at random so that each DNA strand contains some nucleotides derived from the hepatmere sequence → P nucleotides - Addition of N nucleotides at random to each end of the strand by TDT
- Filling in of DNA
− Exonucleases, DNA polymerase and DNA ligase repair the DNA and complete the formation of the join with complete nucleotide sequences on both DNA strands
− The addition of P and N nucleotides has changed the final amino acid sequence of the protein
Summaries the source of diversity in the primary antibody repertoire
• Combinatorial diversity − Multiple gene segments − Any H with any L − 106 • Junctional diversity (localized in CDR3) − P and N nucleotide addition − Exonuclease activity − 1011 − → All junctional diversity is in CDR3, CDR1 and CDR2 diversity comes from selection of different Vs and natural polymorphisms.
Structure of the TCR signalling component
- Antigen binding TCRa:b heterodimer associated with 4 signalling chain (two e, one d and one y) called the CD3 → required for cell surface expression of antigen binding chains and for signaling. Homodimer of z chains also associated
- Each CD3 has one ITAM motif, the z chains have 3
- The TM regions of each chain have either a positive or negative charge – positive charge of a and b chains interact with negative charges on the signaling chains to maintain association.
Structure of the BCR signalling component
- Cell surface Ig with one each of the invariant signaling proteins IgA and IgB
- Ig recognizes and bind antigen directly but cant generate a signal → each IgA and IgB has ITAMs In the tails that allow signaling when B cell is ligated to antigen
- IgA and IgB form a disulphide linked heterodimer that is associated with the heavy chain, but it is not know which binds to the heavy chain
Describe the organisation of the alpha-beta genes
• Separate V, D, J and C regions • TCRa locus (chromosome 14) − Similar to a light chain − 70-80 Va gene segments each preceded by a leader sequence − 61 Ja gene segments − J segments followed by a single C • TCRb locus (chromosome 7) − Similar to heavy chain − 52 V segments located distantly from 2 clusters containing a single D gene, multiple J genes and single C gene
Describe the organisation of the gamma-delta genes
• TCRd locus located within the TCRa V and J segments → V-D-J
− 3 D, 4 J and single C
− 2 V located near the single C, and 6 V interspersed amongst the TCRa V (5 shared with the alpha and can be used by either locus. and one is unique to the delta)
− Because the delta chain is found within the alpha chain, when you get V/J recombination on the alpha chain, you lose the whole delta locus
• TCRy locus → V-J
− Resembles TCRb
− 12 V, and then cluster of J and C
• Rearrangement occurs much the same as the alpha/beta except in the delta rearrangement, two D segments can be used in the same gene, greatly increasing variability
Is RSS diversity generated in the TCR?
- TCR genes also flanked by RSSs
- P and N nucleotides also found in the junctions between the V, D and J segments of the TCRb gene
- However P and N nucleotides also added between V and J of TCRa gene
- Joining of RSS hepatmers and nonamers almost always follows the 12/23 rule, but there is disposition of the RSSs in the TCRb and TCRd loci, so direct V to J joining is in principle allowed but it rarely occurs
Compare the diversity found in the TCR and BCR
• Both assembled by somatic recombination of gene segments
• Combinatorial diversity is similar
• Greater junctional diversity in TCR because:
− D segments are read in all 3 frames often
− High number of J segments in TCRa locus
− TDT activity at all junctions (not really active at light chain in BCR)
• This means total TCR diversity is greater
− NB – TCR diversity concentrated in CDR3 (where junctional diversity affects) – this is which contacts the antigenic peptide, so allows more peptides to be recognized
− With BCR, so much CDR3 diversity isn’t as important as it also uses CDR1 and CDR2 to recognize the whole pathogen
− NB – no somatic hypermutation in the TCR
Summarise the stepwise rearrangement of the TCR
- Double negative stages (no CD4 or CD8)
− DN1 → CD44+ CD35-
− DN2 → CD44+ CD25+
− DN3 → CD44 lower CD25+
− Here we have beta chain rearrangement = V-J recombination then V-DJ, forming the pre-TCR. This is a quality checkpoint.
− If everything is okay, turns the RAG down and you get proliferation
− DN4 → CD44- CD25-
− Proliferation occurring
− RAG then turned on again - Double positive stage (CD4 and CD8)
− Here we have alpha chain rearrangement = V to J recombination
− Gives functioning TCR
− Now have a decision point that decided whether you will be CD4 or CD8 - Single positive
• TDT activity is constant up until functioning TCR made
• Note, allelic exclusion at the alpha chain is not 100%
− In BCR, rearrangement of the heavy chain enough to turn off the RAG gene
− When you’ve proliferated, you express RAG again, make a light chain, then RAG turns off when functional Ab produced
− In the TCR, it isn’t expression of the TCR that turns RAG off → it is positive selection (whether it can bind MHC)
− This gives prolonged RAG activity → up to 30% T cells may have 2 alpha chains
− However as you go through development, one is usually lost so maybe only 1-2% have 2
Describe the anatomy of the thymus
• T cells derive from HSCs in the bone marrow
• Progenitors migrate via the blood to the thymus where they mature
• T cell development parallels B cell in many ways:
− Stepwise rearrangement of genes
− Sequential testing for successful arrangement
− Eventual assemble of heterodimeric antigen receptor
• T cell development does have some distinct features
− Generation of two lineages – a/b and y/d
• Developing T cells are known as thymocytes - undergo selection which depends on interactions with thymic cells
Anatomy:
• Upper anterior thorax
• Numerous lobules differentiated into outer cortical region and inner medulla
• Developing T cell precursors located in the thymic stroma → provides an environment similar to stromal cells in the bone marrow
• Cells enter in the outer cortex and proliferate
• They then head towards the medulla where you get positive and negative selection → deletes 98%
• Passage through thymus takes 3 weeks
• Macrophages present pick up the debris
Describe thymic involution
Thymic Involution
• Transient → stress, eg) infection, malnutrition, pregnancy
• Age associated
− Begins naturally with the first year of life
− 3% loss per year to middle age
− Then 1% loss per year
− Regressing of thymic epithelium and decrease in cellularity
− Increasing reliance on the peripheral pool
• Thymectomising a neonate would be more detrimental than adult → they would be immunodeficient
What instructs the T cell and not B cell lineage choice?
• Notch signal from stromal cells instructs the precursor cells to commit to T cell and not B cell lineage
− Delta binding Notch causes the cytoplasmic domain of Notch to interact with transcription factors, eg Gata 3
− This suppresses B cell development and promotes T cell development
What controls the a:b or y:d T cell lineage commitment?
− T cells expressing y/d receptors differ from a/b in that they are found primarily in epithelial and mucosal sites and lack expression of both CD4 and CD8
− Signals through the y/d T cell receptor and the pre-T cell receptor complete to determine the fate
− During development, DN thymocytes rearrange the gamma/delta and beta TCR loci simultaneously
− If a complete y/d TCR is formed before a successful beta chain re-arrangement has led to a pre-TCR, the thmyocyte receives signals that shurts of rearrangement of the beta gene
➢ Strong ERK signaling commits the cell to the y/d lineage
➢ Many y/d T cells leave with the ability to secrete cytokines → capacity gained by a/b T cells only after antigen encounter in the periphery
− If a functional beta chain is formed before a y/d receptor, it pairs with the pT-alpha to form the pre-TCR
➢ Weak ERK signaling through the pre-TCR leads to commitment to a/b lineage
− During embryonic development. the y/d T cells are the first to appear (first line of defense). After birth, the a:b lineage becomes dominant.
What controls CD4/CD8/Treg lineage choice?
− CD4:
➢ Binding of MHC-II upreglates ThPOK which inhibits Runx3
− CD8:
➢ Binding of MHC-I doesn’t give increase in ThPOK, so Runx3 is retained
➢ Runx3 silences CD4
− Treg:
➢ If you don’t get signaling strong enough tp cause deletion, you get upregulation of Foxp3 and induction of Tregs
What controls iNK T cells lineage choice?
- Invariant NK T cells Lineage Choice:
− Unlike conventional a:b T cells, iNKT
− Have TCR made of nearly invariant TCRa associated with a small set of TCRb
− selected by binding to a non-classical MHC molecule, CD1d, expressed on cTEC
− requires signaling via homotypic SLAM family receptors
− acquire effector properties in the thymus
Describe maturation of T cells and RTEs
• Upregulation of S1P1 receptor required for mature thymocyte to enter the bloodstream → S1P1 present in high concentrations in the blood
• Mature thymocytes express the lymph node homing receptor CD62L
− Klf2 directly promotes S1P1 receptor and CD62L transcription
• Thymocytes exit via blood vessels
• Recent thymic emigrants are not as mature as peripheral naïve T cells
Licensing of Recent Thmyic Emigrants
• Maturing RTEs acquire ability to produce cytokines
− Correlated with epigenetic changes at cytokine gene loci
− Inability of RTE to produce Th1 cytokines on ag stimulation may promote peripheral tolerance
− CD4+ RTEs convert more readily to iTReg than mature naïve T cells
• RTEs mature to acquire improved survival capacity
− IL-7 stimulation required to maintain Bcl2
− Expression of IL-7 receptor induce during positive selection
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Intro to Immunology4
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Complement23
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Innate Like Recognition17
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Pattern Recognition12
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Induced Innate Responses18
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Antigen Presentation via MHC34
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B Cell Development8
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The B Cell Response15
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T Cell Development17
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The Immunological Synapse12
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Molecular Events in T Cell Activation8
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CD4+ T Cell Responses15
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CD8+ T Cell Responses7
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Immunological Memory12
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Tolerance11
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Mucosal Immunity10
