IMMU3201 L6: 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 directly bind 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 hindered antigen (antigen that sits in the binding groove of
MHC)
- B cells see sterically exposed antigen (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 TCR and BCR
- The immunoglobulin fold has a Beta sheet which keeps the structure stable
- Immunoglobulin have hypervariable loops
o The loops provide a way of making a binding site that can be varied







T cell Receptor (TCR)
- TCRs are constitutively expressed by T cells
- They are a Hypervariable heterodimer
- There are 2 types of TCR: ,
o there are 2 chains in 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 specifically binds to antigen in association with MHC molecule
- TCRs bind to antigen in association with either MHC class I or 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 subsets of TCRs expressed by NK T cells recognise non-classical MHC
molecule (e.g. CD1) and glycolipids
- TCRs are structurally more like antibody than TCRs
o TCRs have a long CDR3


















Structure of the T cell receptor

In TCR and TCR:
- and are heavy chains
- and are light chains

- Top half is the variable region
- Bottom half is the constant regions


Crystal structure of TCR (right)
- hypervariable loops at the top form the binding site
- hinge region in the middle
- constant region at the bottom
- the constant regions just above the transmembrane region are disulfide bonded
together

- there are 3 loops in each Ig fold or domain called complementarity determining
regions or CDRs because its the complementarity between the loop and the antigen
structure that determines the affinity Thus CDRs 1, 2, 3
TCR domain

- The dotted boxes indicate where the CDR is within the TCR domain
- CDRs 1, 2, 3 in both and chains are in the same position

- Difference between and chain is the presence of D

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)