Immune Tolerance: from Gene Expression to Drug Discovery

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Immune Tolerance from Gene Expression to Drug
Discovery

• Therapeutic Immunology Group
• (Prof. H. Waldmann)
• Therapeutic Antibody Centre
• (Prof. G. Hale)

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Immune Tolerance and Therapy

• Therapy to reverse breakdown of self tolerance in
  autoimmune diseases
• Tolerance induction rather than non-specific
  immunosuppression to avoid rejection of
  transplanted organs
• Reversal of acquired tolerance to tumour antigens
  and latent viral infections

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Why Start from Gene Expression?
What are the approaches to find new
therapeutics Random screening of chemical
libraries in a surrogate assay (eg. suppression
of antigen specific proliferation in vitro). Look
for monoclonal antibodies that modulate a
function (eg. same assay). Targeted chemical
design/antibodies against specific protein
structures. But How to identify the most
relevant/specific target proteins on possibly
rare cells? Ideally we want targets expressed
ONLY on target cells to avoid potential toxicity
against other tissues. An answer Look for genes
that are specifically expressed in the functional
cell type of interest in our example, Th1 but
NOT T regulatory (Treg) cells.
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Methods for Analysing Gene Expression
Analysis of known genes (RT-PCR/Antibodies/Protein
Gels) There are gt1000 interesting
immunological genes and probably many more
important but unidentified genes. How to
choose? Differential Display and Gene
Cloning Clones genes over-expressed in one cell
compared to one other, but these may be shared
with other cells and you dont know what you are
working with until you have it cloned and
sequenced. How to choose? Gene Chips/Arrays Can
identify patterns of expression from many
(10,000) genes and multiple samples. Genes must
already have been cloned (lt1/3 of genome?), it is
quick, but not very sensitive (or reliable?), and
currently expensive. SAGE (Serial Analysis of
Gene Expression) Can identify almost the entire
pattern of gene expression (the transcriptome)
with no a priori knowledge of the gene sequences.
Multiple samples are directly comparable as a
database. Sensitivity depends on the number of
tags sequenced this can be labour intensive.
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CD4 T cell clones/lines against DBY-Ek male
antigen
Clone Source Polarised in Type Cytokines R2.2
A1(M)xRAG-1-/- IL-2 Th1 IFN-g (graft
primed) R2.4 A1(M)xRAG-1-/- IL-4
Th2 IL-4, IL-10 (graft primed) Tr1D1
A1(M)xRAG-1-/- IL-10 Tr1/Treg IL-10
(IL-4) (naïve) (aCD3 cloned) A1MP A1(M)
naïve Anti-CTLA4 Treg IL-10 (IL-4) DBY-Ek
peptide SKA A1(M) male DBY-Ek peptide
Tskin/Treg IL-10 line skin graft CD4 sorted IELs
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SAGE details
AE Nla-III
TE BsmF1
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Automated DNA Sequencing Machine
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Analysis of SAGE data

• Use SAGEv3.01 software (Velculescu et al) to
  extract numbers of tags from raw sequence files.
• Use Access to link tags to known genes, Unigene
  clusters, and ESTs (from NCBI reliable tag
  list).
• Use Excel to manipulate data tables and
  calculate statistics (custom written function for
  Beysian stats).
• Use custom written cluster analysis and
  presentation program (running on Acorn RISC-PC).

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Clustered Expression Chart of approx. 300 known
genes (CD antigens, cytokines and receptors)
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A close up of a Treg cluster of known genes
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Real Time PCR Machine
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TM4
HPRT
TM4
HPRT
Th1
Th1
Treg
Treg
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Quantitative RT-PCR from rejecting, syngeneic and
tolerant skin grafts
Ratio of tolerant to rejecting skin graft
expression
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Fluorescence Activated Cell Scanner
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The Therapeutic Antibody Centre
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Humanized monoclonal antibodies against cell
surface molecules expressed by effector but not
regulatory T cells
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Monoclonal Antibody Production Bioreactor Schemati
c
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Hollow Fibre Bioreactor for Antibody Production
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Therapeutic antibody purification
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Clinical Trials of Therapeutic Antibody
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For more information see the TIG Web
site www.molbiol.ox.ac.uk/pathology/tig/ Or go
to the Pathology Web site www.path.ox.ac.uk And
click on Herman Waldmann