Dr. Ken West
Research

When undergraduates are taught about antigen presenting cells they are typically told about macrophages and B lymphocytes. While these antigen presenting cells (APC) are involved in the processing and presentation of antigen to T lymphocytes the most important APC is the dendritic cell(DC).

Dendritic cell function is critically dependent on their location and state of maturation. Immature DC reside in all peripheral tissues (areas of high antigen encounter) and are equipped to capture and process antigen. The Langerhans cell of the skin is one of the most studied of these immature DC. In response to activation signals, such as LPS and TNF-alpha, these immature DC undergo maturation. During maturation the DC down-regulate their ability to process antigen and up-regulate MHC class II as well as costimulatory and adhesion molecules developing into potent T cell stimulators. At the same time these DC migrate into the lymph node. In the secondary lymphoid organs, DC express high levels of chemokines which preferentially attract naive (CD45RA⁺) T cells. In addition, DC maturation is associated with morphological changes resulting in the expression of numerous delicate dendrites.

Mature DC are 100 fold more potent than other APC in stimulating resting T cells and are probably the only APC capable of activating a primary immune response. They possess several features which contribute to their superiority in stimulating resting T cells and activating primary immune responses. DC express high levels of costimulatory molecules such as B7-2 and the expression of MHC-peptide complexes is 10-100 fold higher on DC than on other APC such as B cells and monocytes. In addition, DC express high levels of IL-12 upon maturation which further augments innate and acquired immunity.

DC are unique among APC in that they can cluster naive T cells and can interact with many T cells at one time. The majority of T cell activation occurs within these DC-T cell clusters. Clustering facilitates binding of the T cell receptor to MHC Class II and the interaction of various costimulatory receptor ligand pairs resulting in T cell activation. DC unlike other APC are also able to cluster naïve T cells in both an antigen dependant and independent fashion. The antigen independent clustering is presumably important because it allows T cells to "sample" the MHC-peptide complexes on the surface of the DC in order to find specific MHC-peptide complexes that it recognizes.

The unique function of DC in the immune response has placed them at the forefront of immunotherapy for many clinical diseases. DC have been demonstrated to be important for activating immune responses against viruses and organ transplants. DC are currently being evaluated in clinical trials in activating immune responses against various different types of cancer. DC can also be modulated to induce tolerance (or unresponsiveness) to our own tissues and therefore are likely to be critical for our understanding and treatment of autoimmune diseases.

DC and the immunological synapse with T cells
Monther Al-Alwan , Julie Bunker
Collaboration on signaling with David Byers of the Atlantic Research Centre The junction between an APC and a T cell has been nicknamed "the immunological synapse". This specialized contact between the T cell and the APC results from reorganization of the T cell actin cytoskeleton and is characterized by the accumulation of F-actin and other cytoskeletal proteins in the T cell at the contact point with the APC. These active changes in the T cell cytoskeleton result in the dynamic clustering of T cell surface receptors and signaling molecules into a supramolecular activation cluster (SMAC) at the interface with the APC and provides an optimal environment for signaling molecules downstream of the TCR.

It has been widely believed that the APC cytoskeleton is passive in the formation of the immunological synapse; however, previous studies in this area have used activated T cells with B cells as APC. In contrast, my lab has recently demonstrated that the dendritic cell (DC) actin cytoskeleton actively participates in the development of the immunological synapse with resting CD4+T cells.

We have demonstrated, using confocal microscopy, that during interaction with a resting antigen specific T cell the DC actin cytoskeleton focally polarizes towards the T cell. This polarization consists of accumulation of filamentous actin (F-actin) and fascin, an actin bundling protein, at the interface with the T cell. We have shown using actin cytoskeletal inhibitors that DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting CD4+ T cells. This novel observation demonstrates that the establishment of the immunological synapse between APC and resting T cells requires participation of the cytoskeleton in both cell types.

While this establishes the involvement of the DC in formation of the immunological synapse many questions remain unanswered. What are the signaling pathways that result in DC cytoskeletal rearrangement? What proteins are physically involved in the cytoskeletal rearrangement? Does this cytoskeletal rearrangment result in the reorganization and activation of adhesion and costimulatory molecules? Studies to answer these questions are being actively pursued in the lab.

The actin bundling protein, fascin, and its role in dendritic cell function
Monther Al-Alwan, Pat Colp
The name dendritic cell is derived from their morphology. These cells possess many dendrites which are projections from the surface of the cell. Dendrite expression becomes more pronounced with maturation of the dendritic cells. We have recently investigated the expression of fascin, a DC actin bundling protein, during the maturation of DC. Interestingly, fascin expresion is confined to dendritic cells, neurons and tumors and of course the first two both have dendrites. We have demonstrated that fascin is expressed only in mature dendritic cells in vivo and in vitro and its expression correlates highly with the presence of dendrites and the ability of DC to activate T cells. We inhibited the expression of fascin using antisense oligonucleotides and found that it had a marked effect on dendrite formation with 85% of DC losing their dendrites. Importantly, inhibition of fascin expression significantly reduced the ability of DC to activate allogeneic T cells. This study was the first to demonstrate that the dendrites have a role in the ability of the dendritic cell to activate the T cell.

Work from other groups has suggested that fascin expression in other cell types might be involved in motility. We have also shown that fascin is only expressed in vivo in DC that have started to migrate from peripheral tissues to the lymph node indicating that it might also be involved in DC migration. We are currently using a variety of techniques to study the involvement of fascin in migration of DC in vitro and in vivo. We are also undertaking more general experiments on DC migration.

The role of fascin in tumor metastasis
Pat Colp
(in collaboration with Shawn Murray of Pathology and Richard Langley of Dermatology)
In addition to its role in the function and migration of DC there is increasing evidence to implicate the actin bundling protein, fascin, in the motility of other cells. The most striking work came from Yamashiro and colleagues who transfected a non motile epithelial cell line with a cDNA construct for fascin. The cell line became markedly dendritic and was able to migrate across a tissue culture plate. In addition, fascin expression has been documented in a number of tumor cell lines and a recent report suggested that it was also expressed in aggressive forms of breast cancer. We are currently studying the role of fascin in the metastasis of cancer of the skin, breast and genitourinary tract. Fascin is expressed at the leading edge of high grade tumors implicating it as a prognostic factor. Future studies will evaluate the direct role of fascin expression in melanoma and other tumors using transplantable tumor models and antisense fascin inhibition.

Immunosuppressive agents and their effects on Dendritic cell function
Julie Bunker
Immunosuppressive drugs are routinely used in the treatment of clinical disorders such as arthritis and to prevent transplant rejection. Our current understanding of the mechanisms of these agents is based on studies evaluating their effects on T lymphocytes. However, DC function and maturation plays a critical role in the activation of immune responses and the effects of immunosuppressive drugs on DC have not been evaluated. We have demonstrated that immunosuppressive drugs currently in clinical use have pronounced effects on DC maturation and survival influencing their ability to activate immune responses. Ongoing studies are evaluating the basic mechanisms of action of these drugs on gene regulation in DC. We are also evaluating novel immunosuppressive drugs which may affect appropriate DC migration and homing.

Other projects
Other projects in the lab deal with clinical and pathological correlations in transplantation and in disease of the skin.