Andrew C. Chan of HHMI at Washington University School of Medicine has found evidence that a single protein regulates the positive and negative selection of T cells.
Scientists have shown that a single protein plays a key role in eliminating T cells that react with self tissue and educating remaining T cells about the targets they are to recognize and destroy.
In an article published in the October issue of Nature, a team of scientists led by Dennis Loh, a former HHMI investigator at Washington University School of Medicine in St. Louis, who is now working for Hoffmann-LaRoche, found that the immune system fails to develop properly in mice missing both copies of the ZAP-70 gene.
It may be that ZAP-70 is the main signaling pathway controlling thymic differentiation.
ZAP-70 is a signaling molecule that was previously thought to be the cause of a severe combined immunodeficiency (SCID) syndrome noted in several Amish families from the midwest United States. Loh and colleagues, Izumi Negishi, a postdoctoral fellow, and Andrew Chan, a Hughes investigator at Washington University School of Medicine, knocked out both copies of the ZAP-70 gene in mice to assess the molecule's role in immune system development. "This knockout allows future work to focus on the pathways that ZAP-70 is involved in," said Loh. "It may be that ZAP-70 is the main signaling pathway controlling thymic differentiation."
There are two major phases in the life cycle of a T cell. During negative selection, thymocytes (immature T cells) that react with self tissue are eliminated. In positive selection, the remaining thymocytes are "educated" to interact with the appropriate major histocompatability (MHC) molecules on the surface of other cells. "One of the enigmas in immunology has been knowing what molecules are required for positive and negative selection," said Chan. "We have shown that ZAP-70 is required for both positive and negative selection."
Proving ZAP-70's pivotal role in positive selection was relatively straightforward, Chan said. "None of the T cells got out of the thymus when ZAP-70 was knocked out," he said.
Pinning down ZAP-70's contribution to negative selection was made possible by using the DO-10 mouse, a strain created by Loh years ago. The DO-10 mouse was helpful because it gave researchers a mouse whose T cells all expressed the same T cell receptor. "If you present these T cells with the known antigen before they have undergone selection, the T cells are all killed," Chan said.
Negishi bred the ZAP-70 knockout mouse with the DO-10 mouse, creating progeny that had a known T cell receptor, but they did not have ZAP-70. "When challenged with antigen, none of the T cells died. This suggested that ZAP-70 is crucial for negative selection as well," Chan said.
The ZAP-70 knockout mouse model may also help explain the SCID syndrome identified in the Amish population by Chan and Arthur Weiss of HHMI at the University of California, San Francisco. Chan and Weiss noted that this mysterious disorder caused those affected to lose function of half of their immune system. "They had no functional T cells, but their B cells were fine," Chan said. "All of the patients had normal numbers of CD4-positive T cells, but no CD8-positive T cells."
In the thymus, T cells are divided into two populations: those with CD4 on their surface and those with CD8. CD4 and CD8 are proteins that allow T cells to interact with MHC complex molecules. Chan and Weiss reported in Science in 1993 that the SCID was caused by a lack of CD8-positive T cells, which was, in turn, caused by a mutation in ZAP-70.
When Negishi knocked out ZAP-70 in mice, he noted that there were no mature T cells with either CD4 or CD8. "This suggested to us that ZAP-70 was important for both CD4 and CD8 T cell development," Chan said. But the mouse phenotype differed from what Chan and Weiss had seen in the patients because there were no CD4-positive T cells.
Chan said that he and Loh plan to continue their collaboration to explore the differences between the phenotypes exhibited by the ZAP-70 knockout mouse and the patients with SCID caused by ZAP-70 mutations. "It will be very interesting to see what is causing the differences in these phenotypes," Chan said.