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MIIM30015 - Techniques in Immunology > Lecture 8 - Introduction to Unit 3 > Flashcards

Lecture 8 - Introduction to Unit 3 Flashcards

1
Q

Compare antigen recognition in the following cells: • B cells • CD8 T cells • CD4 T cells • NKT cells

A

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

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2
Q

Compare development of conventional and NKT cells

A

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
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3
Q

Compare the TCR of the various types of T cell

A

CD4/8 T cells:

  • α:β TCR
  • Very diverse

NKT cells:

  • α:β TCR
  • Limited diversity
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4
Q

Describe the structure of TCRs

A

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
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5
Q

Describe the nomenclature for CDRs

A

CDR(1-3)α

CDR(1-3)β

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6
Q

Describe the various aspects that contribute diversity to the TCR

A
  1. α and β chain pairings
  • 71 alpha chains
  • 35 beta chains
  1. V, D and J segment pairings
  2. Junctional diversity

• TdT random insertion of nucleotides at the ends of V, D and J segments

  1. Imprecise splicing of gene segments
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7
Q

Describe the TCR of NKT cells

A

α and β TCR α chain:

  • Invariant, only one clone
  • Vα14 Jα18

β chain:

  • Diverse CDR3β
  • Vβ7 / Vβ8 / Vβ2
  • Dβ2
  • Jβ12
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8
Q

Describe the presentation of lipid/glycolipid antigen

A

APCs present glycolipid/lipid antigen in the context of CD1d

β2-microglobin is associated with CD1d

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9
Q

Describe the structure of the TCR signalling complex

A

TCR: α and β chains

CD3:

  • Stabilises the TCR dimer, allowing for presentation on the surface
  • ε, γ, and δ subunits
  • ζ with ITAMs
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10
Q

Describe tetramers:

  • Structure
  • Use in this prac
A

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:

  1. 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)
  1. 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)
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11
Q

How can PCR be used to detect specific T cell clones?

A

PCR of specific TCR genes

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12
Q

What is αGalCer?

Describe the structure

A

A lipid antigen that is specifically recognised by NKT cells

Structure:

  • Galactosyl (sugar) head group
  • Ceramide (lipid) tails
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13
Q

Describe how TCRs interact with MHC and peptide

A

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
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14
Q

Describe how TCRs interact with CD1d/lipid

A

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
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15
Q

Roughly how many clonotypes of T cells are present within an individual?

A

10^7

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16
Q

Compare diversity of MHC and CD1d between individuals

A

MHC:

  • Polymorphisms exist between individuals
  • Means different immune responses between individuals

CD1d:

• Monomorphic

17
Q

Describe the process of T cell development

A
  1. Thymocyte arrives in thymic medulla (vie HEV) from BM
  2. Thymocyte moves up into cortex and interacts with cTEC
  3. DN1
  4. DN2: rearrangement of β chain commences • D → J joining
  5. DN3:

• V → DJ joining of β chain

  1. pTα associates with rearranged β chain
  2. Success signal transducer into DN3 thymocyte
  3. Burst of proliferation
  4. DN4
  5. DP Expression of both CD4 and CD8 11. Rearrangement of α chain

• V → J joining

  1. DP w/ rearranged TCR
  2. Positive selection in cortex

• T cell must engage MHC to some extent

  1. Transient down regulation of CD4 and CD8
  2. TCR interaction with:
  • MHC I engagement → CD8 T cell
  • MHC II engagement → CD4 T cell
  • CD1d engagement → NKT cell
  1. Negative selection
  • In medulla
  • On DCs
  1. Mature T cell leaves the thymus and circulates in the periphery ***
18
Q

Where are the various CDRs?

A

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)
19
Q

How many possible α and β chains are there for conventional T cells?

A

α chain: 71

β chain: 35

20
Q

Characterise the joining of V, D and J segments in rearragenement of the TCR

A

Imprecise:

TdT introduces random nucleotides on the ends of the segments

This generates ‘N regions’

21
Q

Is there any diversity in the TCR of NKT cells?

A

There is some diversity

The CDR3β can be quite diverse

There are three different possible β chains

22
Q

What is the function of the CD3 complex?

A
  • Stabilisation of TCR at T cell surface
  • Involved in signalling
23
Q

Which influenza peptides are the target of T cell responses?

A

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

24
Q

Describe V, D, or J segment bias of T cells specific for PA224

A

Vβ7 / aka TRBV29

Vα6 / aka TRAV21

25
Q
A
26
Q

How many possible clonotypes of T cells are there?

How many are there in reality?

A

1015 possible clonotypes

In reality, only 107, due to positive and negative selection

MIIM30015 - Techniques in Immunology (9 decks)

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