IMMU lecturee notes 6
Transcript of IMMU lecturee notes 6
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IMMU3201L6: TCR
Comparison of antigen recognition by B cells and T cells
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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
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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
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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
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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)
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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
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Junctional combination generates more receptor types
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Pre-T cell receptor
- Cant produce pre-T cellcant produce mature T cell
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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
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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)