Most of us have had a blood test to measure the numbers of the different cells in our blood. Such blood counts are informative because high or low levels of particular cell types can indicate disease, such as infection or cancer.
Blood cells fall into three main categories: red, white (including the immune system's B cells, T cells, and other infection-fighting cells, such as macrophages and granulocytes), and platelets.
To generate the billions of cells in the blood every day, stem cells—or undifferentiated cells—in the bone marrow undergo what is called hematopoiesis. Factors in the marrow stimulate the stem cell to divide and form new types of cells called progenitors, which in turn are transformed by other cell regulators to divide and become increasingly more specialized. As cells mature, different genes are activated or repressed. Stem cells continue to regenerate themselves, thereby providing an inexhaustible reservoir of blood and immune cells for our entire lifetime.
For the past 25 years, Harinder Singh has studied the molecules that control the development of different immune cells during hematopoiesis. In that time, Singh has exploited biochemical, molecular, genetic, and computational methods to discover and analyze key regulatory molecules that direct the maturation of specific immune cells.
Singh's findings about the genetic modulators that act at the beginning of the cell fate determination process have provided fundamental insights about hematopoiesis and the formation of immune system cells. His work may someday lead to new treatments for immune system diseases and cell-based therapies involving genetically engineered immune cells.
Singh began studying the regulation of genes that encode antibodies produced by the immune system's B cells in 1984 as a postdoctoral fellow at the Massachusetts Institute of Technology. At the time, little was known about the control of gene expression in mammals.
Singh developed a new method that scientists could use to isolate or clone the master genes that encode regulators of other genes. "It was important because it made it much easier to take apart and study gene regulatory mechanisms," Singh says.
His technique used a regulatory DNA sequence to find the proteins that bind to that sequence. The DNA sequence acts as a hook that fishes out from a library of thousands of unknown genes the one gene that expresses the protein that specifically binds the DNA element. The method allowed him and other scientists to find many regulators of genes in immune and other cells. But how each of the regulators worked needed further research.
When Singh established his laboratory in 1989 at the University of Chicago, he focused on one of the immune cell regulatory proteins encoded by the PU.1 gene, which another scientist had discovered using the method that Singh had developed. Singh created a mouse that lacked the PU.1 gene to determine what the effect the gene's absence would have on the formation of the animal's immune cells.
By 1994, Singh reported that the mutant mouse made red blood cells and platelets, but that it failed to make white, or immune system, cells. Interestingly, the development of various immune cells was blocked at an early step, suggesting that a common immune system intermediate, a soon-to-be-discovered multiprogenitor cell, was unable to differentiate in the absence of the PU.1 protein.
Singh would later show that introducing the PU.1 gene in culture into the multiprogenitor cell from the mutant mouse restored the immune cell lineages. Using these cultured cells, Singh also systematically added genes that encode different factors to the mutant progenitor cells to generate specific cell lineages. By so doing, he characterized the proteins needed each step of the way to form the different mature white blood cells.
What particularly struck Singh about the PU.1 mutation was that it removed cell lineages from the immune system's innate and adaptive parts. The innate system, which uses macrophages and granulocytes, responds to any invading foreign entity. Simple organisms, like invertebrates, have innate immunity. The more sophisticated adaptive system, involving B cells and T cells, acts only toward specific threats and has a memory that makes it possible to respond to the threat again.
Based on his findings with the PU.1 gene, Singh suggests that the sophisticated adaptive immune system may have evolved by co-opting the genetic circuitry of a cell of the simpler innate system. He is attempting to prove this hypothesis.
In 2000, Singh found that the amount of PU.1 protein in the multiprogenitor cell actually determines cell fate. Lower PU.1 levels favor the generation of adaptive cells, while higher levels lead to the formation of innate cells.
With his combined results about the timing and levels of regulators required to generate immune cells, Singh has begun to collaborate with theoreticians to develop computational models of hematopoiesis. "Mathematical models are very helpful," Singh explains, "because the systems are complex and act beyond the realm a scientist can intuit." Predictions from the models are letting him do experiments he could not have previously imagined.
Singh says that although he is gratified by the fundamental discoveries he has made as a scientist, he is now excited by possible medical applications of his research. Using a method called RNA interference to eliminate the PU.1 gene in human hematopoietic stem cells from umbilical cord blood, he aims to create a human immune cell multiprogenitor, replicating his mouse findings.
By manipulating these human multiprogenitors to become mature immune cells, he hopes the cells might someday be used to treat someone whose blood test has revealed an immune disorder.
