Biomedical Graduate Studies

Description of Research Expertise

My laboratory studies how T lymphocyte activation, differentiation, and tolerance are regulated by components of the cyclin-dependent kinase (CDK) cascade like CDK2 and p27KIP1, and by transcription factors like FOXP3 and IKAROS in cooperation with epigenetic factors like DNMT3A. We have over 15 years of experience in cellular immunology, cell biology, and molecular biology research, including mouse models, in vivo and in vitro lymphocyte function, biochemistry, transcriptional biology and epigenetics. More recently, we have incorporated ‘next generation’ approaches like ATAC-seq, ChIP-seq and methyl-DIP-seq to study transcription factor binding, chromatin remodeling, and DNA methylation genome-wide, and three-dimensional chromosome conformation capture (3C)-based techniques like 4C-seq and Capture C to study long-range interactions between enhancers and promoters. We also use CRISPR/CAS9 genome editing technology to mutate gene regulatory elements in mice and in human cells. Two of the most current projects in the lab are outlined below:

A regulatory T cell-intrinsic role for CDK2 and p27kip1 in acquired immune tolerance:

We previously found that the cyclin-dependent kinase CDK2 phosphorylates the Treg transcription factor Foxp3, targeting it for proteolytic degradation. Treg with defective CDK2 activity express more Foxp3 and are better able to mediate cardiac allograft tolerance in response to costimulatory blockade. We now report that normal Treg development in the thymus and TGFB-mediated iTreg differentiation is associated with marked induction of p27kip1, the natural inhibitor of CDK2. This suggests that effective Treg development or function may require tight control of CDK activity. Indeed, conventional CD4+ T cells genetically deficient for p27kip1 exhibited defective induction of Foxp3 in response to TGFB in vitro, which was associated with CDK2-mediated phosphorylation of Foxp3 as measured by a novel phospho-specific Ab. To determine whether p27kip1 is required for Treg function in vivo, we used p27kip1-fl/fl-Foxp3-YFP-Cre mice in which p27kip1 is specifically deleted in the Treg lineage as recipients of cardiac allografts treated with CD28-CD40 costimulatory blockade. While this treatment led to long-term acceptance of cardiac allografts in p27kip1-sufficient control recipients, recipients with Treg-specific deletion of p27kip1 rejected their allografts. These studies establish a Treg-intrinsic role for p27kip1 in Treg stability and function, and point to the cyclin-dependent kinase pathway as a potential new therapeutic target for promoting transplantation tolerance.

Discovery of new disease-associated regulatory elements controlling inflammatory gene expression in T cells:

Interleukin-2 is a potent T cell growth factor with crucial roles in both immunity and tolerance. Genetic studies in humans and mice demonstrate a role for IL2 in autoimmune disease susceptibility, and for decades the proximal IL2 upstream regulatory region has served as a paradigm of tissue-specific, inducible gene regulation. We have used a combination of ChIP-seq, ATAC-seq and 4C-seq analyses of of chromatin and chromosome remodeling to identify a novel, long-range enhancer of the IL2 gene located 83 kb upstream of the transcription start site. This element can potently enhance IL2 transcription in recombinant reporter assays in vitro, and the native region loops to physically interact with the IL2 gene in vivo in a CD28-dependent manner. We have now edited this ~500 bp element out of the mouse genome using CRISPR/CAS9 in fertilized eggs, and CD4+ T cells from animals homozygous for deletion of the IL2-83 enhancer exhibit a ~80% reduction in IL-2 secretion in vitro. Preliminary analysis of young IL2-83 enhancer-deficient animals suggests largely normal hematopoiesis, but accumulation of activated phenotype CD4+ T cells in the peripheral lymphoid tissues. This cis regulatory element is evolutionarily conserved, and contains human SNPs associated with multiple autoimmune disorders. These results indicate that the regulatory architecture of the IL2 locus is more complex than previously appreciated, and suggest a novel molecular basis for the genetic association of IL2 polymorphism with autoimmune disease. This IL2-83 enhancer knock-out mouse represents a valuable model to study graded effects of this cytokine on activation-induced cell death, CD4 T cell differentiation, CD8 T cell memory, and regulatory T cell homeostasis in vivo.