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in the C chain
o V-variable, D-diversity, J-joining, C-constant
- chain 2 joining needs to be made: V-D and D-J
- chain 1 joining needs to be made: V-J
Human TCR genes
- In human TCR chain locus:
o duplication of DJC
o upstream 75 Vs (V
1
to V
75
)
o downstream repetition of DJC D
1
J
1
C
1
D
2
J
2
C
2
- In human TCR AND chain locus:
o More complicated than chain locus presence of locus in the middle
o If you rearrange chain, you will have removed the locus (unless you do an
inversion)
o In the locus:
upstream many Vs (50), many Js (50-70) and ONE C
middle chain locus
- In human TCR chain locus
TCR gene recombination and transcription
- Every lymphocyte has different DNA due to somatic recombination (changing the
DNA around to produce different receptors)
To produce chain:
1. In Germline DNA: D-J joining via somatic recombination (DNA rearrangement)
2. D-J joins with a V
3. New V-D-J is transcribed into RNA
To produce chain
1. In Germline DNA: V-J joining via somatic recombination
2. New V-J is transcribed into RNA
TCR mRNA processing, translation and protein modification
- with chain RNA
1. RNA is processed (splicing to splice out parts) to produce mRNA
2. mRNA is translated
3. mRNA is processed and glycosylation a chain is produced
- with chain RNA
1. RNA is processed (splicing to splice out parts) to produce mRNA
2. mRNA is translated
3. mRNA is processed an chain is produced
Splicing How is RNA spliced?
- RNA is spliced according to the Heptamer-nonamer rule the rule applies to both
TCR and BCR
The Heptamer-nonamer rule
- The regional genes (V, D, J), used to generate TCRs and Ig molecules, are flanked
by recombination signal sequences or RSSs that 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 5 side
(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).
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
Process of junctional diversity
- Junctional diversity concludes the process of somatic recombination or V(D)J
recombination, during which the different variable segments involved in antigen
recognition of TCRs and immunoglobulins are rearranged and unused segments
removed.
1. A double-strand break between the required segments is introduced.
2. These ends form hairpin loops and must be joined together to form a single strand
(summarised in diagram).
3. This joining is a very inaccurate process that results in the variable addition or
subtraction of nucleotides and, thus, generates junctional diversity.
- Generation of junctional diversity starts as the enzymes, VDJ recombinase, along
with DNA repair proteins, are responsible for single-stranded cleavage of the
hairpin loops and addition of a series of palindromic, 'P' nucleotides.
- An enzyme adds further random N nucleotides.
- The newly synthesised strands anneal to one another, but mismatches are 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.
V region Combination vs. Junctional Combination
- Junctional combination generates more receptor types
Pre-T cell receptor
- Cant produce pre-T cell cant produce mature T cell
o Pre-TCR signal initiate recombination at the TCR chain locus and drive the
transition from double-negative thymocytes to double-positive thymocytes
(next level of T cell maturation)
o Double-positive thymocytes express CD4 and CD8
o Pre-TCR signals also inhibit further TCR chain recombination
o Pre-TCR signals mediate survival of 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, CD3 each have:
o one extracellular Ig domain
o one activating ITAM motif in the cytoplasmic tail
- TCR zeta chain is mainly intracellular and has NO Ig domains but has 3 activating
ITAM motifs
- CD3 complex is responsible for signal transduction i.e. it transmits signals from
or TCR into the T cell
Co-receptor molecules: CD4 and CD8
CD4
- is expressed constitutively
- is a monomer and is invariant (never changing)
- binds to Class II MHC
CD8
- is expressed constitutively
- is made up of and subunits form homo-dimer () or hetero-dimer ()
- binds to Class I MHC
Similarities between CD4 and CD8
- Both increase ability of interaction by stabilising TCR-MHC-peptide (DC-T-cell
interaction)
- Both recruit signalling molecules including Ick to the TCR complex they amplify
TCR recognition
Co-stimulatory Molecules: CD28
- CD28:
o is constitutively expressed but may be downregulated after chronic
stimulation
o is an invariant homodimer
o has ONE Ig domain per chain
o monovalent binding of B7 molecules (CD80, CD86) on APCs
o amplifies the activation signals from TCR
Inhibitory molecules: CTLA-4 (CD152)
CTLA-4
- is a homologue (structurally similar) of CD28
- is an invariant homodimer
- is NOT expressed on nave T cells expressed after T cell activation
- downregulates T 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 adhesion via binding to ICAM-1 (CD54)
- allow strong adhesion between APC and T-cell stabilises interaction
- is involved in forming immunological synapse
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)