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
Immune Tolerance: from Gene Expression to Drug Discovery
Therapeutic Immunology Group
(Prof. H. Waldmann)
Therapeutic Antibody Centre
(Prof. G. Hale)
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.
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.
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?
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
CloneSourcePolarised in TypeCytokines
R2.2A1(M)xRAG-1-/- IL-2 Th1IFN-g
R2.4A1(M)xRAG-1-/- IL-4 Th2IL-4, IL-10
Tr1D1 A1(M)xRAG-1-/- IL-10 Tr1/TregIL-10 (IL-4)
(naïve) (aCD3 cloned)
A1MPA1(M) naïve Anti-CTLA4 TregIL-10 (IL-4)
SKAA1(M) + maleDBY-Ek peptide Tskin/TregIL-10
lineskin graft CD4+
AE = Nla-III
TE = BsmF1
Automated DNA Sequencing Machine
Analysis of SAGE data
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
Quantitative RT-PCR from rejecting, syngeneic and tolerant skin grafts
Ratio of tolerant to rejecting skin graft expression
Fluorescence Activated Cell Scanner
The Therapeutic Antibody Centre
Humanized monoclonal antibodies against cell surface molecules
expressed by effector but not regulatory T cells
Hollow Fibre Bioreactor for Antibody Production
Therapeutic antibody purification
Clinical Trials of Therapeutic Antibody
For more information see the TIG Web site:
Or go to the Pathology Web site:
And click on “Herman Waldmann”