Jennifer A. Punt - US grants

MCB9728332 Punt, Jennifer RUI: Why Can't CD4+ CD8+ Thymocytes be Stimulated to Proliferate? Lay Summary A basic question in immunology is why immature T lymphocytes respond so differently to stimulation compared to their mature descendants. Mature T lymphocytes are important participants in the immune response because they protect the body from viruses and tumors by (a) recognizing and attacking abnormal or infected cells and (b) alerting other parts of the immune system to their presence. T cells recognize threatening cells by binding foreign protein (antigen) to receptors, called T cell receptors, on their surfaces. Every T lymphocyte expresses a different T cell receptor. Therefore the large population of T cells in the body can collectively defend the body against a large range of threatening cells. New T cells with new receptors are generated every day as T cells develop from immature precursors in an organ called the thymus. The receptors expressed by the immature cells (called thymocytes) are randomly generated. Because each new thymocyte expresses a brand new receptor, the cell must be tested for its suitability before being released into the body. The screening for suitable cells occurs at a pivotal stage of development, the "double positive" (DP) stage, so-called because these cells express two key proteins, CD4 and CD8. DP thymocytes are the immediate precursors of mature T cells. However, the vast majority of DP thymocytes are never selected to become mature thymocytes because their receptors are deemed useless or directed against a normal antigen of the body. The fate of the DP cells during this screening process is governed almost entirely by the signals they receive from their new receptors. Interestingly, DP thymocytes interpret receptor signals very differently from mature T cells. When mature T cells receive a strong signal through their receptor, they become activated and start to divide. When immature DP thymocytes receive the same signal, they also b ecome activated but do not divide. Nothing, in fact, seems to induce cells at this early stage of development to multiply. The reason for this is not yet known. To determine why DP thymocytes fail to divide in response to T cell receptor signals, these studies will compare the molecular machinery responsible for cell division in immature DP and mature T cells. These studies will also test the possibility that the inability of a DP thymocyte to divide may be critical to the success of this screening process which fundamentally shapes the immune response.

The atomic force microscope (AFM) can image samples with a resolution of 2 nanometers laterally and 0.2 nanometers vertically, and can also measure extremely small forces (from 100 piconewtons up to 1 micronewton). Laser tweezers extend this force range below 1 piconewton and also allow manipulation of objects on length scales of 50 nanometers. A fluorescence microscope permits imaging of fluorescent tagging molecules, which, in addition to their typical applications, will be used to insure that the objects being studied with AFM and laser tweezers are indeed those of interest, rather than artifacts or contamination. This instrument cluster will be used for a variety of experiments including:

1) the study of novel self-assembling biomaterials based on the coiled-coil protein folding motif
2) the response of both mature and immature T lymphocyte cells to infected cells
3) the effect of naturally-occurring modifications of the microtubule surface on motor protein traction
4) basic experiments to characterize electron transfer in DNA.


With support from the National Science Foundation, an atomic force microscope, a fluorescence optical microscope, and a laser tweezers workstation will be purchased and combined into a single instrument with powerful imaging, manipulation, and force measurement capabilities. The instrument will be the first to integrate these three technologies using commercial components, and should pave the way for other researchers. Auxiliary equipment for vibration isolation, temperature regulation, and the formation of micropipettes, all of which are required for optimal performance and for the force measurements, will also be purchased.

Haverford College has a strong commitment to excellence in the education of undergraduates. At least four graduating students per year will use this instrument cluster for a senior research project. The interdisciplinary nature of the work will spark exciting interactions between the students and also between their mentors.

Mature CD4+CD8- and CD8+CD8- (single positive or SP) T lymphocytes develop from immature CD4+CD8+ (double positive or DP) thymocytes during transit through the thymus. The transition of cells from the DP to the SP stage is governed by signals generated by interactions between the T cell receptor (TCR) and MHC/antigen complexes encountered in the thymus. Depending on the affinity of the interactions, and involvement of select co-stimulatory molecules, DP cells will either mature or undergo cell death (apoptosis). In particular, high affinity signals mediated by the TCR induce the death of immature DP thymocytes. However, once they mature to the SP T cell stage, they respond quite differently to high affinity TCR stimulation. Instead of inducing apoptosis, SP T cells are induced to proliferate. This project reflects an interest in the molecular basis for this difference in response to TCR signals between precursors and product cell populations.


During the previous NSF funding period, a marked cellular difference in the response of immature and mature T cells to TCR signals was demonstrated. The investigators found that, unlike mature SP T cells, immature DP thymocytes failed to recruit lipid rafts to the site of TCR stimulation. Lipid rafts are specialized subdomains in the lipid membrane that are enriched for a variety of critical signaling molecules and targets that can modulate quantitatively and qualitatively TCR signaling. The data further indicated that lipid raft recruitment required PI 3-kinase activity in mature SP T cells and led the investigators to advance a mechanistic model for raft recruitment. The investigators postulated that immature DP thymocytes failed to recruit lipid rafts because they failed to optimally activate the lipid kinase, PI-3 kinase, in response to TCR signals. Further, they suggested that the failure to recruit rafts increased the susceptibility of DP thymocytes to TCR- induced cell death by compromising the activation of a critical regulator of survival and proliferation, Akt/PKB. These hypotheses will be directly addressed by this research.

T lymphocytes (T cells) regulate a body's defense against infection and recognize pathogens through receptors on their surfaces (T cell receptors) that bind to minced pieces of proteins (peptides) from viruses, bacteria, or other invaders. New T cells are produced every day in an organ called the thymus, and every new T cell produced expresses a novel T cell receptor. Because these novel T cell receptors cannot intrinsically discriminate between proteins made by an invading pathogen and proteins made by the body's own cells, autoimmunity is a constant threat for an organism. However, the body has ways to destroy autoreactive T cells as they browse the thymus during their development in the thymus. Specifically, if an immature T cell binds strongly to any thymic peptide it encounters, its T cell receptor will generate signals that induce the cell to die. An immature T cell that does not bind to a self-peptide strongly is permitted to mature into a functional T cell and exit from the thymus. At this point a mature T cell responds to strong T cell receptor signals not by dying, but by dividing and initiating an immune response. The molecular switch(es) responsible for this marked change in interpretation of a strong T cell receptor signal between an immature and mature T cell are not known, yet are the basis for our understanding of how organisms protect themselves against autoimmunity. This project is designed to improve our understanding of the differences in response to T cell receptor stimulation and builds upon findings that a protein that regulates cell death, known as Nur77, is treated differently in mature versus immature T cells. Whereas both cell types respond to T cell receptor signals by increasing production of Nur77, only the mature T cells respond additionally by altering the protein through the attachment of phosphate molecules (phosphorylation). Investigators will determine what internal signals are responsible for these alterations and how and why they differ between the two cells. They will also work to understand the impact of phosphorylation on the function of Nur77 and have specifically hypothesized that the changes disable Nur77, preventing it from turning on a set of genes that coordinate the cell death program. This work will clarify our understanding of how a T cell population that is screened against responsiveness to 'self' proteins develops the ability to respond to pathogens it encounters outside the thymus. The PI has a strong mentoring program in undergraduate laboratory research. Her students co-author papers and present their findings at meetings.

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.

This award from the Major Research Instrumentation (MRI) program will be used to purchase four significant instruments to support a new core imaging facility at Haverford College, a selective liberal arts college with a diverse student body. The new instruments funded by this grant are a transmission electron microscope, a scanning electron microscope, a confocal microscope, and a fluorescence-activated cell sorting (FACS) system. This facility will serve the research and educational missions of the Biology Department and will also be an important resource for faculty and students in the Physics and Chemistry Departments. These instruments will significantly enhance the research capabilities of the faculty, most with external funding to support their research programs. Since the focus of the Haverford Biology Department is in cell and molecular biology, access to these imaging tools will benefit research projects in areas such as biomaterials research, nanotechnology, developmental biology and embryogenesis, neurobiology, immunology, and stem cell biology. Notably, the faculty collaborate frequently in their research, bringing together skills and expertise to develop synergies that advance science in new directions and serving as role models in students' training. Students are immersed in the process of doing science, fostering their ability to think broadly and from interdisciplinary perspectives. Students will use these instruments for their course work and their senior thesis projects, and will also develop state-of-the-art skills that will be helpful in for a career path in STEM (science, technology, engineering, and mathematics).

T lymphocytes, a group of white blood cells, continually scan our tissues for evidence of infection. They can recognize cells that have been infected and initiate powerful immune responses against these cells as well as against the pathogen responsible for infection. Immature T lymphocytes (also known as thymocytes) have to be "trained" to distinguish uninfected from infected cells. This research team has developed an elaborate system to select young T lymphocytes that have potential to make this distinction from among the millions that are generated daily. This selection process occurs in a specialized organ called the thymus and is a critical step in keeping us safe from autoimmune reactions.

In order to mature successfully, thymocytes must interact extensively with specialized cells known as thymic epithelial cells. These interactions determine which immature T cells can safely enter circulation. This project examines the interesting possibility that mature T lymphocytes, themselves, play a role in shaping these all-important interactions. Using genetically modified mouse strains and a set of imaging tools that can be used to identify and trace cells in the thymus, these investigators will test an original hypothesis that a special group of fully mature T lymphocytes stay in the thymus and enhance the ability of epithelial cells to select the most useful and least dangerous immature T lymphocytes for further maturation. By improving our understanding of the molecular and cellular participants in T cell selection, these studies will shed light on what can go awry and contribute to autoimmune disease.

This investigation will also contribute directly and significantly to the training of young scientists. The studies described will be performed exclusively by undergraduates, who were also the inspiration for the project. The investigator has a very strong track record of educating young scientists: 95% of the seventy-five senior undergraduates mentored in her lab over the last sixteen years have pursued post-graduate studies in medicine, research, education, and public health.