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Nobel Laureate Examines Immune Cell Deviations

By Robin Arnette
January 2009

Andrew Fire, Ph.D.
Fire is a professor of Pathology and Genetics at the Stanford University School of Medicine and a member of the National Academy of Sciences and the American Academy of Arts and Sciences. He shared the 2006 Nobel Prize in Physiology or Medicine ( Exit NIEHS with Craig Mello, Ph.D., for the discovery of RNA interference (RNAi) - gene silencing by double-stranded RNA. (Photo courtesy of Linda A. Cicero of the Stanford News Service)

Lymphoma is a term used to describe a group of cancers that affect lymphocytes - the cells involved in the immune system. Pathologists who view these cancerous lymphocytes under a microscope usually see one type of cell, but sometimes two different cell types exist in the same sample. Nobel Laureate Andrew Fire, Ph.D., studies the diversity of these cells and the normal cellular rearrangements that occur in the immune system. His NIEHS Distinguished Lecture on "Cellular Responses to Foreign Nucleic Acids" took place on May 19, and Farhad Imani, Ph.D., a fellow immunologist and principal investigator in the Laboratory of Respiratory Biology, hosted the seminar.

Fire ( Exit NIEHS said that his recent research focus came as a suggestion from a member of his lab, Scott Boyd, M.D. Boyd was looking at lymphoma samples and found one that had both large and small cells. "He wanted to know if the two groups of abnormal cells had a common origin or if they were two separate malignancies that were present in the same individual," Fire added. "He thought that he might be able to address the question by looking at the population of DNA rearrangement present in this individual."

To study the many rearrangements that occur in tumors, Fire said that it was necessary to understand the normal rearrangements that occur in the immune system. He explained that a natural progenitor blood cell that will eventually become an antibody-producing cell population doesn't start out with that capability. It has to combine one of the V, D, J and constant regions to make a functional coding region that can encode an immunoglobin that recognizes something.

The system adds a few nucleotides in the junction spaces to give it more diversity. "Once that happens the cell starts making antibodies and they move to the surface of the cell," Fire continued. "If an organism's immune system recognizes something as ‘self,' it gets rid of that cell, but if the system recognizes something as foreign, it keeps that cell and mounts an immune response."

This recognition triggers cycles of hypermutation and proliferation, but the process has the potential to go awry. Fire explained that a population of cells could "think" that it's recognizing a foreign antigen and starts proliferating when it is really recognizing a self antigen. This scenario occurs in many autoimmune diseases.

Fire took blood samples from patients and extracted DNA from the mononuclear lymphocytes in the blood. Using standard primers that covered the VDJ recombination junction, he employed PCR to generate many copies of the particular sequence. He then utilized high-throughput sequencing technology by attaching little beads that bind one molecule of DNA from one of the recombined cells. By barcoding the samples with small pieces of nucleotides on both sides of the PCR-generated sequences, Fire incorporated 75-100 samples on a single sequencing run.

Fire wrote some of the software that displayed the sequences as images. Each sample was a square panel that had approximately 1000 sequences, which were represented as spots of V and J combinations. Each V and J combination spot had a color and size that corresponded to the number of different instances that particular combination was seen in the sequence set. Normal people have a lot of VJ combinations, while those with lymphomas have a very strong signal in one spot. Each lymphoma has its own individual spot pattern. When Fire looked at the image from the original sample that Boyd examined, he saw that it contained two large spots, which meant that the patient had two immunoglobulin rearrangements. The findings suggest that the lymphoma arose from two distinct cells.

The technique developed by Fire's lab is applicable to other diseases with deviations in the immunome - and these results are just the beginning. "We are interested in understanding people's immune systems, in cases of different histories of both disease and environmental exposure."

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