Immunology Graduate Group

Don L. Siegel, Ph.D., M.D.
Professor and Vice-Chair

Chief, Division of Transfusion Medicine

Pathology & Laboratory Medicine

Address: 510 Stellar-Chance Laboratories
Office Phone: 215-898-9655
Lab Phone: 215-898-1372
Fax: 215-573-3127
Email:  siegeld@mail.med.upenn.edu

Education:
M.D., University of Pennsylvania
Ph.D., Harvard University
Sc.B., Brown University

Research Interests

Production and characterization of pathogenic human monoclonal auto- and alloantibodies

Research Summary

My laboratory is interested in characterizing the human immune response to auto- and alloantigens on a molecular level in order to understand more about human immune repertoire development. Our goal is to use this information to develop specific therapeutic agents that down-regulate immune responses in the settings of autoimmune disease such as immune hemolytic anemias and thrombocytopenias, transfusion reactions, hemolytic disease of the newborn, and acute rejection of solid organ allografts.

To approach these problems experimentally, we have combined Fab/phage-display technology with a novel cell-surface panning method to isolate large arrays of clinically significant human monoclonal antibodies from individual patients. This approach has enabled us to study the genetic and immunological properties of pathogenic auto- and alloantibodies.

Panning phage display libraries using intact red blood cells and magnetically-activated cell sorting.  Antigen-positive cells, precoated with magnetic beads, are mixed with excess antigen-negative cells (A) and then incubated with a Fab/phage-display library (B) constructed from immunoglobulin genes expressed in peripheral blood lymphocytes. Specific Fab/phage (dark particles) are captured by the antigen-positive cells while irrelevant non-specific or "pan-reactive" Fab/phage (clear particles) are absorbed out by the antigen-negative cells. Antigen- positive cells are retrieved with a magnet (C), bound Fab/phage are eluted with acid (D), and the specific phage antibodies are amplified in bacterial culture (E). After two or more rounds of cell-surface panning, Fab/phage preparations comprise ~100% antigen- specific particles. Such particles link together an antibody's phenotype with its genotype and are self-replicative.

Recent Publications

Siegel D.L.:  Cell-surface panning of phage-display libraries.  In Barbas C.F., Burton, D.R., Scott J.K. and Silverman G.J. (Eds.), Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, pp. 23.1-23.32.

 

Siegel D.L.  Research and clinical applications of antibody phage display in transfusion medicine.  Transfusion Medicine Reviews 15:35-52, 2001.

 

Roark J.H., Bussel J.B., Cines D.B., Siegel D.L.:  Genetic analysis of autoantibodies in ITP reveals evidence of clonal expansion and somatic mutation, Blood 100:1388-1398, 2002.

 

Siegel D.L. Selecting immunoglobulins to cell-surface antigens using magnetic sorting techniques.  in Aitken, R. and O’Brien, P.M. (Eds.) Methods in Molecular Biology vol 178:  Antibody Phage Display:  Methods and Protocols, Humana Press, Totowa, NJ, 2002, 219-226.

 

Westhoff C., Siegel D.L. Rh and LW Blood Group Systems.  In Simon T.L., Dzik W.H., Snyder E.L., Stowell C.P, and Strauss R.G. (Eds.), Rossi’s Principles of Transfusion Medicine, 3rd edition, Lippincott Williams & Wilkins, Philadelphia, 2002, 113-122.

Siegel, D.L., Czerwinski M, Spitalnik S.L. Structural/genetic analysis of monoclonal antibodies to blood group antigens.  Transfusion Clinique et Biologique 9:83-97, 2002.

Siegel D.L. Diagnostic and therapeutic applications of phage display technology.  In Stowell C.P. and Dzik W.H. (Eds.), Emerging Technologies and Therapies in Transfusion Medicine, American Association of Blood Banks, Bethesda, 2003.

Cines D.B., McKenzie S.E., Siegel D.L. Mechanisms of action of therapeutics in idiopathic thrombocytopenic purpura.  Journal of Pediatric Hematology/Oncology 25: S52-56, 2003.

Westhoff C.M., Burd C., Siegel D.L., Foskett J.K.: Evidence that the erythrocyte Rh-blood group glycoprotein functions as an NH4+/H+ exchanger, J Biol Chem 279:17443-17448, 2004.

Silverman G.J., Goodyear C.S., and Siegel D.L.:  On the mechanism of Staphylococcal protein A immunomodulation, Transfusion 45:274-280, 2005.

Siegel D.L. Developing phage  display tools for use in Transfusion Medicine. Transfusion 45:100S-108S, 2005.

 

Siegel D.L. Phage-display tools for blood typing. Current Hematology Reports 4:459-464, 2005.

Payne A.S., Ishii K., Kacir S., Lin C., Li H., Hanakawa Y., Tsunoda K., Amagai M., Stanley J.R., Siegel D.L.:  Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display, J. Clinical Invest 115:888-899, 2005.

 

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