SIGNALS REGULATING T CELL DEVELOPMENT
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Paul Love, MD, PhD, Head, Section on Cellular and
Developmental Biology Sandra Hayes, PhD, Senior Fellow LiQi Li, PhD, Visiting Fellow Ki-Duk Song, PhD, Visiting
Fellow Shoji Uehara, MD, PhD, Visiting
Fellow Dalal El-Khoury, Technician Laura Love, Technician |
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Our research
is directed at elucidating the cellular and molecular processes that regulate
mammalian T lymphocyte development. Within this broad context, our studies
focus on three main areas. The first involves characterization of the role of
T cell antigen receptor (TCR) signals and, in particular, individual TCR
signal–transducing subunits and signal-transducing motifs in T cell
development. Our studies employ a number of genetically altered mouse strains
generated by gene targeting and transgenic technology. Second, we have
extended our studies to include analysis of signal-transducing molecules that
function downstream of the TCR (LAT, TLAP) or that inhibit TCR signaling
(CD5). The aim of these studies is to understand how the molecules
participate in TCR-mediated signaling and to determine the roles they and the
signaling pathways play in regulating in T cell maturation and T cell
activation. Third, we have begun to characterize the function of chemokine
receptors that are expressed on developing T cells. These cell surface
proteins mediate chemotaxis in response to specific ligands that are
expressed in discrete regions of the thymus. Chemokine receptors are
candidates for regulating the homing of progenitor cells to the thymus and
for regulating intrathymic migration of thymocytes. TCR
signaling in thymocyte development El-Khoury, Love; in
collaboration with Rosenberg, Shores A
major theme of our research has been the investigation of the role of TCR
signal transduction in thymocyte development. Signal transduction sequences
(termed immunoreceptor tyrosine-based activation motifs;
ITAMs) are contained within four distinct subunits of the multimeric TCR
complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within
ITAMs are phosphorylated upon TCR engagement and function to recruit
signaling molecules, such as protein tyrosine kinases, to the TCR complex,
thereby initiating the T cell activation cascade. Though conserved, ITAM
sequences are nonidentical, raising the possibility that the diverse
developmental and functional responses controlled by the TCR may be
regulated, in part, by distinct ITAMs. To determine if TCR signal-transducing
subunits perform distinct or analogous functions in development, we generated
zeta-deficient and CD3-epsilon–deficient mice by gene targeting,
genetically reconstituted these mice with transgenes encoding wild-type or
signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and
characterized the developmental and functional consequences of these
alterations on TCR signaling. The results of our studies demonstrated that
TCR-ITAMs are functionally equivalent but act in concert to amplify TCR
signals. We found that TCR signal amplification was critical for thymocyte
selection, the process by which potentially useful immature T cells are
instructed to survive and differentiate further (positive selection). We also
found that potentially auto-reactive cells that may cause autoimmune disease
are deleted in the thymus (negative selection). Thus, the multisubunit
structure of the TCR may have evolved to enable complex organisms to develop
a broad, self-restricted yet auto-tolerant T cell repertoire. McFarland HI, Hansal SA, Morris DI, McVicar DW, Mechanism
of CD5-mediated TCR signal inhibition Park, Love The
cell surface protein CD5 negatively regulates TCR signaling and thus
participates in thymocyte selection. Examination of CD5 expression during T
cell development revealed that surface levels of CD5 are regulated by TCR signal
intensity and by the affinity of the TCR for self-peptide ligands in the
thymus that mediate selection. To determine if the ability to regulate CD5
expression is important for thymocyte selection, we generated transgenic mice
that constitutively express high levels of CD5 throughout development.
Overexpression of CD5 significantly impaired positive selection of some
thymocytes (those that would normally express low levels of CD5) but not that
of others (those that would normally express high levels of CD5). The
findings support a role for CD5 in modulating TCR signal transduction and
thereby influencing the outcome of thymocyte selection. The ability of
individual thymocytes to regulate CD5 expression represents a mechanism for
“fine tuning” the TCR signaling response during development. Our
results indicate that this potential for signal modulation may be
particularly useful for generating the maximum possible diversity in the
mature T cell repertoire. Given that a probable mechanism for CD5 function occurs
via the activation-induced binding of regulatory molecule(s) to sequences
within the CD5 cytoplasmic domain, we generated transgenic mice that express
a tail-less form of CD5 (mCD5). We then used the intact and mCD5 transgenes
to reconstitute CD5 surface expression in CD5–/– mice.
The experiments revealed a critical function for the cytoplasmic domain in
CD5 signaling. We are currently attempting to identify molecules that
interact with CD5 in order to determine how CD5 regulates signal transduction
by the TCR. Bhandoola A, Bosselut R, Yu Q, Cowan ML, Feigenbaum L, Love PE,
Singer A. CD5-mediated inhibition of TCR signaling during intrathymic
selection and development does not require the CD5 extracellular domain. Eur
J Immunol 2002;32:1811-1817. McFarland HI, Hansal SA, Morris DI, McVicar DW, Role
of LAT in T cell development Park, Love; in collaboration
with Samelson, Shores, Sommers Linker
for Activation of T cells (LAT) is an integral membrane protein that
functions as a critical adaptor linking the TCR to multiple downstream
signaling pathways required for T cell activation. The distal four tyrosines
in LAT (tyr136, tyr175, tyr195,
tyr235) are necessary and sufficient for LAT activity in T
cells, which includes activation of the calcium and MAP kinase (MAPK)
downstream signaling pathways. A large number of other receptors activate
these signaling pathways, which are required for the development and function
of many cell types. Thus, their inactivation in all cells would likely result
in embryonic lethality. However, by mutating specific LAT tyrosines, we have
been able to uncouple the TCR from downstream signaling pathways in T cells
without affecting the ability of other receptors or cells to use these
pathways. We generated knockin mutant mice that express LAT proteins
containing single or multiple tyrosine-phenylalanine mutations of the four
critical tyrosine residues. Knockin mice that express the wild-type version
of the protein exhibited normal T cell development, thereby validating the
targeting strategy. Inactivation of all four distal LAT tyrosines yielded a
null phenotype (identical to the LAT knockout), demonstrating the critical
role of these residues for T cell development. Surprisingly, knockin mutation
of the first tyr residue (tyr136) resulted in a fatal
lymphoproliferative disorder characterized by expansion and multitissue infiltration
of CD4+ T cells. Consistent with previous data demonstrating that
tyr136 preferentially binds to phospholipase C-gamma, examination
of the signaling response of T cells from these mice revealed a severe defect
in TCR-induced/phospholipase C-gamma–mediated calcium flux. However,
MAPK signaling was intact in these cells, indicating that the TCR was
selectively uncoupled from the calcium but not from the MAPK pathway. The
results reveal a critical role for LAT in coordinating downstream signals
initiated by TCR engagement and demonstrate that such function is essential
for normal T cell homeostasis. We are currently studying the role of calcium
signaling in thymocyte selection by using the LAT tyr136 knockin
mice and analyzing other LAT tyr knockin mutants generated in our laboratory. Sommers CL, Park CS, Lee J, Feng C, Fuller CL, Grinberg A,
Hildebrand JA, Lacana E, Menon RK, Shores EW, Samelson LE, Love PE. A LAT
mutation that inhibits T cell development yet induces lymphoproliferation. Science
2002;296:2040-2043. Sommers CL, Samelson LE, Structure
and signaling potential of the gamma/delta TCR complex Hayes, Love; in
collaboration with Fowlkes, Laky Most
vertebrate species contain two separate lineages of T cells that are
distinguished by the antigen-binding, clonotype-specific chains contained
within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the
more abundant alpha/beta TCR has been extensively characterized, much less is
known about the structure or function of the gamma/delta TCR, which is
expressed on the smaller subset of gamma-delta T cells. We found that the
subunit composition of the gamma/delta TCR differs from that of the
alpha/beta TCR in that a component of the alpha/beta TCR, the CD3delta chain,
is not present in gamma/delta TCRs. The results revealed a major difference
in the subunit structure of the alpha/beta and gamma/delta TCRs.
Interestingly, we found signal transduction by the gamma/delta TCR to be
superior to that of the alpha/betaTCR as assessed by several criteria. Our
data suggest that the structural difference between alpha/beta and
gamma/delta TCRs may influence the signaling potential of the TCR complex and
may have important functional consequences on T cell activation. Current
studies involve further analysis of the effect of TCR subunit structure on
signaling responses and determining if TCR subunit composition influences T
cell development and T cell lineage commitment. Hayes SM, Laky K, El-Khoury D, Kappes DJ, Fowlkes BJ, Hayes SM, Hayes SM, Shores EW, Role
of the chemokine receptor CCR9 in T cell development Uehara, Love; in
collaboration with Farber T
cell development continues into adulthood and requires the periodic migration
of T-progenitor cells from the bone marrow to the thymus. The ordered
progression of thymocytes through distinct stages of development is also
associated with migration into and between different thymus
microenvironments, where thymocytes are exposed to growth factors and
signals. Chemokines are a group of small, structurally related molecules that
regulate trafficking of leukocytes through interactions with a subset of
seven-transmembrane, G protein–coupled receptors. The chemokine CCL25
is highly expressed in the thymus and small intestine, the two known sites of
T lymphopoiesis. CCR9, the receptor for CCL25, is expressed on the majority
of thymocytes, raising the possibility that CCR9 and its ligand may play an
important role in thymocyte development. To investigate the role of CCR9
during lymphocyte development, we generated CCR9-deficient (CCR9–/–)
and CCR9 transgenic mice. Surprisingly, both T cell and B cell development
appeared normal in CCR9–/– mice. However, bone marrow
transplantation experiments demonstrated that lymphocyte progenitors from
CCR9–/– mice had a markedly reduced capacity to
repopulate the thymus when forced to compete with progenitor cells from CCR9+/+
mice. In other experiments, overexpression of CCR9 in transgenic mice
inhibited early thymocyte development and blocked the normal migration of
immature thymocytes within the thymus. The results indicate that CCR9
participates in regulating both the migration of progenitor cells to the
thymus and the migration of developing thymocytes within the thymus. However,
CCR9 is not essential for normal T cell development, suggesting a functional
redundancy. We are currently testing this hypothesis by generating mice
deficient in both CCR9 and CXCR4, a second chemokine receptor highly
expressed on developing thymocytes. Park MK, Amichay D, Love PE, Wick E, Liao F, Grinberg A, Rabin
RL, Zhang HH, Gebeyehu S, Wright TM, Iwasaki A, Weng Y, DeMartino JA, Elkins
KL, Farber JM. The CXC chemokine murine monokine induced by IFN-gamma (CXC
chemokine ligand 9) is made by APCs, targets lymphocytes including activated
B cells, and supports antibody responses to a bacterial pathogen in vivo. J
Immunol 2002;169:1433-1443. Uehara S, Farber JM, Uehara S, Grinberg A, Farber JM, Uehara S, Song K, Farber JM, Function
of developmental transcription factors in T cell development Li, Love Lhx genes
encode a conserved family of proteins that function as transcription factors
during embryonic development (see report by Westphal). Although these genes
have been shown to play critical roles in the development of various organ
systems, their possible role in lymphopoiesis has not been systematically
examined. In addition, the Wnt signaling pathway has recently been found to
contribute to T cell maturation, suggesting that Wnt proteins as well as
their receptors and inhibitors have important functions during thymocyte
development. We initiated an RT-PCR–based screening for expression of
these genes in fetal and adult lymphoid tissues. An attractive feature of our
study is that knockout mice and embryonic stem (ES) cells are already
available for many of the genes. Thus, if the expression pattern of specific
genes suggests a role in lymphopoiesis, the results can be rapidly extended
by analyzing the lymphoid phenotype of knockout mice or, in the case of
embryonic lethality, generating chimeric mice by using knockout ES cells to
study lymphopoiesis specifically. Woodside KJ, Shen H, Muntzel C, Daller JA, Sommers CL, Love PE.
Expression of Dlx and Lhx family homeobox genes in fetal thymus
and thymocytes. Gene Expr Patterns 2004;4:315-320. COLLABORATORS Joshua Farber, MD, Laboratory of Clinical
Investigation, NIAID, Bj Fowlkes, PhD, Laboratory of Cellular and
Molecular Immunology, NIAID, Karen Laky, PhD, Laboratory of Cellular and
Molecular Immunology, NIAID, Amy Rosenberg, MD, Division of Therapeutic
Proteins, Center for Biologics Evaluation and Research, FDA, Alfred Singer, MD, Experimental Immunology
Branch, NCI, Connie L. Sommers, PhD, Laboratory of
Cellular and Molecular Biology, NCI,
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