Intellectual merit: Each new T lymphocyte produced in the body expresses a novel T cell receptor (TCR) that has the potential to react to self-proteins. Fortunately, organisms have devised a way to avoid autoimmunity, by 'testing' immature T cells (thymocytes) for self-reactivity as they mature in the protected environment of the thymus. If the T cell receptors expressed by an immature thymocyte engage self-antigens encountered in the thymus with high avidity, the TCRs will generate signals that kill the cell. A proportion of cells that pass this test mature into CD4+ and CD8+ single-positive T cells that populate the lymph nodes and spleen (the 'periphery'). Once in the periphery, T cells respond very differently to strong T cell receptor engagement. Instead of dying, they proliferate and differentiate into helper and killer lymphocytes. It is still a mystery how immature thymocytes and their immediate descendants, mature T cells, respond so differently to the identical TCR signal. Published findings funded by a previous NSF-RUI grant revealed a provocative distinction between the signaling cascades initiated by T cell receptors in immature and mature T cells. Whereas both cell populations respond to TCR stimulation by upregulating the expression of Nur77 - a transcription factor known to induce cell death - only mature T cells heavily phosphorylate Nur77, an event that banishes Nur77 to the cytosol of a cell. The investigators have also shown that the failure of immature T cells to phosphorylate Nur77 in response to TCR signals is in part due to their failure to activate (phosphorylate) a key upstream regulator of cell survival, Akt. The goal of the project is to move closer to the origin of the differences in TCR signaling between immature and mature T cells. The PI describes an unexpected finding, that immature T cells fail to phosphorylate Akt not because of a problem with the activity of upstream kinases, but because of an enhancement in the activity of an upstream phosphatase. The project will explore a novel hypothesis, that this phosphatase activity and holoenzyme composition differ between immature and mature T cells, and that these differences are the basis for the distinct response of immature and mature T cells to TCR stimulation.
Broader impact: The work will be performed entirely by undergraduates at Haverford College, and the experiments that led to the hypothesis were motivated directly by their original pursuits. The NSF RUI (Research at Undergraduate Institutions) funding not only allows students to be exposed to the most current research performed in the immunological discipline, but also permits them to be direct contributors to it. Funding of this project directly supports the efforts of senior Biology majors, all of whom are expected to perform an original research project. It will also support the development of younger students (many of whom are in Haverford's Multicultural Scholar Program) who join the lab in the summer or as work-study students during the academic year. All students are exposed to an educational approach that asks them to raise a question of importance, test a hypothesis experimentally, and rigorously critique data. NSF funds allow the students to engage in bench research at a sophisticated level and enhances the expectations that students and faculty have of each other as they collaborate in an effort that inspires the development a serious intellectual commitment, a willingness to engage literature, critique evidence, and the courage to speculate. Funds also permit students to follow their experimental 'noses' and wander off narrow trajectories to test an original thought. Some students produce publishable data and participate in writing papers, most will present their work at national and local scientific meetings, and some will discover something novel that will inspire the next grant and the next 'generation' of students. And those who may not pursue research as a career will still have experienced what it is like to be a participant rather than an observer in the generation of knowledge - and will leave with a more developed sense of responsibility for the quality of information that they will one day shape and share.
Project Outcome Report (Jenni Punt, 2012) Intellectual Merit: T lymphocytes, vertebrate white blood cells that coordinate the attack on pathogens, continually make decisions about their fate. Depending on the signals they receive through receptors as they browse other cells for signs of infection, T cells will divide and initiate an immune response, differentiate and tailor that immune response to the type of pathogen, or die and help prevent inappropriate immune reactions. How T cells discriminate among the signals they receive is still poorly understood. This NSF award supported work that advanced our understanding of the intracellular players behind these decisions. Specifically, it allowed my undergraduates at Haverford College and I to pursue an unexpected finding that a phosphatase (PP2A) - a protein that strips other proteins of modifications (phosphate groups) - is responsible, in part, for r the differences in response of immature and mature T cells to receptor stimulation. Young T cells expressed very active phosphatases, which made them more sensitive to T cell receptor signals, whereas more mature T cells were able to suppress the activity of their phosphatases. The award also supported the generation of a new genetic tool and a new set of investigations into one of the targets of the phosphatase, Nur77, which is likely to play a central role in determining cell fate by regulating how a cell generates and utilizes energy. We (and others) noted that the stronger a signal that T cells receive, the more Nur77 protein was made. In order to understand how strength of signal might determine cell fate, we generated a mouse whose cells would fluoresce green whenever Nur77 was expressed (a Nur77(GFP) reporter mouse). Live cells that were stimulated to make Nur77 could then be traced with equipment that could detect fluorescence – the brighter the fluorescence, the more Nur77 they were making, and the stronger the stimulus they had received. This effort inspired two new collaborations with two other immunologists (with the University of Minnesota and the La Jolla Institute for Allergy and Immunology and has generated three publications in top journals already, two of which include a Haverford undergraduate as a co-author. Finally, the award supported the development of an entirely new focus for our laboratory. While examining the blood cells in these Nur77(GFP) reporter mice, two undergraduate senior thesis students discovered that a small subset of hematopoietic stem cells (cells that give rise to all blood cells) fluoresced green. They have gone on to characterize this subset, showing that it is the most immature and most quiescent of the hematopoietic stem cell subpopulations. They have presented this work at national meetings and have inaugurated an exciting new line of investigation (and new set of collaborations) at Haverford College. Broader impacts: All the work described above was performed with and by Haverford College undergraduates, who were full colleagues in the research effort. NSF funding from this award supported twenty-three undergraduate thesis students (five per year) some of whom worked in the laboratory during the summers prior to their senior year and all of whom worked 10-20 hours a week throughout their senior year. Twenty-one of these twenty-three students are (or shortly will be) pursuing careers in the biomedical sciences (thirteen are in medical school, five in PhD or MD/PhD programs, the most recent three graduates as research assistants (two of whom will apply to medical school and one to a PhD program)). The other two are working in health related fields (one as a clinical research administrator, the other on toxicity assessments for a company in China). Five students were authors on published peer reviewed articles and twenty presented their work at regional and/or national meetings. Four of the students supported by NSF funds were African American, a group under-represented in the science professions. One of these very talented students, Thomas Beck (HC’10), is finishing his second year in the Immunobiology PhD program at Yale. He received an NSF Graduate fellowship in 2011. NSF-RUI funding enables me to provide undergraduate students with the resources to ask sophisticated questions, to master and develop cutting edge experimental approaches, interpret data, present their results publicly and write them up for peer review. Perhaps most importantly, the funds give the students the liberty to make mistakes and learn from them. The support has allowed us to develop a lab and atmosphere where we teach each other, discussing results and honing approaches continually, as colleagues. Students gain mastery over experimental approaches and, most importantly, gain ownership of and responsibility for ideas. These extraordinary undergraduates, who have and will contribute to the best of our biomedical advances, will probably never fully realize how much of their wonderful education they owe to the vision and support of the NSF. Thank you.
