Assays for antigenspecific T cells

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Assays for antigen-specific T cells

• Proliferation
• Cytotoxicity
• Cytokine release
• ELISPOT
• Intracellular cytokine expression

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Lessons from structural biologists and biochemists
Soluble molecules for crystallography and
biochemical studies Required high purity and
homogeneity AND yields of gt20mg
Expression systems for recombinant proteins
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HLA-class I heavy chain for mammalian and insect
cell expression systems
Signal Sequence
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• Mammalian expression systems
• Secrete properly folded MHC molecules
• Heterogeneous peptides

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Insect cell expression systems

• Good yields of soluble class I molecules.
• Growth at 22-280C allows the production of empty
  class I molecules

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Bacterial Expression Systems

• Large quantities of protein-denatured
• Quick/cheap
• Association of b2microglobulin with class I heavy
  chain is peptide dependent
• Class I heavy chains produced in bacteria are
  insoluble in the absence of peptide and
  b2microglobulin.

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HLA-class I heavy chain for bacterial expression
systems
Signal Sequence
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Inclusion body preparations of recombinant H-2Kb
heavy chain and b2microglobulin.
H-2Kb
b2m
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HLA-B8/EBV
H-2Kb/Ova
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Are there uses for refolded MHC molecules?
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• MHC molecules complexed to a specific peptide
  potentially provide a reagent to identify
  Ag-specific T cells by their Ag-specific
  receptor?

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Problem

• The affinity of MHC/TcR interactions is weak
  (usually 5-20µM).
• (Too weak for stable binding)
• Stability of MHC/peptide complexes

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The solution... increased avidity through the use
of multimeric complexes

• Potential approaches
• Ig fusion proteins
• Biotinylation
• amine-coupling
• site-specific

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BirA (biotin ligase)

• BirA catalyses the addition of a biotin molecule
  to specific substrate sequences.
• These substrate sequences can be as small as 12
  amino acids
• These sequences can be used as tags to allow
  site-specific biotinylation of proteins

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BirA substrate peptide
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Modified HLA-class I heavy chain for bacterial
expression system
Signal Sequence
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BirA substrate peptide
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Cautionary notes

• Not all HLA class I alleles appear to refold
  equally well.
• The efficiency of refolding is dependent on the
  affinity of the peptide for class I.
• The sequence of the peptide can be problematic.

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Uses for tetrameric MHC/peptide complexes

• Quantitation of antigen-specific T cells.
• Correlation of Ag-specificity with the expression
  of other cell surface receptors (eg activation
  markers)
• Analysis of T cell receptor affinity/avidity.
• Histochemistry?

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Staining cells with tetramers

• Choice of fluorochromes
• Temperature
• Cell numbers

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LC13
MS01
Events
Events
RAKFKQLL FLRGRAYGL
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HLA-B8-, EBV
HLA-B8, EBV
HLA-B8, EBV -
(a)
CD8 - TRI COLOR
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Stability of TCD8 estimates
of Tetramer staining TCD8 cells (Donation 2)
of Tetramer staining TCD8 cells (Donation 1)
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H-2Kb/mb2m/gB tetramers stain Ag-specific cells
from herpes virus infected mice.
Infected
Naive
CD8
gB/Kb tetramer
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CD25 HSV-specific Tcells are found predominantly
in the draining lymph node.
gB-specific cells that express CD25
Effector CTL expand in the periphery
gB-specific cells that express CD43
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Specificity and function?

• Combining the use of tetramers with intracellular
  cytokine staining offers the possibility of
  assaying both specificity and function.
• Problem Downregulation of TcR following
  stimulation

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Whats next?

• Better characterisation of Ag-specific T cells
  (by staining with additional mAb specific for
  activation markers, memory markers, homing
  receptors, etc)
• Class II tetramers
• Ex-vivo functional assays-tetramer staining
  combined with intracellular cytokine assays.
• In situ staining of tissue sections??
• Higher order oligomers?
• Specificity?
• Mutation of CD8 binding site.

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Conclusions

• Tetramers have enhanced our ability to analyse T
  cell responses
• Simultaneous assessment of Ag-specific T cells
  with phenotypic markers
• Heterogeneous populations of memory CD8 T cells
• In vivo CTL assays are more sensitive than
  tetramers

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Acknowledgements

• Whitney Macdonald
• Adam Winterhalter
• Jim McCluskey
• Scott Burrows
• Richard Coles
• Frank Carbone