Interests

The laboratory focuses on signaling in the immune system and study of host processes that HIV-1 exploits. Control of apoptosis, autoimmunity, angiogenesis, retrovirology, and blockade of HIV-1 infection are prominent in our studies.

We use advanced Flow Cytometric analysis (FACS) of phosphoproteins in single cells and dominant effector genetics to achieve many of our goals. For this we have developed a range of FACS assays, cDNA and peptide expression systems using viruses, and single-cell genetic selections, to study pathways of interest to us.

Proteomics: Multiparameter phosphoprotein analysis in single cells by Flow Cytometry and FACS

Signaling systems can now be analyzed directly by flow cytometry and Fluorescence Activated Cell Sorting. We have developed a series of methods for following multiple phosphoproteins in complex populations of primary cells.
Up to 11 simultaneous parameters can be followed in single cells including multiple kinases, phosphoproteins, cell cycle, and other parameters allow for exacting resolution of cellular activation states.
We are using these techniques to study B and T cell signaling, dendritic cell function, and other immune parameters by analysis of biochemical functions at the single cell level.

High complexity retroviral libraries of cDNAs and peptides

Recombinant retroviruses are a primary tool by which this laboratory studies signaling mechanisms, HIV-1 transcriptional control, oncogenesis, and cell cycle control. As a research tool retroviruses allow for highly efficient gene transfer into mammalian cells.
We can create high-complexity libraries of a variety of different types of molecules and stably deliver them to nearly any dividing cell type at extremely high efficiency for molecular genetic and biochemical characterization of structure function and epistatic analysis.
Termed DOMINANT EFFECTOR genetics, the approaches have a variety of utilities in basic and biomedical arenas. We use the systems for studies of NFAT/Rel signaling and developmental progression in the immune system. Retroviral library approaches allow for the genetic selection of molecules that have dominant effects on intracellular signaling and disease pathogenesis. Other systems under study include apoptosis, NFAT, HIV-1 attachment, TNF-alpha and Fas signaling, oncogenic progression, and differentiation inhibitors.
Check out the Genetic Screens and Library Systems sections for more information on these topics.

HIV-1 Transcriptional Control

HIV-1 transcriptional activation is mediated in large part by a family of transcriptional activators that include the NF-kappa B and NFAT Rel family of regulators.

We utilize whole virus and standard recombinant virus in our studies. Aspects of the work focus on the development of novel inhibitors for aspects of the viral life cycle, the study of HIV-1 regulatory proteins, and the biochemistry of transcriptional initiation in HIV-1 at the initiators.

NFAT (nuclear factor of activated T cells) was hypothesized first by this laboratory to be a true ancestral relation to the NF-kappa B family of transcriptional regulatory factors, which has pointed to a number of important research venues for NFAT.

Angiogenesis

Angiogenesis is the process of blood vessel formation in reponse to normal or pathological intercellular signals. Normal angiogenesis is required for appropriate oxygenation and of target tissues, among other requirements.
The laboratory has developed novel angiogenic inhibitors as well as embarked on a search for new regulators of angiogenic regulators using a combination of tools: retroviral library tools (cDNAs and peptides) as well as gene chip resources using our own gene chip spotting and reading facility.

We have developed a series of ScFV and peptides through genetic selection techniques that appear, in vitro to inhibit angiogenic processes. We are currently determining the nature of these agents and the target mechanisms upon which they operate.

Apoptosis: Signaling and Regulation

It is now understood that apoptosic pathways are not just simple, one-way death signals, but a complicated mixture of pro-life and pro-death stimuli. Critical to maintaining this balance are a host of cell surface molecules that very often send two contradictory signals. Molecules such as TNF and Fas are classical examples. Genes discovered by our group to play roles in apoptotic blockade, such as toso and icam-2, play roles in the immune system and beyond.

Toso: Toso is a cell surface molecule which prevents Fas-induced apoptosis in T Cells. Toso was discovered in the Nolan lab using retroviral delivery of cDNA molecules to Jurkat T cells followed by Fas selection. The model for the Toso pathway and details of its discovery are published in Immunity: toso98.pdf [fulltext link]
Currently we are interested in how Toso and other molecules of the apoptosis machinery maintain the balance between proliferation, cell cycle arrest, and cell death. Members working on this project are Jonathan Irish and Jeff Fortin. For the most current information on this project check out out the Toso Project website (http://toso.stanford.edu).

ICAM-2: ICAM-2 was found to elicit an anti-apoptotic signal by activation of AKT/PKB dependent cell survival. ICAM-2 is of physiological relevance because its overexpression is observed in a variety of lymphoproliferative diseases that include B-cell chronic leukemia. The elucidation of the signaling mechanism for ICAM-2 attribute a novel role for immunoglobin superfamily members in actively participating in the cumulative process of cancer and metastatic spread. Current work is invovled in delineating signaling pathways for leukocyte adhesion mediated events.

FLUORETTES, and single cell studies of intracellular signaling

FACS-Gal and associated techniques, published in 1987, are areas in which the laboratory continues an interest for studies of transcriptional events and other events at the single cell level.

New Ways to Detect or Perturb Intracellular Events: The lab is also using genetic selection to develop a new toolset to detect and perturb intracellular events. Kevin Marks and Michael Rozinov have used phage display of peptide libraries to discover short, constrained peptides that bind to fluorescent dyes. Selections of phage-displayed peptide libraries yielded peptides with nanomolar binding to the fluorophore Texas Red These fluorophore-binding peptides (or "fluorettes") were used to target Texas Red inside of mammalian cells. Thus, this short peptide can be used as a tag to monitor proteins in vivo, and it provides proof-of-principle that peptides can be used to target small molecules inside of living cells.

Having shown that we can target small molecules to specific proteins inside of living cells, we are a creating a generalized system which uses bifunctional small molecules: one part of the chemical binds to the targeting peptide, and the second part of the small molecule functions as a sensor of relevant properties- potentially ranging from presence of ions or a specific protein, membrane localization, pH etc. This modular system will allow the us to routinely target a wide variety of chemicals to any locale or protein within cells.

Autoimmunity: Diabetes and Arthritis

Autoimmunity is a pathological state in which the immune system turns against host tissues. It encompasses diseases such as insulin dependent diabetes mellitus, rheumatoid arthritis, multiple sclerosis and lupus. In collaboration with the laboratories of Garrison Fathman, Ed Engleman and Larry Steinman, we are using our retroviral technologies to deliver dominant negative effectors and cytokines to block autoimmune inflammation.

The T helper response can be considered to have two different arms known as Th1 (pro-inflammatory, promoting cytotoxic T cell activity) and Th2 (non-inflammatory, promoting B cell activity). Although today this is generally considered to be an oversimplistic view, it serves as a framework in which we can consider mechanisms of blocking inflammation. Th1 cytokines (such as interferon (IFN) g and tumour necrosis factor (TNF) a) inhibit Th2 cell activity, whilst Th2 cytokines (such as interleukins (IL) 4, 5 and 10) block Th1 activity.

We are particularly interested in using scFvs against other immune system molecules in an effort to block inappropriate immune responses. In work being carried out by Dr. Richard Smith, targets of current interest include CD3, CD40, CD80 and CD86. We are also starting to use scFv backbones to present intracellular and secreted peptide libraries, in the context of the hypervariable regions, in an effort to identify novel factors involved in antigen presentation, costimulation and T cell activation. Further information relating to our work with scFvs and autoimmunity can be found here.

Gene Transfer/Therapy via Retroviruses: Oncoretrovirus and Lentivirus

The laboratory has developed several rapid, stable high-titre retroviral production systems. Major collaborations include provision of viral systems for IDDM (diabetes) with Dr. Gary Fathman and Edgar Engleman, skin diseases due to genetic deficiency (collaborations with Dr. Paul Khavari), and HIV-1 gene Therapy in a multicenter gene therapy trial involving Stanford, the NIH, University of Michigan, and UCLA.

Phoenix: Second Generation 293T-based Retroviral Producer Cells: Phoenix Cells, Phoenix-GP, Phoenix-A, Phoenix-E (gag-pol, Ecotropic, Amphotropic, respectively) were created to overcome a number of obstacles that limit the utility of retroviruses. The cells allow for rapid (3 days) production of high-titre retrovirus with human lipid and polysacchararide viral coat proteins. They can be obtained by downloading the MTAs by clicking here.
FELIX: Fourth generation Feline Immunodeficiency Virus based lentiviral vectors. Developed by Dr. Mike Curran during his graduate work in the laboratory these vectors have shown wide utility in delivery of genes to non-dividing cells. They are meant as a complement to the HIV-1 based retroviral systems for safety and other biological applications. They show some positive differences in target cell tropism and efficiency compared to HIV-1 based vectors. They can be obtained by downloading the MTAs by clicking here.
HELIX: Fourth generation Human Immunodeficiency Virus based vectors. Developed by Dr. Roland Wolkowicz in the lab, these vectors are high efficiency, self-inactivating retroviral vectors for delivery to non-dividing cells. They are available in a variety of formats with a variety of promoters and reporter elements through downloading of the MTAs by clicking here.

The cells and vector systems are helper-virus free and useful for production of high-titre retroviruses and lentiviruses. The systems have shown application in delivery of clonal virus and high complexity viral libraries for a variety of therapeutic and research purposes.

FLUORETTES, and single cell studies of intracellular signaling

Related links: