Research ArticleImmunology and Cancer

Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing

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Science Translational Medicine  23 Dec 2009:
Vol. 1, Issue 12, pp. 12ra23
DOI: 10.1126/scitranslmed.3000540

Editor's Summary

Like a reporter who serially unearths fragments of a story until a plausible picture of the latest scandal emerges, scientists have over time gathered pieces of the vast amount of information inherent in the highly recombined genes of the human immune system—probing their complexity, seeking a disease diagnosis, or hunting for evidence of remission. Back in 1987, Susumu Tonegawa won the Nobel Prize in Physiology or Medicine for discovering the genetics behind the diversity of human antibodies—a process called V-D-J recombination. Now, more than 20 years later, scientists at Stanford University and 454 Life Sciences have used powerful next-generation DNA sequencing technology to comprehensively characterize the products of V-D-J recombination in both cancer patients and healthy volunteers. Indeed, this ability to exhaustively profile the human immune response will help to untangle some of biomedicine’s most knotty problems—cancer, autoimmune disease, and vaccine development.

B and T lymphocytes, cells of the adaptive immune system, build the blueprints for myriad antigen-recognizing proteins—immunoglobulins (Ig) and T cell receptors—by recombination within variable (V), diversity (D), and joining (J) gene segments to rearrange the intervening highly variable DNA sequences that can specify numerous antigen recognition domains. All of this reassortment creates a repertoire of receptors that recognizes scads of molecules from foreign invaders (antigens), a process that spurs the immune system to respond to the threat. When an immune cell sporting a particular antigen receptor finds and binds its matching antigen, the cell divides repeatedly, giving rise to many genetically identical lymphocytes that target a particular antigen for elimination. In contrast to this vibrant diversity of healthy immune systems, those of people with B lymphocyte– or T lymphocyte–based cancers (lymphomas or leukemias) generate cells that express a single dominant (clonal) receptor.

In the new work, Boyd et al. performed massively parallel DNA sequencing of rearranged IgH gene loci in blood and tissue samples from cancer patients and healthy people to examine the diversity of their B cells, the immune cells that make antibodies. To this end, they amplified the rearranged IgH B cell DNA with a series of primers and the polymerase chain reaction to generate bar-coded, amplified DNA mixtures. These samples were then sequenced and the information was analyzed to determine which DNA segments had been joined to generate the blueprints for the IgH immune molecules. The experimental design used by Boyd et al. employs a high-throughput deep sequencing machine and can accommodate up to 150 samples at a time, providing an intricate snapshot of the immune repertoire. From healthy individuals, the authors were able to estimate the normal complexity of the B cell repertoire. With samples from the cancer patients, they obtained disease-specific signatures of clonal B cell proliferation events. For example, in a lymph node sample from one patient, deep sequencing detected two distinct V-D-J rearrangements. This finding indicates that there were two separate clonal B cell populations in this specimen and, therefore, two different B cell lymphomas.

Such signatures could be obtained at the time of disease diagnosis and then monitored on an ongoing basis and thereby used to assess the effects of anticancer therapies that target these clonal populations or for early detection of disease relapse. Characterization of immune cell populations by deep sequencing also may illuminate fundamental aspects of infectious and autoimmune diseases as well as the body’s response to vaccination, gene and cell therapies, and other surgical procedures.

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