Transcript Document

Immune Tolerance: from Gene
Expression to Drug Discovery
Therapeutic Immunology Group
(Prof. H. Waldmann)
Therapeutic Antibody Centre
(Prof. G. Hale)
Immune Tolerance and
Therapy
• Therapy to reverse breakdown of self
tolerance in autoimmune diseases
• Tolerance induction rather than nonspecific immunosuppression to avoid
rejection of transplanted organs
• Reversal of acquired tolerance to tumour
antigens and latent viral infections
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.
Methods for Analysing Gene Expression
Analysis of known genes (RT-PCR/Antibodies/Protein Gels):
There are >1000 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 don’t 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 (<1/3 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.
CD4+ T cell clones/lines against DBY-Ek male antigen
Clone
Source
R2.2
A1(M)xRAG-1-/(graft primed)
R2.4
A1(M)xRAG-1-/(graft primed)
Tr1D1
A1(M)xRAG-1-/- IL-10
(naïve)
(aCD3 cloned)
A1MP A1(M) naïve
SKA
line
Polarised in
Type
Cytokines
IL-2
Th1
IFN-g
IL-4
Th2
IL-4, IL-10
Tr1/Treg
IL-10 (IL-4)
Treg
IL-10 (IL-4)
Tskin/Treg
IL-10
Anti-CTLA4
DBY-Ek peptide
A1(M) + male DBY-Ek peptide
skin graft CD4+
sorted IELs
SAGE details
AE = Nla-III
TE = BsmF1
Automated DNA Sequencing Machine
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).
DC cluster
T cell clone cluster
Spleen CD4 cell cluster
Clustered
Expression Chart
of approx. 300
known genes (CD
antigens, cytokines
and receptors)
A close up of a Treg
cluster of known genes
Real Time PCR Machine
HPRT
TM4
HPRT
TM4
Th1
Treg
Th1
Treg
Quantitative RT-PCR from rejecting, syngeneic and tolerant skin grafts
Ratio of tolerant to rejecting skin graft expression
Normalized mRNA expression
14.8** 624** 32.6** 2.8* 0.14
0.5
3.4*
0.6
9.9
1.1
0.7
0.3
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
RST RST RST RST RST RST RST RST RST RST RST RSTN
TGFb2 ENK GM2A GITR Cyp11a CysF IL1R2 GRZMa LEF1 Ly116 RNTS CD3
Fluorescence Activated Cell Scanner
Tr1D1
A1MP
Tskin
Th2
Th1
Chandra/Ly116 ->
Live cells
(7AAD neg)
Permeabilized
Chandra/Ly116 ->
Peptide
inhibition
CD25-
CD4+CD25+
Chandra/Ly116 ->
CD25 ->
CD103 ->
CD4 ->
Th1 (R2.2)
CD103 ->
Treg(Tr1D1)
CD103 ->
Chandra/Ly116 ->
Th2 (R2.4)
CD103 ->
Tskin(SkA)
CD103 ->
The Therapeutic Antibody Centre
Humanized monoclonal antibodies against cell surface molecules
expressed by effector but not regulatory T cells
Monoclonal
Antibody
Production
Bioreactor
Schematic
Hollow Fibre Bioreactor for Antibody Production
Therapeutic antibody purification
Clinical Trials of Therapeutic Antibody
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”