2Generating Evidence for and isolating the TCR Generation of cytotoxic T lymphocytes (CTLs)Zinkernagel and Doherty – 1974 demonstrated role of another molecule, MHC got Nobel Prize 1996Isolation of the TCR using monclonal antibodies that were clonotypic i.e recognized single T cell cloneIdentification of genes of the TCR
5Discovery of the T cell antigen receptor (TcR) Polyclonal T cellsfrom an immunised strain A mouseGrow and clone a single antigen-specific T cell in-vitro with antigen, IL-2 and antigen presenting cellsMonoclonal (cloned) T cellsIn vitro “clonal selection” means each daughter cell has the same antigen specificity as the parent cellMost molecules present on the monoclonal T cells will be identical to the polyclonal T cells EXCEPT for the antigen combining site of the T cell antigen receptor
6Making anti- clonotypic TcR antibodies T cell clonefrom a strain A mouseNaïve strain A mouseMake monoclonal antibodies by hybridisation of the spleen cellswith a myeloma cell lineThe strain A mouse will not make antibodies to the hundreds of different molecules associated with strain A T cells due to self toleranceBUTThe naïve mouse has never raised T cells with the specificity of the T cell clone,SOthe only antigen in the immunisation that the A strain mouse has never seen will be the antigen receptor of the monoclonal T cells
7Making anti- clonotypic TcR antibodies Screen the supernatant of each cloned hybridoma against a panel of T cell clones of different specificity(i.e.cells with subtly different antigen-binding structures)YMonoclonal antibodiesYYClone used forimmunisationYYYYYYT cell clonesYYYAnti-TcR Abs that recognise only one clone of T cells are CLONOTYPICHypothesise that anti-clonotype Abs recognise the antigen receptor
8Discovery of the T cell antigen receptor (TcR) Lyse cells and add anti-clonotype Abthat binds to unique T cell structuresYCapture anti-clonotype Ab-Agcomplex on insoluble supportIMMUNOPRECIPITATIONWash away unbound proteinYElute Ag from Ab and analyse the clonotypically-expresssed proteins biochemicallyPrincipal component was a heterodimeric 90kDa proteincomposed of a 40kDa and a 50kDa molecule ( and chains)Several other molecules were co-immunoprecipitated.
9Structure of the TcR polypeptides T cell clone AT cell clone BT cell clone CIntact TcR chain polypeptidesCyanogen bromide digestion of the and proteinsBiochemical analysis of digestion productsCVPolypeptides contain a variable, clone-dependent pattern of digestion fragments and a fragment common to all TcR
10Cloning of the TcR genes BTThe experimental strategyThe majority of genes expressed by T and B lymphocytes will be similarGenes that greatly differ in their expression are most likely to be directly related to the specialised function of each cellSubtract the genes expressed by B cells from the genes expressed by T cells leaving only the genes directly related to T cell function
11Cloning of TcR genes by subtractive hybridisation AAAAAmRNAHybridise thecDNA and mRNA sharedbetween T and B cellsAAAAAT cell singlestranded cDNAAAAAADiscard hybridsAAAAADigest unhybridised B cell mRNAClone andsequence T cell-specific genesIsolate non-hybridising material specific to T cells
12Analysis of T cell-specific genes Of the T cell-specific genes cloned, which cDNA encoded the TcR?Assumptions made after the analysis of Ig genes:TcR genes rearrange from germline configurationIg gene probes can be used as TcR genes will be homologous to Ig genesRestrictionenzyme sites32PGERMLINEDNAVDJC32PVDJCREARRANGEDDNAFind two restriction sites that flank the TcR regionCut the T cell cDNA and placental (i.e. germline) DNAand Southern blot the fragments
14TCREach a and b chain consists of one ‘Ig-like’ N-terminal variable region (V), one Ig-like constant (C) domain, a hydrophobic transmembrane region, and a short cytoplasmic region.Thus the extracellular portion of the ab heterodimer is structurally similar to the antigen-binding fragment (Fab) of an Ig, which is made up of the V and C regions of a light chain and the V region and one C region of a heavy chain.
15The T cell antigen receptor combining siteResembles an Ig Fab fragmentFabVHVLFcCLCHVaVbDomain structure: Ig gene superfamilyCarbohydratesMonovalentCaCbNo alternative constant regionsNever secretedHingeHeterodimeric, chains are disuphide-bondedTransmembrane region+++Very short intracytoplasmic tailPositively charged amino acids in the TM regionCytoplasmic tailAntigen combining site made of juxtaposed Va and Vb regions30,000 identical specificity TcR per cell
16T cell antigen receptor diversity TcR are highly variable in the individualDiversity focused on small changes in the charge & shape presented at the end of the T cell receptor.TcR diversity to the peptide antigens that bind to MHC moleculesMechanisms of diversity closely related to T cell developmentRandom aspects of TcR construction ensures maximum diversityMechanisms of diversity generation similar to immunoglobulin genes
17Generation of diversity in the TcR COMBINATORIAL DIVERSITYMultiple germline segmentsIn the human TcRVariable (V) segments: ~70, 52Diversity (D) segments: 0, 2Joining (J) segments: 61, 13The need to pair and chains to form a binding sitedoubles the potential for diversityJUNCTIONAL DIVERSITYAddition of non-template encoded (N) and palindromic (P) nucleotides atimprecise joints made between V-D-J elementsSOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR
18Estimate of the number of human TcR and Ig Excluding somatic hypermutationImmunoglobulin TcRElementHVariable segments405952~70Diversity segments272D segments inall 3 frames--YesYesJoining segments691361Joints with N & Pnucleotides2(1)*2123603640No. of V gene pairsJunctional diversity~1013~1013Total diversity~1016**~1016* Only half of human k chains have N & P regions**No of distinct receptors increased further by somatic hypermutation
19Organisation of TcR genes L & Vx70-80J x 61CTcR L & Vx52D1Jb1 x 6C1D2Jb2 x 7C2TcR TcR genes segmented into V, (D), J & C elements(VARIABLE, DIVERSITY, JOINING & CONSTANT)Closely resemble Ig genes (a~IgL and b~IgH)This example shows the mouse TcR locus
20TcR a gene rearrangement by SOMATIC RECOMBINATION Germline TcR VnJCV2V1Rearranged TcR1° transcriptSpliced TcR mRNARearrangement very similar to the IgL chains
21TcR a gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frameVnJCV2V1Vn+1a chain tries for a second time to make a productive join using new V and J elementsProductivelyrearranged TcR1° transcript
22TcR b gene rearrangement SOMATIC RECOMBINATION L & Vx52D1JC1D2C2Germline TcR D-J JoiningV-DJ joiningRearranged TcR 1° transcriptC-VDJ joiningSpliced TcR mRNA
23TcR b gene rearrangement RESCUE PATHWAY There is a 1:3 chance of productive D-J rearrangement and a 1:3 chance of productive D-J rearrangement(i.e only a 1:9 chance of a productive b chain rearrangement)D1JC1D2C2Germline TcR D-J JoiningV-DJ joiningV2nd chance atV-DJ joiningNeed to remove non productiverearrangementUse (DJC)b2 elements
24V, D, J flanking sequences Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides.Va7239Ja7129Db7129Vb7239Jb
25Recombination signal sequences (RSS) HEPTAMER - Always contiguous with coding sequenceNONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacerVb7239Db12Jb√√Vb7239Db12Jb12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS
26Molecular explanation of the 12-23 rule 23-mer = two turns12-mer = one turnIntervening DNAof any length23Vb9712DbJb79
27Molecular explanation of the 12-23 rule V1DJV2V3V4V8V7V6V5V9V1V2V3V4Loop of interveningDNA is excisedV8V7V6V5Heptamers and nonamers align back-to-backThe shape generated by the RSS’s acts as a target for recombinases7923-mer12-merV9DJAn appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the rule)
28Mini-circle of DNA is permanently lost from the genome Junctional diversity712923712923Mini-circle of DNA is permanently lost from the genomeSignal jointCoding jointVDJVDJImprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding joint.
29Non-deletional recombination V1V2V3V4V9DJLooping out works if all V genes are in the same transcriptional orientationV17239D12JHow does recombination occur when a V gene is in opposite orientation to the DJ region?V4V1V2V3V9DJDJ7129V423
30Non-deletional recombination J7129V423V4 and DJ in opposite transcriptional orientationsDJ7129V4231.DJ7129V4232.DJ7129V4233.DJ7129V4234.
31Fully recombined VDJ regions in same transcriptional orientation 7129V4231.DJ7129V4232.Heptamer ligation - signal joint formationDJV47129233.V to DJ ligation - coding joint formationDJV4712923Fully recombined VDJ regions in same transcriptional orientationNo DNA is deleted4.
32Steps of TcR gene recombination Recombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSSV7239D7129JV7239The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ regionD7129JThe recombinase complex makes single stranded nicks in the DNA, the ends of each broken strand.The nicks are ‘sealed’ to form a hairpin structure at the end of the V and D regions and a flush double strand break at the ends of the heptamers.The recombinase complex remains associated with the break723912723912VDJ
33Steps of TcR gene recombination VDJ723912A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends.VDJThe hairpins at the end of the V and D regions are opened, and exonucleases and transferases remove or add random nucleotides to the gap between the V and D regionVDJ723912DNA ligase IV joins the ends of the V and D region to form the coding joint and the two heptamers to form the signal joint.
34Junctional diversity: P nucleotide additions 7129JV237V239TC CACAGTGAG GTGTCACAT GTGACACTA CACTGTG7D129JThe recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA.DJVTCAGATTAU7D129JV23CACAGTGGTGTCACGTGACACCACTGTGTCAGATTAThe 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D region.
35V2V3V4V8V7V6V5V97239CACAGTGGTGTCAC12GTGACACCACTGTGHeptamers are ligated by DNA ligase IVVTCAGUDJATTAVTCAGUDJATTAV and D regions juxtaposed
36Generation of the palindromic sequence VTCAGUDJATTARegions to be joined are juxtaposedEndonuclease cleaves single strand at random sites in V and D segmentVTCAGUDJATTAThe nicked strand ‘flips’ outVTC~GAAGDJATTA~TAThe nucleotides that flip out, become part of the complementary DNA strandIn terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic.The nucleotides GA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.
37Junctional Diversity – N nucleotide additions Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNAVTC~GAAGDJATTA~TACACTCCTTATTCTTGCAAVTC~GAAGDJATTA~TACACACCTTATTCTTGCAAComplementary bases annealDJTA~TAExonucleases nibble back free endsVTC~GACACACCTTATTCTTGCAAVDJDNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strandsTC~GAAGATTA~TACACACCTTATTCTTGCAAVTCDTAGTT AT ATAG C
38V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity Germline-encoded nucleotidesPalindromic (P) nucleotides - not in the germlineNon-template (N) encoded nucleotides - not in the germlineCreates an essentially random sequence between the V region, D region and J region in beta chains and the V region and J region in alpha chains.
39How does somatic recombination work? How is an infinite diversity of specificity generated from finite amounts of DNA?Combinatorial diversity and junctional diversityHow do V region find J regions and why don’t they join to C regions? ruleHow does the DNA break and rejoin?Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity.
40Why do V regions not join to J or C regions? VbDbJbCIF the elements of the TcR did not assemble in the correct order, diversity of specificity would be severely compromisedDIVERSITY2x1xFull potential of the beta chain for diversity needs V-D-J-C joining - in the correct orderWere V-J joins allowed in the beta chain, diversity would be reduced due to loss of the imprecise join between the V and D regions
41Location of junctional diversity TcR chainTcR chainCDR3CDR1CDR2V-JJoinV-DJoinD-JjoinVariabilityAmino acid No.of TcR chainCDR = Complemantarity determining region
42T-cell ReceptorT cells also express other membrane receptors that do not recognize antigen but participate in responses to antigens: these are collectively called accessory molecules.2/9/04
43T cell co-receptor molecules Lck PTKCD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHCcomplexes by ~100 foldTcRCD8CD4MHC Class IMHC Class II32
44CD8 and CD4 contact points on MHC class I and class IIMHC class IMHC class IICD8 binding siteCD8 binding site
45TcR-CD3 complex TcR CD3 The intracytoplasmic region of the TcR chain is too shortto transduce a signalThe CD3or (zeta)chainsare required for cell surfaceexpression of the TcR-CD3complex and signallingthrough the TcRSignalling is initiated by aggregation of TcR by MHC-peptide complexes on APC
46Transmission of signals from the cell surface to the nucleusAlmost identical to transmission in B cellsT cell-specific parts of the signalling cascade are associated with receptors unique to T cells - TcR, CD3 etc.Subsequent signals that transmit signals to the nucleus are common to many different types of cell.The ultimate goal is to activate the transcription of genes, the products of which mediate host defence, proliferation, differentiation etc.Once the T cell-specific parts of the cascade are complete, signalling tothe nucleus continues via three common signalling pathways via:The mitogen-activated protein kinase (MAP kinase) pathwayAn increase in intracellular calcium ion concentration mediated by IP3The activation of Protein Kinase C mediated by DAG
48Simplified scheme linking antigen recognition with transcription of T cell-specific genes MAP Kinase cascadeSmall G-protein-activated MAP kinases found in all multicellular animals - activation of MAP kinases ultimately leads to phosphorylation of transcription factors from the AP-1 family such as Fos and Jun.Increases in intracellular calcium via IP3IP3, produced by PLC-g, binds to calcium channels in the ER and releases intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++] activate a phospatase, calcineurin, which in turn activates the transcription factor NFAT.Activation of Protein Kinase C family members via DAGDAG stays associated with the membrane and recruits protein kinase C family members. The PKC, serine/threonine protein kinases, ultimately activate the transcription factor NFkBThe activated transcription factors AP-1, NFAT and NFkB induce B cell proliferation, differentiation and effector mechanisms
49Estimate of the number of human TcR and Ig Excluding somatic hypermutationImmunoglobulin TcRElementHVariable segments405952~70Diversity segments272D segments inall 3 frames--YesYesJoining segments691361Joints with N & Pnucleotides2(1)*2123603640No. of V gene pairsJunctional diversity~1013~1013Total diversity~1016**~1016* Only half of human k chains have N & P regions**No of distinct receptors increased further by somatic hypermutation
50Why do TcR not undergo somatic mutation? Antigen presentationYBT cell helpSelf AntigenForeign antigenAPCAntibodyAnergy or deletionof anti-self cellsYTYBNoT cell helpAffinity maturation due to somatic mutation
51Why do TcR not undergo somatic mutation? BYBYBOccasional B cellthat somatically mutatesto become self reactiveAffinity maturation due to somatic mutation
52The lack of somatic mutation in TcR helps to prevent autoimmunity T cell helpYTYBOccasional B cellthat somatically mutatesto become self reactiveXNo T cell helpYTT cell that doesn’t mutatecan not help theself reactive B cellT cell that mutates can may help the self reactive B cellAutoantibody production
53If TcR did undergo somatic mutation: TcR interacts with entire top surface of MHC-peptide antigen complexSomatic mutation in the TcR could mutate amino acids that interact with the MHC molecule causing a complete loss of peptide-MHC recognition
54If TcR did undergo somatic mutation: TcR-MHC interaction is one of many between the T cell and APCOn-off rate of TcR determines rate of ‘firing’ to give qualitatively different outcomesMust be of relatively low affinity as cells with high affinity TcR are deleted to prevent self reactivity.If TcR underwent affinity maturation, they would be deleted
55Why do B cell receptors need to mutate? Neutralisation ofbacterial toxinsY`Ab-Ag interaction must be of high affinity to capture and neutralise toxins in extracellular fluidsThere is a powerful selective advantage to B cells that can somatically mutate their receptors to increase affinitySOMATIC MUTATIONY`Toxin bindingblockedPreventstoxicity
56An alternative TcR: Discovered as Ig-homologous, rearranging genes in non TcR T cellsVVJC3x D3x J1x CHuman locus3x JC12x JC212x V1The locus is located between the V and J regionsV to J rearrangement deletes D, Jand CTcR cells can not express g TcRFew V regions, but considerable junctional diversity as chain can use 2 D regions
57 T cells Distinct lineage of cells with unknown functions 1-5% of peripheral blood T cellsIn the gut and epidermis of mice, most T cells express TcRLigands of TcR are unknownPossibly recognise:Antigens without involvement of MHC antigens - CD1Class IB genes
60Summary • The TcR was discovered using clonotypic antibodies • Antibodies and TcR share many similarities, but there are significant differences in structure and function• The structure and organisation of the TcR genes is similar to the Ig genes• Somatic recombination in TcR genes is similar to that in Ig genes• The molecular mechanisms that account for the diversity of TcR include combinatorial and junctional diversity• TcR do not somatically mutate• The highly variable CDR loops map to the distal end of the TcR• The most variable part of the TcR interacts with the peptide