The T Cell Antigen Receptor Complex

Generating 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 1996 Isolation of the TCR using monclonal antibodies that were clonotypic i.e recognized single T cell clone Identification of genes of the TCR

Discovery of the T cell antigen receptor (TcR) Polyclonal T cells from an immunised strain A mouse Grow and clone a single antigen-specific T cell in-vitro with antigen, IL-2 and antigen presenting cells Monoclonal (cloned) T cells In vitro “clonal selection” means each daughter cell has the same antigen specificity as the parent cell Most 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

Making anti- clonotypic TcR antibodies T cell clone from a strain A mouse Naïve strain A mouse Make monoclonal antibodies by hybridisation of the spleen cells with a myeloma cell line The strain A mouse will not make antibodies to the hundreds of different molecules associated with strain A T cells due to self tolerance BUT The naïve mouse has never raised T cells with the specificity of the T cell clone, SO the only antigen in the immunisation that the A strain mouse has never seen will be the antigen receptor of the monoclonal T cells

Making 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) Y Monoclonal antibodies Y Y Clone used for immunisation Y Y Y Y Y Y T cell clones Y Y Y Anti-TcR Abs that recognise only one clone of T cells are CLONOTYPIC Hypothesise that anti-clonotype Abs recognise the antigen receptor

Discovery of the T cell antigen receptor (TcR) Lyse cells and add anti-clonotype Ab that binds to unique T cell structures Y Capture anti-clonotype Ab-Ag complex on insoluble support IMMUNOPRECIPITATION Wash away unbound protein Y Elute Ag from Ab and analyse the clonotypically-expresssed proteins biochemically Principal component was a heterodimeric 90kDa protein composed of a 40kDa and a 50kDa molecule ( and  chains) Several other molecules were co-immunoprecipitated.

Structure of the TcR polypeptides T cell clone A T cell clone B T cell clone C Intact TcR chain polypeptides Cyanogen bromide digestion of the  and  proteins Biochemical analysis of digestion products C V Polypeptides contain a variable, clone-dependent pattern of digestion fragments and a fragment common to all TcR

Cloning of the TcR genes B T The experimental strategy The majority of genes expressed by T and B lymphocytes will be similar Genes that greatly differ in their expression are most likely to be directly related to the specialised function of each cell Subtract the genes expressed by B cells from the genes expressed by T cells leaving only the genes directly related to T cell function

Cloning of TcR genes by subtractive hybridisation AAAAA mRNA Hybridise the cDNA and mRNA shared between T and B cells AAAAA T cell single stranded cDNA AAAAA Discard hybrids AAAAA Digest unhybridised B cell mRNA Clone and sequence T cell- specific genes Isolate non-hybridising material specific to T cells

Analysis 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 configuration Ig gene probes can be used as TcR genes will be homologous to Ig genes Restriction enzyme sites 32P GERMLINE DNA V D J C 32P V D J C REARRANGED DNA Find two restriction sites that flank the TcR region Cut the T cell cDNA and placental (i.e. germline) DNA and Southern blot the fragments

TCR Each 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.

The T cell antigen receptor combining site Resembles an Ig Fab fragment Fab VH VL Fc CL CH Va Vb Domain structure: Ig gene superfamily Carbohydrates Monovalent Ca Cb No alternative constant regions Never secreted Hinge Heterodimeric, chains are disuphide-bonded Transmembrane region + + + Very short intracytoplasmic tail Positively charged amino acids in the TM region Cytoplasmic tail Antigen combining site made of juxtaposed Va and Vb regions 30,000 identical specificity TcR per cell

T cell antigen receptor diversity TcR are highly variable in the individual Diversity 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 molecules Mechanisms of diversity closely related to T cell development Random aspects of TcR construction ensures maximum diversity Mechanisms of diversity generation similar to immunoglobulin genes

Generation of diversity in the TcR COMBINATORIAL DIVERSITY Multiple germline segments In the human TcR Variable (V) segments: ~70, 52 Diversity (D) segments: 0, 2 Joining (J) segments: 61, 13 The need to pair  and  chains to form a binding site doubles the potential for diversity JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR

Estimate of the number of human TcR and Ig Excluding somatic hypermutation Immunoglobulin  TcR Element H    Variable segments 40 59 52 ~70 Diversity segments 27 2 D segments in all 3 frames - - Yes Yes Joining segments 6 9 13 61 Joints with N & P nucleotides 2 (1)* 2 1 2360 3640 No. of V gene pairs Junctional diversity ~1013 ~1013 Total diversity ~1016** ~1016 * Only half of human k chains have N & P regions **No of distinct receptors increased further by somatic hypermutation

Organisation of TcR genes L & V x70-80 J x 61 C TcR  L & V x52 D1 Jb1 x 6 C1 D2 Jb2 x 7 C2 TcR  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

TcR a gene rearrangement by SOMATIC RECOMBINATION Germline TcR  Vn J C V2 V1 Rearranged TcR 1° transcript Spliced TcR mRNA Rearrangement very similar to the IgL chains

TcR a gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frame Vn J C V2 V1 Vn+1 a chain tries for a second time to make a productive join using new V and J elements Productively rearranged TcR 1° transcript

TcR b gene rearrangement SOMATIC RECOMBINATION L & V x52 D1 J C1 D2 C2 Germline TcR  D-J Joining V-DJ joining Rearranged TcR 1° transcript C-VDJ joining Spliced TcR mRNA

TcR 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) D1 J C1 D2 C2 Germline TcR  D-J Joining V-DJ joining V 2nd chance at V-DJ joining Need to remove non productive rearrangement Use (DJC)b2 elements

V, D, J flanking sequences Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides. Va 7 23 9 Ja 7 12 9 Db 7 12 9 Vb 7 23 9 Jb

Recombination signal sequences (RSS) HEPTAMER - Always contiguous with coding sequence NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer Vb 7 23 9 Db 12 Jb √ √ Vb 7 23 9 Db 12 Jb 12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

Molecular explanation of the 12-23 rule 23-mer = two turns 12-mer = one turn Intervening DNA of any length 23 Vb 9 7 12 Db Jb 7 9

Molecular explanation of the 12-23 rule V1 D J V2 V3 V4 V8 V7 V6 V5 V9 V1 V2 V3 V4 Loop of intervening DNA is excised V8 V7 V6 V5 Heptamers and nonamers align back-to-back The shape generated by the RSS’s acts as a target for recombinases 7 9 23-mer 12-mer V9 D J An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule)

Mini-circle of DNA is permanently lost from the genome Junctional diversity 7 12 9 23 7 12 9 23 Mini-circle of DNA is permanently lost from the genome Signal joint Coding joint V D J V D J Imprecise 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.

Non-deletional recombination V1 V2 V3 V4 V9 D J Looping out works if all V genes are in the same transcriptional orientation V1 7 23 9 D 12 J How does recombination occur when a V gene is in opposite orientation to the DJ region? V4 V1 V2 V3 V9 D J D J 7 12 9 V4 23

Non-deletional recombination J 7 12 9 V4 23 V4 and DJ in opposite transcriptional orientations D J 7 12 9 V4 23 1. D J 7 12 9 V4 23 2. D J 7 12 9 V4 23 3. D J 7 12 9 V4 23 4.

Fully recombined VDJ regions in same transcriptional orientation 7 12 9 V4 23 1. D J 7 12 9 V4 23 2. Heptamer ligation - signal joint formation D J V4 7 12 9 23 3. V to DJ ligation - coding joint formation D J V4 7 12 9 23 Fully recombined VDJ regions in same transcriptional orientation No DNA is deleted 4.

Steps of TcR gene recombination Recombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSS V 7 23 9 D 7 12 9 J V 7 23 9 The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region D 7 12 9 J The 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 break 7 23 9 12 7 23 9 12 V D J

Steps of TcR gene recombination V D J 7 23 9 12 A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends. V D J The 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 region V D J 7 23 9 12 DNA 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.

Junctional diversity: P nucleotide additions 7 12 9 J V 23 7 V 23 9 TC CACAGTG AG GTGTCAC AT GTGACAC TA CACTGTG 7 D 12 9 J The recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA. D J V TC AG AT TA U 7 D 12 9 J V 23 CACAGTG GTGTCAC GTGACAC CACTGTG TC AG AT TA The 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D region.

V2 V3 V4 V8 V7 V6 V5 V9 7 23 9 CACAGTG GTGTCAC 12 GTGACAC CACTGTG Heptamers are ligated by DNA ligase IV V TC AG U D J AT TA V TC AG U D J AT TA V and D regions juxtaposed

Generation of the palindromic sequence V TC AG U D J AT TA Regions to be joined are juxtaposed Endonuclease cleaves single strand at random sites in V and D segment V TC AG U D J AT TA The nicked strand ‘flips’ out V TC~GA AG D J AT TA~TA The nucleotides that flip out, become part of the complementary DNA strand In 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.

Junctional Diversity – N nucleotide additions Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA V TC~GA AG D J AT TA~TA CACTCCTTA TTCTTGCAA V TC~GA AG D J AT TA~TA CACACCTTA TTCTTGCAA Complementary bases anneal D J TA~TA Exonucleases nibble back free ends V TC~GA CACACCTTA TTCTTGCAA V D J DNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands TC~GA AG AT TA~TA CACACCTTA TTCTTGCAA V TC D TA GTT AT AT AG C

V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity Germline-encoded nucleotides Palindromic (P) nucleotides - not in the germline Non-template (N) encoded nucleotides - not in the germline Creates 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.

How does somatic recombination work? How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity and junctional diversity How do V region find J regions and why don’t they join to C regions? 12-23 rule How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity.

Why do V regions not join to J or C regions? Vb Db Jb C IF the elements of the TcR did not assemble in the correct order, diversity of specificity would be severely compromised DIVERSITY 2x 1x Full potential of the beta chain for diversity needs V-D-J-C joining - in the correct order Were 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

Location of junctional diversity TcR  chain TcR  chain CDR3 CDR1 CDR2 V-J Join V-D Join D-J join Variability Amino acid No. of TcR chain CDR = Complemantarity determining region

T-cell Receptor T 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

T cell co-receptor molecules Lck PTK CD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHC complexes by ~100 fold TcR   CD8 CD4 MHC Class I MHC Class II 3 2

CD8 and CD4 contact points on MHC class I and class II MHC class I MHC class II CD8 binding site CD8 binding site

TcR-CD3 complex         TcR CD3 The intracytoplasmic region of the TcR chain is too short to transduce a signal    The CD3or  (zeta)chains are required for cell surface expression of the TcR-CD3 complex and signalling through the TcR Signalling is initiated by aggregation of TcR by MHC-peptide complexes on APC

Transmission of signals from the cell surface to the nucleus Almost identical to transmission in B cells T 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 to the nucleus continues via three common signalling pathways via: The mitogen-activated protein kinase (MAP kinase) pathway An increase in intracellular calcium ion concentration mediated by IP3 The activation of Protein Kinase C mediated by DAG

Simplified scheme linking antigen recognition with transcription of T cell-specific genes MAP Kinase cascade Small 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 IP3 IP3, 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 DAG DAG stays associated with the membrane and recruits protein kinase C family members. The PKC, serine/threonine protein kinases, ultimately activate the transcription factor NFkB The activated transcription factors AP-1, NFAT and NFkB induce B cell proliferation, differentiation and effector mechanisms

Estimate of the number of human TcR and Ig Excluding somatic hypermutation Immunoglobulin  TcR Element H    Variable segments 40 59 52 ~70 Diversity segments 27 2 D segments in all 3 frames - - Yes Yes Joining segments 6 9 13 61 Joints with N & P nucleotides 2 (1)* 2 1 2360 3640 No. of V gene pairs Junctional diversity ~1013 ~1013 Total diversity ~1016** ~1016 * Only half of human k chains have N & P regions **No of distinct receptors increased further by somatic hypermutation

Why do TcR not undergo somatic mutation? Antigen presentation Y B T cell help Self Antigen Foreign antigen APC Antibody Anergy or deletion of anti-self cells Y T Y B No T cell help Affinity maturation due to somatic mutation

Why do TcR not undergo somatic mutation? B Y B Y B Occasional B cell that somatically mutates to become self reactive Affinity maturation due to somatic mutation

The lack of somatic mutation in TcR helps to prevent autoimmunity T cell help Y T Y B Occasional B cell that somatically mutates to become self reactive X No T cell help Y T T cell that doesn’t mutate can not help the self reactive B cell T cell that mutates can may help the self reactive B cell Autoantibody production

If TcR did undergo somatic mutation: TcR interacts with entire top surface of MHC-peptide antigen complex Somatic mutation in the TcR could mutate amino acids that interact with the MHC molecule causing a complete loss of peptide-MHC recognition

If TcR did undergo somatic mutation: TcR-MHC interaction is one of many between the T cell and APC On-off rate of TcR determines rate of ‘firing’ to give qualitatively different outcomes Must 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

Why do B cell receptors need to mutate? Neutralisation of bacterial toxins Y ` Ab-Ag interaction must be of high affinity to capture and neutralise toxins in extracellular fluids There is a powerful selective advantage to B cells that can somatically mutate their receptors to increase affinity SOMATIC MUTATION Y ` Toxin binding blocked Prevents toxicity

An alternative TcR:  Discovered as Ig-homologous, rearranging genes in non  TcR T cells V V J C 3x D 3x J 1x C Human  locus 3x J C1 2x J C2 12x V1 The  locus is located between the V and J regions V to J rearrangement deletes D, Jand CTcR cells can not express g TcR Few V regions, but considerable junctional diversity as  chain can use 2 D regions

 T cells Distinct lineage of cells with unknown functions 1-5% of peripheral blood T cells In the gut and epidermis of mice, most T cells express  TcR Ligands of  TcR are unknown Possibly recognise: Antigens without involvement of MHC antigens - CD1 Class IB genes

Summary • 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