August 5, 2004

Fundamental Change to Immunology 101
Penn Researchers Pinpoint Identity of Early-Stage T-cells Circulating in Blood

(Philadelphia) – T cells are critically important for human immunological defenses against pathogens, yet little is known about their early development. T cells are made in the thymus, but ultimately come from hematopoietic stem cells in the bone marrow, from which all blood-cell types begin. A progenitor cell must leave the bone marrow to seed the thymus, eventually giving rise to T cells. The identity of this cell has long been sought and might help correct disorders of T-cell production, says Avinash Bhandoola, MD, PhD, Assistant Professor of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine.

Bhandoola and Benjamin Schwarz, a fifth year MD/PhD student, identified such a cell. “Our work really provides the tools,” says Bhandoola. “Everyone can now study this cell, and a better understanding of early steps in T-cell development should follow.” They describe their findings in the current advance online publication of Nature Immunology.

Hematopoietic stem cells (HSCs) are the ultimate progenitors of all blood cell types, from platelets and red blood cells (erythrocytes) to immune cells like T cells and B cells. But T-cell development differs from other cell lineages in that it occurs in the thymus, a small organ situated under the breastbone near the heart, rather than the bone marrow. To do this, though, the thymus periodically imports marrow T-cell progenitor cells via the circulatory system. The cell types that travel from the marrow to the thymus were not exclusively pinned down, but researchers have suggested HSCs themselves, multipotent progenitor (MPPs) cells, or a common lymphoid progenitor (CLPs) cell from the marrow itself.

In a previous study, Bhandoola along with David Allman, PhD, an Assistant Professor in Penn’s in the Department of Pathology and Laboratory Medicine, identified the earliest T-lineage progenitor in the thymus and demonstrated that it was not derived from CLPs, as was widely assumed. (Click on thumbnail image above to view the current and revised models). “I had learned in class that T cells develop from CLPs,” explains Schwarz. “The textbook figures would always show an early split in hematopoiesis between the lymphoid lineages [T cells, B cells, NK cells], developing from a CLP, and the myeloid lineages. This is a very elegant model, and I was surprised to find how little direct evidence there was to support the role of a CLP as a physiological T-cell progenitor. That’s why I took on this project, to better understand where T cells actually come from.”

To figure out which type of progenitor cell reaches the thymus to make T-cells, Schwarz and Bhandoola analyzed the blood of adult mice for early progenitor cell populations. The only way to resolve the exact cell type was to correctly identify progenitors present in the blood, which is a tall order. “It’s known that they must be there, but at very low frequencies,” explains Bhandoola.

“Our lab has previously shown that the early T-cell progenitor, found in the thymus, looks like the MPP and HSC in the marrow, so we assumed that the cell in the middle – in the blood stream – would look exactly the same,” says Schwarz. And this is what they found. The team used flow cytometry to detect cell types present in low frequencies. Flow cytometry squeezes cells one-by-one past a bank of lasers, detecting which cells fluoresce in a certain way based on a prescribed molecular tag. The suspected early progenitors (HSCs, MPPs, and CLPs) had known differences in cytokine receptors, so the team used these as molecular tags to characterize the different cells present.

What Schwarz found in the blood was cells with a common HSC-MPP-early T-cell lineage progenitor phenotype and none with the CLP phenotype. This implies that there is not a lymphoid stem cell, or CLP, that leads to all lymphoid lineages but has no myeloid potential. Although another research group found a cell in the bone marrow that they named the CLP, this is most likely a misnomer, say Bhandoola and Schwarz. The so-called CLP never physiologically gives rise to T cells, but remains in the bone marrow and develops into NK and B lineage cells.

To further characterize the cell population they isolated from mouse blood, Schwarz transferred these cells into mice whose bone marrow had been destroyed. For 16 weeks Schwarz determined what blood cell types were being made in these mice and found all lineages – T-cells, B-cells, and myeloid cells. From this he inferred that the circulating cell type is either an HSC or the MPP. They concluded that both were present in blood, but it is still unclear which of these can enter the thymus.

Finally being able to pinpoint what cell type connects the bone marrow to the thymus in T-cell production may help researchers understand what happens when this part of hematopoiesis goes awry. “If you want to understand where T cells come from and what goes wrong when you stop making T cells, we need to know exactly what this cell type is,” says Bhandoola. As humans age, the thymus makes fewer T cells. “To understand that you have to know what cell actually is a T-cell progenitor.”

In bone-marrow-transplant patients, every blood-cell lineage comes back relatively rapidly after the new marrow is received, except for T-cells, which tend to be difficult to reconstitute, particularly in older patients. What about this process of T-cell production happens less efficiently after a transplant? “Now we can ask these questions. Does this process change as we get older and what can we do about it?,” asks Bhandoola.

The National Institutes of Health and Concern Foundation funded this research.

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This release is available online at http://www.uphs.upenn.edu/news/News_Releases/aug04/HSCs.htm