Lecture 8 - Introduction to Unit 3 Flashcards
Compare antigen recognition in the following cells: • B cells • CD8 T cells • CD4 T cells • NKT cells
B cells:
• Recognise whole molecules or pathogens with surface Ig
CD8 T cells:
• Recognise antigen peptide presented in the context of MHC I with α:β TCR
CD4 T cells:
• Recognise antigen peptide presented in the context of MHC II with α:β TCR
NKT cells:
• Recognise lipid and glycolipid antigen presented in the context of CD1d with invariant α:β TCR
Compare development of conventional and NKT cells
Both develop in the thymus
Differentiation into NKT cells and conventional T cells occurs at the DP stage
- NKT cells are selected on CD1d
- CD8 T cells are selected on MHC I
- CD4 T cells are selected on MHC II
Compare the TCR of the various types of T cell
CD4/8 T cells:
- α:β TCR
- Very diverse
NKT cells:
- α:β TCR
- Limited diversity
Describe the structure of TCRs
Two chains:
α and β
Two domains per chain:
- V: variable
- C: constant
CDRs (complimentarily determining region)
- 3 per variable region
- Located around the peptide binding cleft
- Contribute the majority of the diversity of the TCR
Describe the nomenclature for CDRs
CDR(1-3)α
CDR(1-3)β
Describe the various aspects that contribute diversity to the TCR
- α and β chain pairings
- 71 alpha chains
- 35 beta chains
- V, D and J segment pairings
- Junctional diversity
• TdT random insertion of nucleotides at the ends of V, D and J segments
- Imprecise splicing of gene segments
Describe the TCR of NKT cells
α and β TCR α chain:
- Invariant, only one clone
- Vα14 Jα18
β chain:
- Diverse CDR3β
- Vβ7 / Vβ8 / Vβ2
- Dβ2
- Jβ12
Describe the presentation of lipid/glycolipid antigen
APCs present glycolipid/lipid antigen in the context of CD1d
β2-microglobin is associated with CD1d
Describe the structure of the TCR signalling complex
TCR: α and β chains
CD3:
- Stabilises the TCR dimer, allowing for presentation on the surface
- ε, γ, and δ subunits
- ζ with ITAMs
Describe tetramers:
- Structure
- Use in this prac
Four soluble MHC or CD1d molecules, each conjugated to biotin
One streptavidin molecule binds the four biotinylated MHC/CD1d tetramer
Streptavidin conjugated to a fluorophore that allows it to be detected with a laser
Use:
- Identification of T cells
- The MHC/CD1p + Antigen tetramers bind TCRs
- The TCRs are then detected by flow cytometry (the fluorophore on streptavidin bound to the tetramer)
- Identification of NKT cells
- CD1d tetramers + αGalCer Ag
- NKT Cells bind the tetramer through their TCR
- NKT cells acquired with flow cytometry (fluorophore on streptavidin on tetramer)
How can PCR be used to detect specific T cell clones?
PCR of specific TCR genes
What is αGalCer?
Describe the structure
A lipid antigen that is specifically recognised by NKT cells
Structure:
- Galactosyl (sugar) head group
- Ceramide (lipid) tails
Describe how TCRs interact with MHC and peptide
The TCR recognises both the MHC and the peptide being presented α and β chains are equally important for recognition
The CDR3α loop dominates the interaction with the peptide
Docking mode:
- Diagonal / orthogonal
- TCR sits diagonal to MHC
Describe how TCRs interact with CD1d/lipid
αGalCer lipid tails bind deep in CD1d
Sugar head group of αGalCer presented to TCR of NKT cell
NKT cell TCR α chain dominates the binding with CD1d/lipid (binds αGalCer)
NKT cell TCR β chain only binds CD1d
Docking mode:
- Parallel
- TCR docks parallel to CD1d/lipid
Roughly how many clonotypes of T cells are present within an individual?
10^7
Compare diversity of MHC and CD1d between individuals
MHC:
- Polymorphisms exist between individuals
- Means different immune responses between individuals
CD1d:
• Monomorphic
Describe the process of T cell development
- Thymocyte arrives in thymic medulla (vie HEV) from BM
- Thymocyte moves up into cortex and interacts with cTEC
- DN1
- DN2: rearrangement of β chain commences • D → J joining
- DN3:
• V → DJ joining of β chain
- pTα associates with rearranged β chain
- Success signal transducer into DN3 thymocyte
- Burst of proliferation
- DN4
- DP Expression of both CD4 and CD8 11. Rearrangement of α chain
• V → J joining
- DP w/ rearranged TCR
- Positive selection in cortex
• T cell must engage MHC to some extent
- Transient down regulation of CD4 and CD8
- TCR interaction with:
- MHC I engagement → CD8 T cell
- MHC II engagement → CD4 T cell
- CD1d engagement → NKT cell
- Negative selection
- In medulla
- On DCs
- Mature T cell leaves the thymus and circulates in the periphery ***
Where are the various CDRs?
β chain:
- CDR1 and CDR2 are in V region
- CDR3 is across V-D-J joining (thus highly diverse)
α chain:
- CDR1 and CDR2 in V region
- CDR3 across V-J joining (huge diversity)
How many possible α and β chains are there for conventional T cells?
α chain: 71
β chain: 35
Characterise the joining of V, D and J segments in rearragenement of the TCR
Imprecise:
TdT introduces random nucleotides on the ends of the segments
This generates ‘N regions’
Is there any diversity in the TCR of NKT cells?
There is some diversity
The CDR3β can be quite diverse
There are three different possible β chains
What is the function of the CD3 complex?
- Stabilisation of TCR at T cell surface
- Involved in signalling
Which influenza peptides are the target of T cell responses?
NP366
• A nuclear protein
PA224
• An acid polymerase protein When B6 mice are infected with H3N2 influenza, one observed large populations of T cell specific for these two antigens
Describe V, D, or J segment bias of T cells specific for PA224
Vβ7 / aka TRBV29
Vα6 / aka TRAV21
How many possible clonotypes of T cells are there?
How many are there in reality?
1015 possible clonotypes
In reality, only 107, due to positive and negative selection
