IMMU lecturee notes 6

8/10/2019 IMMU lecturee notes 6

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IMMU3201L6: TCR

Comparison of antigen recognition by B cells and T cells

-

T cells bind to antigen in association with MHC molecule

o i.e TCR binds to MHC-Ag (peptide) complex

- B cell can directlybind to free antigen therefore B cells have higher affinities

o i.e. Antigen directly binds to BCR or mIg (membrane bound Ig or antibody)

- T cells can only see peptides that bind to MHC

- B cells can see almost anything with mass

- B cells see shapes (conformations)

- T cells see peptides (linear fragment of original protein)

- T cells see sterically hinderedantigen (antigen that sits in the binding groove of

MHC)

- B cells see sterically exposedantigen (free floating antigen)

The Immunoglobulin Fold in TCR and BCR

- The immunoglobulin fold is a recurring motif in immune activation molecules

- Immunoglobulin fold is present in both TCRand BCR

- The immunoglobulin fold has a Beta sheetwhich keeps the structure stable

- Immunoglobulin have hypervariable loops

o The loops provide a way of making a binding site that can be varied


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T cell Receptor (TCR)

- TCRs are constitutively expressed by T cells

- They are a Hypervariable heterodimer

-

There are 2 typesof TCR: ,

o there are 2 chainsin each TCR type: chain, chain

o there are 2 Ig folds (or domains) in each chain (i.e. 4 Ig domain in each TCR)

- TCR specificallybinds to antigenin association with MHC molecule

- TCRs bind to antigen in association with either MHC class Ior MHC class II

o TCRs recognising MHC class I with peptide MHC class I-restricted

o TCRs recognising MHC class II with peptide MHC class II-restricted

-

Some subsetsof TCRs expressed by NK T cellsrecognise non-classical MHC

molecule(e.g. CD1) and glycolipids

- TCRs are structurally more like antibodythan TCRs

o TCRs have a long CDR3


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Structure of the T cell receptor

In TCRand TCR:

- and are heavy chains

- and are light chains

- Top half is the variable region

- Bottom half is the constant regions

Crystal structureof TCR(right)

- hypervariable loopsat the top form the binding site

-

hingeregion in the middle

- constant region at the bottom

- the constant regions just above the transmembrane regionare disulfide bonded

together

- there are 3 loopsin each Ig foldor domaincalled complementarity determining

regionsor CDRs because its the complementarity between the loop and the antigen

structure that determines the affinityThus CDRs 1, 2, 3


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Human TCR genes

- In human TCR chain locus:

o duplication of DJC

o upstream75 Vs (V1to V75)

o

downstreamrepetition of DJC D1J1C1 D2J2C2

- In human TCR AND chain locus:

o More complicated than chain locuspresence of locus in the middle

o If you rearrange chain, you will have removedthe locus(unless you do an

inversion)

o In the locus:

upstreammany Vs (50), many Js (50-70) and ONE C

middle chain locus

- In human TCR chain locus


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TCR gene recombination and transcription

- Every lymphocyte has different DNA due to somatic recombination(changing the

DNA around to produce different receptors)


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To produce chain:

1. In Germline DNA: D-Jjoining via somatic recombination(DNA rearrangement)

2. D-Jjoins with a V

3.

New V-D-Jis transcribedinto RNA

To produce chain

1. In Germline DNA: V-Jjoining via somatic recombination

2. New V-Jis transcribed intoRNA

TCR mRNA processing, translation and protein modification

- with chain RNA

1.

RNA isprocessed (splicingto splice out parts)to produce mRNA

2. mRNA is translated

3. mRNAis processed and glycosylationa chain is produced

- with chain RNA

1. RNA isprocessed (splicingto splice out parts)to produce mRNA

2. mRNA is translated

3.

mRNAis processedan chain is produced


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Splicing How is RNA spliced?

- RNA is spliced according to the Heptamer-nonamerrulethe rule applies to both

TCR and BCR

The Heptamer-nonamer rule

- The regional genes (V, D, J), used to generate TCRsand Ig molecules, are flanked

by recombination signal sequencesor RSSsthat are recognized by a group of

enzymes known collectively as the VDJ recombinase.

- RSSs are composed of seven conserved nucleotides (a heptamer) that reside next to

the gene encoding sequence followed by a spacer (containing either 12 or 23

unconserved nucleotides) followed by a conserved nonamer(9 base pairs).

- The RSSs are present on the 3 side(downstream) of a V region and the 5side

(upstream) of the J region. These are the sides that will be involved in the joining.

- Only a pair of dissimilar spacer RSSs are efficiently recombined (i.e. one with a spacer

of 12 nucleotides will be recombined with one that has a spacer containing 23

nucleotides). This is known as the 12/23 rule of recombination (or the one-turn/two-

turn rule).


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Two

ways the V and J can combine: Deletion (splicing) or Inversion

- Deletion is the common mechanism

Generating Junctional diversity

- Junctional diversity allows many more different receptors to be generated

- Nucleotide are added or deleted at the junction


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Process of junctional diversity

- Junctional diversity concludes the process of somatic recombinationor V(D)J

recombination, during which the different variable segments involved in antigen

recognition of TCRs and immunoglobulinsare rearranged and unused segments

removed.

1. A double-strandbreak between the required segments is introduced.

2. These ends form hairpin loopsand must be joined together to form a single strand

(summarised in diagram).

3. This joining is a very inaccurate processthat results in the variable additionor

subtraction ofnucleotidesand, thus, generates junctional diversity.

- Generation of junctional diversity starts as the enzymes, VDJ recombinase, along

with DNA repair proteins, are responsible for single-strandedcleavage of the

hairpin loopsand addition of a series of palindromic, 'P' nucleotides.

- An enzyme adds further random N nucleotides.

- The newly synthesised strands anneal to one another, but mismatchesare common.

- Enzymes remove these unpaired nucleotides and the gaps are filled by DNA

synthesis and repair machinery.

- Enzymes may also cause shortening of this junction

- Junctional diversity is liable to cause frame-shift mutations (out of frame sequence)

and thus produce non-functional proteins.

- Therefore, there is considerable waste involved in this process.


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V region Combination vs. Junctional Combination

-

Junctional combination generates more receptor types


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Pre-T cell receptor

- Cant produce pre-T cellcant produce mature T cell

o

Pre-TCR signal initiate recombination at the TCR chainlocus and drive the

transitionfrom double-negative thymocytes to double-positive thymocytes

(next level of T cell maturation)

o Double-positive thymocytes express CD4and CD8

o Pre-TCR signals also inhibit further TCR chainrecombination

o Pre-TCR signals mediate survivalof pre-T cells and contribute to pre-T cell

proliferation

CD3 complex

- CD3 complex is:

o constitutively expressed

o required for surface expression of or TCR

- CD3, CD3, CD3each have:

o one extracellularIg domain

o one activating ITAM motif in the cytoplasmic tail

- TCR zetachain is mainly intracellular and has NO Ig domainsbut has 3activating

ITAM motifs

- CD3 complex is responsible for signal transductioni.e. it transmits signals from

or TCRinto the T cell


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Co-receptor molecules: CD4 and CD8

CD4

- is expressed constitutively

-

is a monomerand is invariant(never changing)

- binds to Class II MHC

CD8

- is expressed constitutively

- is made up of and subunitsform homo-dimer() or hetero-dimer()

- binds to Class I MHC

Similarities between CD4 and CD8

- Both increaseability of interaction by stabilising TCR-MHC-peptide(DC-T-cell

interaction)

- Both recruitsignalling molecules including Ickto the TCR complexthey amplify

TCR recognition


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Co-stimulatory Molecules: CD28

- CD28:

o is constitutively expressed but may be downregulatedafter chronic

stimulation

o is an invariant homodimer

o has ONE Ig domainper chain

o monovalentbinding of B7 molecules (CD80, CD86) on APCs

o amplifies the activation signals from TCR

Inhibitory molecules: CTLA-4 (CD152)

CTLA-4

- is ahomologue (structurally similar)of CD28

- is an invariant homodimer

- is NOT expressed on nave T cellsexpressed after T cell activation

- downregulatesT cell activation

- Expressed by Tregs(regulatory T cells)

Adhesion molecules: LFA-1

LFA-1

- is an 2 integrin

- is a dimer of L (CD11a) and 2 (CD18)

- mediates leukocyte adhesionvia binding to ICAM-1 (CD54)

- allow strong adhesion between APCand T-cellstabilises interaction

- is involved in forming immunological synapse


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Immunological synapse

- in a mature immunological synapse:

o between LFA-1/ICAM-1 and TCR/MHC there is a zone of co-stimulatory

molecules: (CD28/CD80, CD86)

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IMMU lecturee notes 6