Immunology Graduate Group
Virginia Smith Shapiro, Ph.D.
Assistant Professor,
Pathology and Laboratory Medicine
Address: 288 John Morgan Building/6082
Office Phone: (215) 573-9260
Lab Phone: (215) 573-9261
Fax: (215) 898-4227
Email: shapirov@mail.med.upenn.edu
Education:
Post-doctoral fellow, University of California, San Francisco (lab of Art Weiss, M.D., Ph.D.)
Ph.D., University of California, Berkeley
A.B., Harvard University
Research Summary
My laboratory is interested in:
- Understanding how T cells upregulate NF-kB and AP-1 by CD28 costimulation.
- The role of an adaptor molecule ALX in T cell activation.
The activation of T cells is critical in immune responses, such as the elimination of tumor cells. Minimally, two signals are required to initiate a T cell response: an antigen specific signal through the TCR, and a second “costimulatory” signal, usually provided by CD28. One consequence of T cell activation by TCR/CD28 stimulation is the production of cytokines including interleukin-2 (IL-2). The biochemical events downstream of CD28 activation, and how these signals synergize with TCR stimulation to induce transcription of cytokine genes, is poorly characterized, although NF-kB activation has been shown to play a critical role. The identification of signalling pathways downstream of CD28 will reveal potential targets to manipulate T cell activation and the immune response. The focus of my research is CD28 signal transduction and its role in lymphocyte activation.
Examination of CD28 signalling through analysis of Jurkat mutant cell lines
Mutated cell lines that did not respond to TCR stimulation were instrumental in elucidating the immediate events downstream of TCR engagement. To perform a similar analysis of CD28 signalling, I generated a panel of 15 different Jurkat T cell lines that do not activate an RE/AP reporter (a composite element from the IL-2 promoter containing NF-kB and AP-1 sites) in response to TCR/CD28 stimulation, the J.REM's ( J urkat RE /AP m utants). However, TCR stimulation of a different transcriptional element from the IL-2 promoter, NFAT, is largely unaffected in these cell lines. Therefore, the defect is specific to CD28-mediated costimulation, rather than a generic block in TCR signaling. The generation of these cell lines is described in Greene and Shapiro, 2003 (reference below). We have undertaken a genetic approach to identify the genes responsible for these defects. Two of the J.REM mutant cell lines were infected with a retroviral leukocyte expression library, and screened for their ability to activate the RE/AP reporter. From this screen, we have isolated two cell lines in which RE/AP activation is either partially or totally restored. Of course, in this genetic screen we may re-express a wild-type copy of the mutated gene in the J.REM cell lines, or we may overexpress a protein which can compensate for the original defect; we are working on differentiating between these possibilities.
Role of the ALX and RIBP adaptor proteins in lymphocyte activation
We recently identified a novel adaptor, ALX (Adaptor in Lymphocytes of Unknown Function, ‘X'). ALX was cloned by homology to another adaptor in T cells, RIBP (also known as TSAd, Lad or VRAP). RIBP had been isolated by two hybrid screen for proteins which associated with kinases involved in the proximal events of T cell activation: Lck, Itk, Rlk and MEKK2. However, the RIBP knockout had a mild phenotype, with an approximately 2/3 decrease in IL-2 production after TCR/CD28 stimulation. We reasoned that the lack of strong phenotype in the RIBP knockout may have been due to redundancy with a similar adaptor in T cells. Our initial cloning and characterization of ALX confirmed its functional relationship to RIBP. We have recently generated ALX deficient mice, as well as ALX/RIBP double knockout mice. Interestingly, T cells from ALX-deficient mice produced increased amounts of IL-2 as compared to their wild-type littermates. Therefore, ALX appears to be a negative regulator of T cell activation. While RIBP and ALX are related, it appears that their functions are not redundant, but rather may have opposite functions. We have acquired the RIBP-deficient mice, and have crossed them to our ALX-deficient mice and are examining the phenotype.
Possible rotation projects:
- Continue to screen our retrovirally-infected J.REM cell lines to identify novel molecules involved in the regulation of NK-kB and CD28 signalling during T cell activation.
- Assist in the analysis of clones already identified by this screen and the role they play in regulating signaling pathways during T cell activation.
- Assist in the analysis of the ALX/RIBP/double knockout animals and the role these adaptors have in lymphocyte development and activation.
Current Lab Members (Fall, 2005):
Michael Shapiro, Ph.D. Research Associate
Claire Perchonock, Graduate Student, IGG
Tony Pajerowski, Graduate Student, IGG (rotating student)
Melissa Fernando, Research Specialist
Chau Nguyen, Research Specialist
Recent Publications
Perchonock, C., Fernando, M., Chen, Y.-Y., Shapiro, M.J., and Shapiro, V.S.: Enhanced T cell activation in ALX-deficient mice, manuscript in preparation.
Shapiro, M.J., Chen, Y.-Y., and Shapiro, V.S .: Regulated nuclear export of ALX during T cell activation, submitted.
Shapiro, M.J., Powell, P., Ndubuizu, A., Nzerem, C., and Shapiro, V.S .: The ALX Src homology 2 domain is both necessary and sufficient to inhibit T cell receptor/CD28-mediated up-regulation of RE/AP. J. Biol. Chem. 279 : 40647-40652, 2004.
Greene, T.A., Powell, P., Nzerem, C., Shapiro, M.J. and Shapiro, V.S .: Cloning and characterization of ALX, an adaptor downstream of CD28. J. Biol. Chem. 278 : 45128-45134, 2003.
Greene, T.A., Shapiro, V.S : Genetic Analysis of CD28 signalling. Immunologic Research 27 : 513-520, 2003.
Shapiro, V.S ., Mollenauer, M.N. and Weiss, A: Endogenous CD28 expressed on myeloma cells upregulates IL-8 production: implications for multiple myeloma progression. Blood . 98 : 187-193, 2001.
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