An important mechanism that defends us from infection is the ability of our immune cells to recognize pathogens through Toll-like Receptors. Our lab has focused on the response to bacterial DNA where natural or synthetic DNA containing a motif centering on CGTT stimulates B lymphocytes to divide and make antibody, and also stimulates cell-mediated immunity indirectly through its effect on antigen-presenting cells. We have discovered that oligonucleotide DNA fragments (ODN) closely related to stimulatory sequences can competitively inhibit their action. Careful analysis of structure-function relationship has identified 3 pairs of nucleotides that are important for inhibitory ODN activity. Pair 1 is shared between stimulatory (ST-) and inhibitory (IN-) ODN, and is postulated to bind to the receptor TLR9. Pair 2 is a regulatory site where CC renders either ST- or IN-ODN inactive. Pair 3 must be TT in ST-ODN and GGG in IN-ODN, and thus determines whether a signal is sent to activate the cell, or not.

Immediate future plans include: 1) Determine the sequence requirements for IN-ODN in human cells. Early data confirms many of the IN-ODN sequences developed for mouse cells also work in human cells, though they sometimes differ radically in order of potency. 2) Can we use IN-ODN in the whole animal (mice) to block the stimulatory effects of ST-ODN? There is evidence that simultaneous engagement of TLR9 with the B cell receptor greatly augments stimulation, and that this mechanism may operate in rheumatoid arthritis and lupus, so that learning the optimal sequences of IN-ODN for humans, and strategies for using them in vivo, may have important therapeutic implications. 3) Regarding mechanism, does the avidity of ODN binding to TLR9 determine its biologic activity? Do IN-ODN bind TLR9 directly, and can they displace ST-ODN from TLR9? In collaboration with the Golenbock laboratory at Univ. of Massachusetts, we plan to obtain binding constants for a selected group of ST-ODN and IN-ODN to test these hypotheses. 4) Confocal microscopy techniques already used by Golenbock to track ST-ODN migration between intracellular compartments will also be used to track our IN-ODN. 5) Do the active ODN sequences self-aggregate? Do they aggregate TLR9? These questions can be answered by various techniques that separate molecules by size. By answering these questions, we hope to better understand the molecular interactions that occur in cells exposed to ODN and also explore potential applications of IN-ODN to medicine.

Medical School:
Columbia University

Residency:
Peter Bent Brigham Hospital

Fellowship:
Research Associate, NIAMD, NIH
Helen Hay Whitney Fellow,
National Institute for Medical Research, London, England
University of California, LA