The T Cell AntigenReceptor 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 cellsfrom an immunised

strain A mouse

Monoclonal (cloned) T cells

Grow and clone a single antigen-specific T cell in-vitro with antigen, IL-2 and antigen presenting 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 clonefrom a strain A mouse Naïve strain A mouse

The strain A mouse will not make antibodies to the hundreds of different molecules associated with strain A T cells due to self tolerance

BUTThe 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

Make monoclonal antibodies by hybridisation of the spleen cellswith a myeloma cell line

Anti-TcR Abs that recognise only one clone of T cells are CLONOTYPICHypothesise that anti-clonotype Abs recognise the antigen receptor

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)

Making anti- clonotypic TcR antibodies

YY Y

Y

YYYYYYYYMonoclonal antibodies

T cell clones

Clone used for immunisation

Lyse cells and add anti-clonotype Abthat binds to unique T cell structures

YYYYCapture anti-clonotype Ab-Agcomplex on insoluble supportIMMUNOPRECIPITATIONWash away unbound protein

Elute Ag from Ab and analyse the clonotypically-expresssed proteins biochemically

Principal component was a heterodimeric 90kDa proteincomposed of a 40kDa and a 50kDa molecule ( and chains)

Several other molecules were co-immunoprecipitated.

Discovery of the T cell antigen receptor (TcR)

Y YYYY YYYY

YYYY

Structure of the TcR polypeptides

Cyanogen bromide digestion of the and proteins

Biochemical analysis of digestion products

T cell clone A T cell clone B T cell clone C

Polypeptides contain a variable, clone-dependent pattern of digestion fragments and a fragment common to all TcR

Intact TcR chain polypeptides

C C CV VV

Cloning of the TcR genes

BT

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

AAAAA

AAAAA

Isolate non-hybridising material specific to T cells

T cell singlestranded cDNA

Cloning of TcR genes by subtractive hybridisation

BTAAAAAAAAAA

mRNA

Discard hybridsAAAAA

Digest unhybridised B cell mRNA

Clone andsequence T cell-specific genes

Hybridise the cDNA and mRNA shared

between T and B cellsAAAAA

Analysis of T cell-specific genes

GERMLINEDNAV D J C

32P

V DJ CREARRANGEDDNA

Restrictionenzyme sites

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

Find two restriction sites that flank the TcR region

Ig gene probes can be used as TcR genes will be homologous to Ig genes

Cut the T cell cDNA and placental (i.e. germline) DNAand Southern blot the fragments

32P

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

Va Vb

Ca Cb

Carbohydrates

Hinge

Monovalent

Resembles an Ig Fab fragment

Fab

VHVL

Fc

CL

CH

VLVH

CH CL

CH CH

CHCH

No alternative constant regions

Transmembrane region

Never secreted

Domain structure: Ig gene superfamily

Heterodimeric, chains are disuphide-bonded

Cytoplasmic tail

Very short intracytoplasmic tail++

+Positively charged amino acids in the

TM region

Antigencombining site

Antigen combining site made of juxtaposed Va and Vb regions

30,000 identical specificity TcR per cell

• 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

T cell antigen receptor diversity

Generation of diversity in the TcR

COMBINATORIAL DIVERSITYMultiple germline segments

In the human TcR

Variable (V) segments: ~70, 52Diversity (D) segments: 0, 2Joining (J) segments: 61, 13

The need to pair and chains to form a binding sitedoubles the potential for diversity

JUNCTIONAL DIVERSITYAddition 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

ElementImmunoglobulin TcR

Variable segments

Diversity segments

D segments inall 3 frames

Joining segments

Joints with N & Pnucleotides

No. of V gene pairs

Junctional diversity

Total diversity

H

40

27

Yes

6

22360 3640

~1013 ~1013

~1016** ~1016

59

0

-

9

(1)*

52 ~70

2 0

Yes -

13 61

2 1

* Only half of human k chains have N & P regions**No of distinct receptors increased further by somatic hypermutation

Estimate of the number of human TcR and IgExcluding somatic hypermutation

TcR

Organisation of TcR genes

L & Vx70-80 C

TcR

D1 Jb1 x 6 C1 D2 Jb2 x 7 C2

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

J x 61

L & Vx52

TcR a gene rearrangement bySOMATIC RECOMBINATION

Spliced TcR mRNA

Germline TcR

Vn J CV2 V1

Rearranged TcR1° transcript

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

Vn J CV2 V1Vn+1

a chain tries for a second time to make a productive join using new V and J elements

Productivelyrearranged TcR

1° transcript

Rearranged TcR 1° transcript

Spliced TcR mRNA

L & Vx52 D1 J C1 D2 J C2

Germline TcR

TcR b gene rearrangementSOMATIC RECOMBINATION

D-J Joining

V-DJ joining

C-VDJ joining

D1 J C1 D2 J C2

Germline TcR

D-J Joining

V-DJ joining

V

TcR b gene rearrangement RESCUE PATHWAYThere 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)

2nd chance atV-DJ joining

Need to remove non productiverearrangement

Use (DJC)b2 elements

V, D, J flanking sequences

Va 7 23 9

Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides.

Ja7129

Db7129 7 12 9

Vb 7 23 9 Jb7239

Recombination signal sequences (RSS)

12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

Vb 7 23 9

Db7129 7 12 9

Jb7239

HEPTAMER - Always contiguous with coding sequence

NONAMER - Separated fromthe heptamer by a 12 or 23

nucleotide spacer

Vb 7 23 9

Db7129 7 12 9

Jb7239

√ √

23-mer = two turns 12-mer = one turn

Molecular explanation of the 12-23 rule

Intervening DNAof any length23

Vb 97

12

DbJb79

23-mer

12-mer

Loop of intervening

DNA is excised

• Heptamers and nonamers

align back-to-back

• The shape generated by the

RSS’s acts as a target for

recombinases

7

9

97

V1 V2 V3 V4

V8V7

V6V5

V9 D J

V1 D J

V2

V3

V4

V8

V7

V6

V5

V9

• An appropriate shape can not be formed if two 23-mer flanked elements

attempted to join (i.e. the 12-23 rule)

Molecular explanation of the 12-23 rule

V D J712

9

723

9

7 12 97239

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.

Junctional diversity

Mini-circle of DNA is permanently lost from the

genome

Signal jointCoding joint

V1 V2 V3 V4 V9 D J

Looping out works if all V genes are in the same transcriptional orientation

V1 V2 V3 V9 D J

Non-deletional recombination

D J7129V47239

V1 7 23 9 D7129 J

How does recombination occur when a V gene is in opposite orientation to the DJ region?

V4

D J7129V47239V4 and DJ in opposite transcriptional orientations

DJ

712

9V47239

1.

DJ

712

9

V47239

3.

DJ7

129

V47239

2.

D J7129

V472394.

Non-deletional recombination

D J7129

V47239

1.

D J

V4

7129

7239

3.

V to DJ ligation - coding joint formation

D J7129

V47239

2.

Heptamer ligation - signal joint formation

D JV47 12 97239

Fully recombined VDJ regions in same transcriptional orientationNo DNA is deleted

4.

V 7 23 9

D7 12 9J

V 7 23 9

7 23 9

7 12 9D7129 J

7 23 9

7 12 9

V

DJRecombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSS

The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region

• 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

Steps of TcR gene recombination

V

DJ

7 23 9

7 12 9

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 72

39

71

29

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.

Steps of TcR gene recombination

7D 12 9J

Junctional diversity: P nucleotide additions

7V 23 9

D7 12 9J

V 7 23 9TC CACAGTGAG GTGTCAC

AT GTGACACTA CACTGTG

The recombinase complex makes single stranded nicks at random sites close to the

ends of the V and D region DNA.

7D 12 9J

7V 23 9CACAGTGGTGTCAC

GTGACACCACTGTG

TCAG

ATTADJ

V TCAG

ATTA

UU

The 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D

region.

V2V3

V4

V8

V7V6

V5

V9

7 23 9CACAGTGGTGTCAC

7 12 9GTGACACCACTGTG

V TCAG U

DJ ATTA U

Heptamers are ligated by DNA ligase IV

V and D regions juxtaposed

V TCAG U D JAT

TA

U

V TCAG U D JAT

TA

U Endonuclease cleaves single strand at random sites in V and D segment

V TC~GAAG D JAT

TA~TAThe nucleotides that flip out, become part of the complementary DNA strand

Generation of the palindromic sequence

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.

V TCAG U D JAT

TA

U

Regions to be joined are juxtaposed

The nicked strand ‘flips’ out

Junctional Diversity – N nucleotide additions

V TC~GAAG D JAT

TA~TA

Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA

CACTCCTTA

TTCTTGCAA

V TC~GAAG D JAT

TA~TA

CACACCTTA

TTCTTGCAA Complementary bases anneal

V D JDNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands

TC~GAAG

ATTA~TA

CACACCTTA

TTCTTGCAA

D JTA~TAExonucleases nibble back free endsV TC~GACACACCTTA

TTCTTGCAA

VTC

DTA

GTT AT AT

AG C

V D JTCGACGTTATATAGCTGCAATATA

Junctional Diversity

TTTTTTTTTTTTTTT

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?

1. How is an infinite diversity of specificity generated from finite

amounts of DNA?

Combinatorial diversity and junctional diversity

2. How do V region find J regions and why don’t they join to C regions?

12-23 rule

3. 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?

IF the elements of the TcR did not assemble in the correct order, diversity of specificity would be severely compromised

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

DIVERSITY

2x

DIVERSITY

1x

Vb Db Jb C

V-DJoin

D-Jjoin

TcR chain

V-JJoin

TcR chain

Location of junctional diversity

Amino acid No.of TcR chain

Var

iabi

lity

CDR1CDR2

CDR3

CDR = Complemantarity determining region

2/9/04

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.

T cell co-receptor molecules

CD8

MHC Class I MHC Class II

3 2

TcR TcR

CD4

Lck PTK Lck PTK

CD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHCcomplexes by ~100 fold

MHC class II

CD8 and CD4 contact points on MHCclass I and class II

CD8 binding site

MHC class I

CD8 binding site

TcR-CD3 complex

TcR

CD3CD3

The intracytoplasmic regionof the TcR chain is too short

to transduce a signal

Signalling is initiated by aggregation of TcR by MHC-peptide complexes on APC

The CD3or (zeta)chainsare required for cell surfaceexpression of the TcR-CD3

complex and signallingthrough the TcR

Transmission of signals from the cellsurface to the nucleus

• 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 tothe nucleus continues via three common signalling pathways via:

1.The mitogen-activated protein kinase (MAP kinase) pathway2.An increase in intracellular calcium ion concentration mediated by IP3

3.The activation of Protein Kinase C mediated by DAG

Almost identical to transmission in B cells

• 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 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 DAGDAG 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

Simplified scheme linking antigen recognition with transcription of T cell-specific genes

ElementImmunoglobulin TcR

Variable segments

Diversity segments

D segments inall 3 frames

Joining segments

Joints with N & Pnucleotides

No. of V gene pairs

Junctional diversity

Total diversity

H

40

27

Yes

6

22360 3640

~1013 ~1013

~1016** ~1016

59

0

-

9

(1)*

52 ~70

2 0

Yes -

13 61

2 1

* Only half of human k chains have N & P regions**No of distinct receptors increased further by somatic hypermutation

Estimate of the number of human TcR and IgExcluding somatic hypermutation

Self Antigen

Foreign antigen

APC

YT

Antigen presentation

YB

T cell help

YT

Anergy or deletionof anti-self cells

YB

NoT cell help

Affinity maturation due to somatic

mutation

Antibody

Why do TcR not undergo somatic mutation?

YB

Occasional B cellthat somatically mutatesto become self reactive

Why do TcR not undergo somatic mutation?

YB

Affinity maturation due to somatic mutation

YBY

B

YB

YB

YB

YB

Occasional B cellthat somatically mutatesto become self reactive

T cell that doesn’t mutatecan not help the

self reactive B cell

YT

XNo T cell help

YT

T cell that mutates can may help the self reactive

B cell

T cell help

Autoantibody production

The lack of somatic mutation in TcR helpsto prevent autoimmunity

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

Y`

`

Y` `

Toxin bindingblocked

Preventstoxicity

Why do B cell receptors need to mutate?

Neutralisation ofbacterial toxins

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

An alternative TcR:

Discovered as Ig-homologous, rearranging genes in non TcR T cells

The locus is located between the V and J regions

V to J rearrangement deletes D, Jand CTcR cells can not express g TcR

Few V regions, but considerable junctional diversity as chain can use 2 D regions

3x D 3x J 1x C

Human locus

3x J C1 2x J C212x V1

V V VVV J C

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

• 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

Summary