Department of Biomedical Engineering Final Oral Examination | 3:00pm June 23, 2008 | The James & Anne Duderstadt Center

Daniel Joseph Estes

High-Throughput Profiling of Ion Channel Activity in Lymphocytes for Quantifying Activity of Human Autoimmune Disease

The voltage-gated potassium ion channel, Kv1.3, in human lymphocytes is a promising target for treatment of several autoimmune diseases, including multiple sclerosis (MS) and rheumatoid arthritis (RA). Despite the relevance of this ion channel for disease, current techniques to measure Kv1.3 activity are low-throughput, laborious, and require significant expertise. Consequently, studying ion channels in cells of the immune system is not accessible to most clinicians and immunologists.

This thesis describes the development of a high-throughput assay to measure Kv1.3 activity in lymphocytes. The method is automated, specific for Kv1.3 channels, and able to measure Kv1.3 activity in 100-200 lymphocytes within 1 h. This throughput is over 20-fold higher than the throughput of manual patch clamp techniques.

Using this high-throughput assay enabled profiling Kv1.3 activity in T cells from peripheral blood of patients with MS and RA. Patients with chronic progressive (CP) MS exhibited significantly higher Kv1.3 activity compared to healthy controls or MS patients in remission. Development of metrics to quantify the percentage of T cells with high Kv1.3 activity made it possible to distinguish between CP-MS patients and controls with 100% sensitivity and 94% specificity. In addition, patients with an active form of RA exhibited higher Kv1.3 activity than patients with inactive RA. These results suggest that Kv1.3 activity may be a useful clinical marker for quantifying activity of inflammatory autoimmune disorders.

Moreover, the assay developed here enabled immunological experiments to study the changes in Kv1.3 activity upon T cell stimulation. Kv1.3 activity increased ~3-fold in T cells following stimulation. We showed that this upregulation was driven by signaling through the interleukin (IL)-2 receptor. Interestingly, inflammatory cytokines IL-2 and IL-15 increased Kv1.3 activity even in the absence of signaling through T cell receptor pathways. These studies suggest that both direct activation of T cells and inflammatory cytokines lead to high Kv1.3 activity in vivo.

High-throughput electrophysiology introduces a promising new strategy for clinical applications such as diagnosis and therapeutic monitoring of autoimmune disease. This work also provides a general methodology that makes the study of ion channels in primary cell types accessible to laboratories not specialized in electrophysiology.

Click here to see Dan's profile and view his publication list.