Jennifer A. Punt
Research Interests: 2004-2005
Over the past eight years in my laboratory at Haverford College, I have continued a long standing interest in examining the molecular 'reasoning' behind decisions made by developing T cells. We have focused specifically on determining why immature thymocytes interpret T cell receptor costimulation as a death signal but their mature descendants interpret the costimulatory signal as a proliferation signal. Our work has revealed novel molecular differences between the responsiveness of CD4+CD8+ (double positive) thymocytes and mature single positive T cells that should ultimately contribute to our understanding of the molecular mechanism behind thymocyte selection.
T lymphocytes develop in the thymus from cells with no functional capacity antigen-specific regulators and effectors of the adaptive immune response. As they mature, immature T cells (thymocytes) pass through discrete developmental stages characterized by changes in expression of CD4, CD8 and the antigen specific T cell receptor (TCR). Double negative (DN) cells expressing neither CD4 nor CD8 develop into double positive (CD4+CD8+;DP) cells expressing both CD4 and CD8 which in turn develop into cells expressing either CD4 or CD8 (single positive or SP). At the DN stage of development, cells manufacture a unique T cell receptor (TCR) and begin to express receptor components on their surface. A complete TCR is first expressed at the CD4+CD8+ thymocyte stage, which is typically considered an important target of selection events that screen developing cells against autoreactive TCR specifities (negative selection) and for specificities restricted to thymic MHC (positive selection).
The fate of CD4+CD8+ thymocytes appears to differ depending on the "type" of TCR signal they receive. It is generally believed that CD4+CD8+ thymocytes will die (by apoptosis) if they receive either (i) too strong a signal through the TCR (negative selection) or (ii) no signal at all (death by neglect). However, if they receive a signal of "intermediate" strength through the TCR they will differentiate into SP T cells (positive selection). Coreceptors and costimulatory molecules also play a role in guiding a cell through the thymus. In particular, it is becoming more generally accepted that TCR mediated cell death of developing thymocytes also requires second signal(s) that can be provided by CD28, CD5, CD43 and, in some cases, fas.
It is estimated that the overwhelming majority of DP thymocytes die because they don't receive adequate TCR stimulation to maintain a survival program; approximately 5% receive a TCR signal that permits them to mature and another 5% die as a consequence of negative selection. Mature SP thymocytes are ultimately exported to the "periphery" of the organism and populate the spleen and lymph nodes where T cell receptor signals usually results in activation and proliferation.
Therefore, TCR stimulation of maturing T cells can induce death, differentiation or proliferation depending on receptor/ligand avidity, the developmental stage of a cell and the participation of costimulatory partners. The molecular basis for the variation in interpretation of TCR signals is still not fully understood and a topic of intense investigation.
Current research interests in the laboratory.
TCR signals, alone, are not sufficient to induce T cells to divide, differentiate, or die. Instead, they must be accompanied by signals from costimulatory molecules, of which CD28 is an example. In mature cells, TCR/CD28 costimulation results in activation, enhanced survival, sustained IL-2 production, and proliferation. In immature DP thymocytes, however, TCR/CD28 costimulation results in apoptosis.
Although CD28 is not unique in its ability to cooperate with TCR to induce thymocyte death, in vivo, it is a potent costimulator of TCR-induced apoptosis in vitro. Because in vitro TCR/CD28 costimulation results in such different outcomes in immature and mature T cells, it is a particularly useful probe for developmentally regulated differences in TCR signaling. Using this system, we have specifically assessed differences in the consequences of TCR costimulation of immature and mature T cells. Although we typically use immobilized antibody (bead bound or plate bound) to stimulate our cells in our efforts to understand negative selection events, we are also beginning to work with a TCR transgenic system that will allow us to examine more physiologic interactions between thymocytes and antigen presenting cells that can mediate negative selection in vivo.
Specific research questions that are currently being asked in the laboratory
Š Why don't DP thymocytes proliferate in response to mitogenic stimuli? Instead of asking why DP thymocytes are so apt to die when stimulated (a topic many are interested in), we have asked why they are so unable to proliferate (a topic fewer are interested in) - the theme of the first NSF grant I received (NSF-RUI #MCB-9728332). Briefly, we have found that DP thymocytes progress into mid-G1 phase of the cell cycle, but fail to upregulate cyclin E and progress into S phase. We have evidence that the failure to upregulate cyclin E is due not to a failure to phosphorylate the cell cycle regulator, Rb, but rather to a developmentally regulated difference in expression of the E2F family of transcription factors which associate with Rb. We also have evidence that the cell cycle machinery is being usurped to induce apoptosis in thymocytes.
Jones, K., Bliwise, H., Nish, S., Zauber, S. E., Brinner, K., Rahman, M., Odessey, E., Tolat, A., and J. A. Punt. Cell cycle regulation of immature CD4+CD8+ thymocytes. Manuscript in preparation (Note: this has been in preparation for quite some time – just as we get ready to write, data and perspectives evolve. Suffice it to say that the work is still ongoing –and a manuscript is still in the future.)
Our work on negative selection has also inspired a renewed interest in Nur77, a molecule that regulates apoptosis in developing T cells and is likely responsible for the apoptotic events inspired by TCR/CD28 costimulation of CD4+CD8+ thymocytes. Our preliminary data confirm previous observations that Nur77 is rapidly upregulated in response to TCR-mediated apoptotic stimuli and also shows that Nur77 expression patterns differ between immature and mature T cells. Not only do the kinetics of expression differ, but Sam Edmondson ('03), MacLean Sellars ('04), Steve Artim ('04) and Allison Tan ('03) have recently shown that the phosphorylation status and cellular localization of Nur77 also appears differs according to developmental stage. Our data also indicate that Nur77 expression may be an unusually useful in vivo marker for cells just targeted for negative selection and has helped us to resolve questions about the precise phenotype of thymocytes that receive TCR-mediated apoptotic signals in vivo.The role of Nur77 in T cell fate decisions.
Nur77 is also a downstream target of multiple signaling routes that are of particular interest to us (as described above), including Ca2+, Notch, cell cycle kinases, MAPK, and Akt. Grant Scott ('06) confirmed that the differences in expression pattern of Nur77 between immature and mature T cells was due to phosphorylation events and Steve Artim ('04) determined that MAPK and Akt cooperate to phosphorylate Nur77 in mature T cells.We are currently working to determine how changes in phosphorylation alter Nur77 function and compartmentalization in developing T cells, a project that will be pursued by Rachel Gormley ('05) and Nicole Monatesti (Research technician), with the help of some new tools that Scott Gordon ('06) is developing. This is the theme of our third and recently funded NSF grant (#MCB-0343479).
Š Do proximal TCR signaling events differ between immature and mature T cells?
a. Lipid rafts. Pete Ebert ('00) showed that unlike mature T cells, immature CD4+CD8+ cells fail to reorganize lipid microdomains (lipid rafts) upon TCR/CD28 costimulation. We are working to determine the reasons for this failure and the consequences of this failure to cap lipid rafts. This research is the topic of our last NSF grant (NSF-RUI #MCB-0090921), which foucses on the role of the PI-3 kinase and AKT (PKB) signaling pathways in developing T cell responses. These findings also inspired a collaboration with a Biophysics investigator at Haverford College, Dr. Suzanne Amador and an equipment grant that resulted in the purchase of a confocal microscope with colleagues at Bryn Mawr College.
b. Akt/PI3K signaling pathways. Our most recent data suggest that the failure of immature DP thymocytes to recruit lipid rafts (see paper by Pete Ebert ('03) above) may compromise their ability to activate the key survival and pro-proliferative signaling pathway mediated by Akt. Allison Tan ('03), Christen Fornadel ('04), and Shawn Alexander ('03) have been instrumental in demonstrating that DP thymocytes do not optimally activate Akt and have generated data indicating several reasons for the depression in activity. This work is being prepared as a manuscript. We are currently investigating the hypothesis that differences in Akt activity underlie the differential phosphorylation of Nur77 that we observe in stimulated immature and mature T cells and, in a broader context, may underlie overall differences in vulnerability to TCR mediated apoptosis.
c. Ca2+ signaling. We have collaborated with Dr. Bruce Freedman at the University of Pennsylvania to examine the role of Ca2+ signaling in T cell fate decisions. We have found that Ca2+ fluctuations are regulated differently in immature and mature T cells and can be modulated depending on the avidity of TCR signals. This work is the subject of a manuscript Bruce and I co-wrote in 1999.
Freedman B.D., Liu Q.-H., Somersan S., Kotlikoff, M.I., and J.A. Punt. (1999). Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4+CD8+ thymocytes. J. Exp. Med. 190: 943-952.
d.MAPK's. We haverevived an interest in the MAPK, p38, whose activity appears to be required for negative selection. Frank Albino ('05) has been working with Dan Grant ('06) to establish a flow based assay for p38 activity and we are evaluating where it fits in the signaling pathways responsible for TCR-mediated death of thymocytes. Nathan Singh ('06) has also been involved in establishing these phosphoflow assays.
e. Cytoskeleton reorganization. We have recently been intrigued when reevaluating data generated by graduate John Szumowski ('02) and a rotating student, Emily Nietzreba ('04) suggesting that both immature and mature T cells polarize actin and actin associated proteins (ERMs) normally after TCR-stimulated but only mature T cells polarize their microtubules to the site of stimulation. Mary Chang ('05) is working to confirm these data and understand differences in the regulation of MTOC in immature thymocytes.
What regulates early caspase activity during negative selection?
Scott Cho (Research technician, now grad student at University of Utah) developed a flow based caspase-3 cleavage assay and showed that cells activate caspase-3 within 2 hours of TCR/CD28 stimulation. His preliminary data suggest that this activation is caspase-8 dependent yet death receptor independent, an observation that is intriguing but consistent with the work of Mike Lenardo and Astar Winoto. Darren Hirsch ('05) and Nicole Monatesti are currently working to confirm the involvement of caspase, determine whether caspase-9 plays a role, and test the hypothesis that Nur77 is involved in early caspase-8 and caspase-3 activation.
What is the basis for the ability of Notch to regulate T cell fate decisions?
Izon, D*., Punt, J.*, Karnell, F., Xu, L-W, and W. Pear. Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. (2001). Immunity 14: 253-264. *shared first authorship
A comment on research at a Haverford College, an undergraduate institution:
Although Haverford College is strictly an undergraduate institution, it has long been committed to supporting original research among its faculty and student body. Teaching is a very important component of our jobs, but the integration of teaching and research efforts are the cornerstone of our undergraduate educational program in the sciences, social sciences and humanities, alike. In the classroom, we emphasize experimental evidence behind textbook ‘fact’ and in the laboratory, students are introduced to cutting edge biological techniques in the context of a significant biological problems. In their senior year, all Biology majors critique the primary literature and generate their observations, hypotheses and evidence by performing research in our laboratories.
All tenure-line faculty in the Biology Department are currently supported by relatively large individual (typically NSF-RUI) grants. Our natural science research program is also supported by multiple grants that enhance our resources and curriculum. We recently received HHMI funding for a fourth round to sustain our program for young research scholars and to foster interdisciplinary research efforts in the area of Bioinformatics, Ethics, and Computer Science. We have also received a Beckman Foundation grant to promote the scientific development of talented students committed to a research career, and a Packard Foundation grant for a particularly promising interdisciplinary research project. We continue to receive funding from other agencies that support student research endeavors, including the Merck foundation and the Sherman Fairchild Foundation. Through a combination of the efforts of our undergraduates (which can be remarkable, but also more slowly paced) and select collaborations with colleagues at other institutions, we are able to make significant progress in our research goals.
Our efforts are also enthusiastically supported by our administration, which awards us teaching credit for our senior research students, supports summer research students, and provides the Department with a healthy budget that allows us to maintain and update equipment and facilities. The College has just completed the first phase of construction of an interdisciplinary science center (INSC) that will bring all the natural science departments together under one facility designed to foster and inspire interdisciplinary teaching and research. The opportunity to interact with scientists in diverse fields is a remarkable 'perk' of the research undergraduate institution and has already resulted in a fruitful collaborations between our laboratory and the laboratory of two biophysics professors (Drs. Suzanne Amador and Walter Smith).
Students trained in our laboratory:
We have no graduate students at Haverford College. However, during their senior year, our undergraduates operate at the level of graduate students in the laboratory and I am continually impressed by their energy, devotion and insights. Many spend the summer in the laboratory prior to their thesis work and are able to contribute a great deal ot our progress.
Students are intimately involved in the experimental process and design of the work, which is funded by grants from NSF, NIH, and the HHMI. They discuss their work and current, relevant literature at weekly lab meetings, present their work formally at a final poster/presentation session and at national meetings. The ability of the students and their continuing interest in the biomedical sciences are manifest by their accomplishments and choice of careers, as shown below. Those students asterixed contributed directly to manuscripts that have been published or are in the late stages of preparation.
Š Alex Baldwin Graduate School, BU
Š Elizabeth Crane Masters in Education, Harvard U.
Š Leah Watson Research technician, NIH
Š Kate Steiner MPH, UC Berkeley
Š Brian Bean Graduate School in International Policy
Š Caroline Bishop* Graduate Student, U. of Washington
HHMI scholar, graduated Summa Cum Laude (Bryn Mawr College), High Honors in Biology
recipient of NSF and HHMI graduate fellowships
Š Jesse Goldmark Graduate Student, U. of Washington
graduated with Honors in Biology
Š Rachel Kowal Medical School, U. of Rochester
graduated with Honors in Biology
Š Mariam Rahman* Medical School, MCP, Philadelphia
graduated Magna Cum Laude (Bryn Mawr College) (deferred)
Š Princess Toussaint Medical School, MCP, Philadelphia
Š Jon Willis Computer technology, Boston
graduated with Honors in Biology
Caroline Bishop attended the FASEB sponsored AAI meeting in San Francisco, April 1998 and presented a poster entitled: “Ca++ response patterns in immature versus mature T cells”. She also won first prize in the undergraduate category for a distinct poster entitled “Expression of major bcl-2 family members in T-cell development” at the Thomas Jefferson University Research Day competition in March, 1998. Brian Bean also presented a poster entitled “JNK is differentially activated in immature versus mature T cells” at this session.
Š Sam Floyd Graduate School in Philosophy, U. Pitt.
HHMI scholar, graduated Magna Cum Laude, Honors in Biology, High Honors in Philosophy
Š Amy Jost* Medical School, U. of Chicago
graduated Magna Cum Laude, High Honors in Biology
Š Eric Odessey* Residency in Reconstructive Surgery, U. Chicago.
Š Selin Somersan* MD/PHD program, MIT/Harvard U.
Merck scholar, graduated with Honors in Biology
Š S. Elizabeth Zauber* Residency in Neurology, Wash. U., St. Louis
Amy Jost, Selin Somersan and Liz Zauber accompanied me to the FASEB sponsored AAI meeting in Washington, D.C. in April, 1999. Amy Jost presented a poster entitled: “Life and Death in the Immune System: The expression of bcl-2 family members in thymocyte development”. Liz Zauber was a winner in the undergraduate category for her poster entitled: “p27 is required for bcl-2 induced cell cycle inhibition” at the Thomas Jefferson University Research Day competition in March, 1999.
2000 (on sabbatical)
Š Peter Ebert* Graduate School, Immunology, Stanford U.
HHMI scholar, graduated Magna Cum Laude with High Honors in Biology and Mathematics
Recipient of NSF and HHMI graduate fellowships
Š Kathryn Brinner* Residency, Pediatrics, Harvard U.
HHMI scholar, graduated Magna Cum Laude with High Honors in Biology
Pete Ebert and Katie Brinner accompanied me to the Keystone Symposium on T cell activation, differentiation, and death at Keystone Colorado in January/February 2000 and were invited to present their work orally. Pete presented a poster entitled “Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated stimulation”. Katie presented a poster entitled “Bcl-2 mediated inhibition of T cell proliferation is p27-dependent”.
Š Josh Adelman* (with Suzanne Amador) ('01) Graduate student, UC Berkeley ,
Biophysics concentrator, Honors in Biology and Physics, Fulbright Scholarship
Developed a mathematical model for lipid raft polarization
Š Josh Baker* ('01) U. Penn. School of Medicine
Examined the polarization of other TCR signaling molecules (including LAT and tyrosine phosphorylated proteins) after stimulation of immature and mature T cells) after TCR stimulation. Co-author on our publication in J. of Immunology (2000).
Š Tamara Coley ('01) Columbia University, Public Health
Minority Scholar Performed library research on the basis for subtype dominance in HIV infections in sub-Saharan Africa
Š Chris Cornell ('01) Traveling with his band in California
Examined the subcellular localization of Akt in immature and mature T cells using confocal microscopy
Š Simone Nish* ('01) Graduate student, Yale University
Minority Scholar, Graduated with Honors in Biology, Ariel Loewy Prize for Senior Research
Assessed Rb phosphorylation status of immature versus mature T cells after TCR stimulation. Gave oral and poster presentation at AAI meeting (Orlando, FA 2001).
Š Jessica Nguyen ('01) Medical School, U. Pittsburgh
Minority Scholar, High Honors in Biology Examined the expression and subcellular localization of the Akt-regulated protein, Nur77, in immature and mature T cells.
Š Allison Teed ('01) Research Assistant, Mass. Gen., Boston
Examined activation status of Akt in immature versus mature T cells.
Š Kate Gentry* ('02) U. Washington School of Medicine
Magna cum laude, High Honors in Biology Examined the activity of Akt in immature and mature T cells and the phosphorylation of downstream substrates of Akt
Š Brie Mahoney ('02) University of Connecticut School of Medicine
Examined the role of LFA-1 in immature T cell stimulability (in collaboration with Dr. Erik Peterson and Dr. Gary Koretzky).
Š Arden Miller* ('02) Consultant in Health Care industry
Honors in Biology Examined the expression of Nur77 during thymocyte development
Shared first authorship on Cutting Edge publication in J. of Immunology (2003).
Š John Szumowski* ('02) Harvard Medical School
Magna cum laude, High Honors in Biology, HHMI Scholar Examined changes in polarization of ERM family members in immature and mature thymocytes. Won third prize at the Thomas Jefferson University Student Research Day for his poster entitled "Exclusion of Inhibitors of T Cell Activation from the Immunological Synapse in CD4+CD8+ Thymocytes" (Spring, 2002).
Š Shawn Alexander* ('03) Research technician, Wistar Institute
MSP Scholar Examined the cause of the failure of lipid rafts to polarize after costimulation of immature T cells. Focusing on PI 3-K activity. Presented poster at AAI Conference, Denver, Colorado, May, 2003. Shared third prize with Chris Bennett (HC '03) for their poster entitled "The generation of a retroviral construct for overexpression of m-survivin in lymphocytes" (Fall, 2001). Co-author on Cutting Edge publication in J. Immunol. (2003).
Š Mari Davis ('03) English teacher in Milan, Italy
Examined the role of Nur77 in immature B cell deletion.
Š Sam Edmondson* ('03) Musician, San Francisco
Summa cum laude, High Honors in Biology, Ariel Loewy Prize for Senior Research, Beckman Scholar Examined the role of Nur77 in negative selection of immature CD4+CD8+ thymocytes. Shared first authorship on Cutting Edge publication in. J. Immunol. (2003). Presented poster entitled "Tracing the role of Nur77 in the negative selection of autoreactive thymocytes" at the Midwinter Conference of Immunologists, Asilomar, CA, January, 2003.
Š Kevin Jones* ('03) Graduate student, UCSF
HHMI Scholar, Honors in Biology, recipient of NSF Graduate Fellowship. Examined the role of the cell cycle kinase cdk2 in the negative selection of CD4+CD8+ thymocytes. Won award at the Thomas Jefferson University Student Research Day for his poster entitled "Differential compartmentalization of cdk2 in immature thymocytes and mature T cells" Spring, 2002.
Š Allison Tan* ('03) Research Assistant, UCSF
Ariel Loewy Prize for Senior Research. Examined the activity of Akt in immature and mature T cells to test hypothesis that Akt is suboptimally activated in immature thymocytes after TCR/CD28 costimulation. Won second prize at the Kimmel Cancer Center Research Day for poster entitled "The role of suboptimal Akt Activation in immature thymocytes apoptosis" in Fall, 2002. Presented poster at the Midwinter Conference of Immunologists, Asilomar, CA January, 2003.
Š Steve Artim* ('04) Research Assistant, MIT
HHMI Scholar, Honors in Biology, Recipient of Koshland Prize for contributions to Biology Department. Examined the molecular basis for differential phosphorylation of Nur77 in immature and mature T cells and shared award with MacLean Sellars at the Thomas Jefferson University Research Day undergraduate poster competition (Spring, 2004) for poster entitled Nur77: Living with Death, which they also presented at the T cell development Keystone Symposium at Banff, Alberta (February, 2004).
Š Christen Fornadel* ('04) Fulbright Fellowship, Australia
Beckman Scholar, High Honors Biology, Fulbright Scholar. Evaluated the hypothesis that suboptimal activation of phosphoinositide 3-kinase (PI3K) and the downstream kinase Akt is responsible for the failure of immature T cells to hyperphosphorylate Nur77 and presented poster entitled “Examination of PI3-kinase in developing T lymphocytes” at the Beckman Symposium in Irvine, Ca (2003). She also received an award for her poster entitled “Akt is differentially activated in immature and mature T cells” at the Thomas Jefferson University Research Day undergraduate poster competition (Spring, 2004), which she also presented at the T cell development Keystone Symposium at Banff, Alberta (February, 2004).
Š MacLean Sellars* ('04) Research technician, IGMBC, Strasbourg
HHMI Scholar, High Honors Biology, double major in Biology and Sociology. Examined the functional consequence of Nur77 phosphorylation in immature and mature T cells. Won Grand prize at the Thomas Jefferson Research Day undergraduate poster competition (Fall, 2001) for his poster entitled "What's the difference? Nur77 and costimulation in TCR mediated apoptosis of immature T cells and proliferation of mature T cells". Shared award with Steve Artim at the Thomas Jefferson University Research Day undergraduate poster competition (Spring, 2004) for poster entitled Nur77: Living with Death, which they also presented at the T cell development Keystone Symposium at Banff, Alberta (February, 2004).
2004-2005 Academic Year
Frank Albino (’05), HHMI Scholar, is working on the role of p38 in thymocyte negative selection.
Mary Chang (’05), MSP scholar, is evaluating differences in cytoskeletal organization between mature and immature T cells.
Darren Hirsch (’05) is working to determine the molecular events responsible for early activation of caspase 3 in TCR./CD28 stimulated thymocytes.
Rachel Gormley (’05) is assessing the subcellular compartmentalization of Nur77 and its function in immature versus mature T cells
Susannah Locke (’05), HHMI Scholar, is evaluating the activation of Notch in developing T cells.