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Virginia
Smith Shapiro
Assistant Professor, Dept of Pathology and Laboratory Medicine
Cancer Biology Program
Address
288 John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104-6082
Office tel.: 215 573-9260
Lab tel.: 215 573-9261
Fax: 215 898-4227
E-mail: shapirov@mail.med.upenn.edu
EDUCATION
Harvard University: AB (Biochemistry), 1989.
University of California, Berkeley: PhD (Molecular and Cell
Biology), 1994.
University of California, San Francisco: Postdoctoral Research
in the Lab of Art Weiss, M.D., Ph.D. (CD28 signalling in T
cell activation), 1995-2000.
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Research Interests
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.
Key
words: CD28 TCR T cell activation
ALX IL-2 AML myeloma.
Search PubMed for articles
Description of Research
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 (Jurkat RE/AP mutants).
The generation of these cell lines is described in Greene
and Shapiro, 2003 (reference below). 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. 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 and we are working
on differentiating between these possibilities. Rotation projects
for CGC/CAMB students would involve characterizing one of
the genes isolated on the screen, or to continue screening
for additional components involved in regulating NF-kB/AP-1
downstream of CD28 stimulation.
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. Interestingly,
ALX overexpression primarily affects CD28-mediated costimulation
of IL-2, rather than TCR-mediated activation. We have recently
generated ALX deficient mice, as well as ALX/RIBP double knockout
mice. Our initial results demonstrate that this family functions
in T and B cell development, activation, survival and cytokine
production.
Recent Publications
Perchonock, C., Fernando, M., Chen, Y.-Y., Shapiro,
M.J., and Shapiro, V.S.: Enhanced T cell activation
in ALX-deficient mice, to be submitted July, 2005.
Shapiro, M.J., Chen, Y.-Y.,
and Shapiro, V.S.: Regulated nuclear export of ALX during
T cell activation, to be submitted July, 2005.
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.
Lab
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, including:
- Lab Personnel
- Michael Shapiro, Ph.D. Research Associate
Claire Perchonock, Graduate Student, IGG
Tony Pajerowski, Graduate Student, IGG
Melissa Fernando, Research Specialist
Yen-yu Tina Chen, Research Specialist
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last updated 6/2005
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