1. Gene expression analyses of anergic and suppressive T cells: identifying new players
in regulatory T cell (Treg) biology.
Deborah Hopkins, Mindy Zhang, Dapei Liu, Brenda Richards, Steve Madden, Bruce
Roberts, and Srinivas Shankara.
Genzyme Corporation, Framingham, MA 01701
Several recent publications have described a statistically significant increase in
CD4+CD25+ regulatory T cells (Tregs) in both peripheral blood and at the tumor site in
various malignancies, which correlate with late stage disease and poor prognosis. From
these data, Tregs are emerging as both a biomarker for clinical evaluation and as a
potential therapeutic intervention point. The identification of FOXP3 as a marker for and
potent modulator of Treg function is an example of how differential gene expression
analysis has improved the existing knowledge of Treg biology. Mutation of FOXP3 is
responsible for IPEX or Immune dysregulation, polyendocrinopathy, enteropathy and X
linked inheritance in humans, a fatal recessive autoimmune syndrome. Additionally,
ectopic transfer of FOXP3 to T cells has been shown to potentiate both an anergic and
suppressive phenotype in the cells. These are very convincing arguments for the
indispensable role of FOXP3 in Treg biology. However, still more distinct genes and/or
cell surface markers are needed for diagnostic markers as well as to better understand
how these cells regulate and are regulated. Towards this end, we have undertaken gene
expression analyses of Tregs isolated directly from peripheral blood, after a 10 day in
vitro expansion phase and a more artificial system wherein a Jurkat T cell line was stably
transfected with the huFOXP3 gene. Prior to gene expression analysis, Tregs isolated
from the periphery demonstrate a clear suppressive activity when cocultured with
responsive T cells and stimulus, both directly and following expansion. In the Jurkat
system, we demonstrated that FOXP3 dramatically alters protein and gene expression
upon PMA/PHA stimulation when compared with empty vector controls. Human whole
genome arrays from Affymetrix (Hu133plus2.0) were employed to analyze samples from
a time course study following PMA/PHA stimulation of FOXP3 transfected and control
cells. From initial microarray data, it is clear that FOXP3 acts in part as a transcriptional
regulator, shutting of pathways and gene sets associated with T cell activation such as IL-
2, IFNγ, and NFAT regulated signaling. Central to our goals for the study we have also
identified individual genes that are upregulated in response to FOXP3 expression from
which to identify a list of candidate biomarkers. Using the strength of the Genzyme
SAGE technology together with microarray techniques we hope to gain a better
understanding of Treg identification and function by evaluating gene expression in Treg
cells as compared to CD4+ CD25- as well as a result of FOXP3 expression. Any new
genes could feed into identification of potential therapeutic targets as well as the growing
need to identify biomarkers for clinical evaluation of patients.