By
From the Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom
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Abstract |
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The tyrosine kinase p56lck regulates the differentiation and proliferative expansion of pre-T
cells. However, nothing is known about other signaling molecules that operate with p56lck to
mediate the pleiotropic changes that occur at this stage of thymocyte development. We used a
genetic strategy to examine the requirement for the GTPase Rho in p56lck-mediated signals in
the thymus. By generating mice double transgenic for a constitutively activated form of p56lck
(p56lckF505) and the Rho inhibitor C3 transferase we were able to compare thymocyte development in mice expressing active p56lck on a wild-type or Rho background. Thymocytes
expressing active p56lck show enhanced proliferation of pre-T cells resulting in increased numbers of late pre-T cells, however, this dramatic effect on pre-T cell proliferation is lost when
the p56lck transgene is expressed in thymocytes lacking endogenous Rho GTPase function.
Expression of active p56lck also generates double positive (DP) thymocytes with low levels of
CD2 antigen expression. Again, p56lck cannot prevent expression of CD2 when expressed on
a Rho
background. CD4+CD8+ DP cells expressing active p56lck have been shown to lack
functional
/
-T cell receptor (TCR) complexes due to p56lck-mediated inhibition of TCR
gene V
-D
rearrangement. This inhibition of TCR expression by active p56lck is unimpaired in the absence of Rho function. The signaling pathways that are mediated by p56lck and
control thymocyte proliferation,
/
-TCR and CD2 antigen expression can thus be distinguished by their dependency on Rho function.
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Introduction |
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Lymphocyte development in the thymus is dependent
on signaling pathways generated by antigen receptors
and cytokine receptors (1, 2). Different developmental
stages within the thymus can be defined by expression of
CD4 and CD8 molecules which act as receptors for MHC
class II and class I molecules, respectively. The earliest T
cell progenitors are found within the CD4CD8
double
negative (DN)1 population that comprises 2-3% of the total
thymocyte population. These progenitor cells can be further subdivided into four developmental stages on the basis
of expression of CD44 and CD25 molecules: the earliest
progenitors are CD44+CD25
, followed sequentially by
CD44+CD25+, CD44
CD25+, and CD44
CD25
stages.
At the CD44
CD25+ stage, cells that successfully rearrange
their TCR-
locus proliferate rapidly, downregulate CD25
and differentiate into CD4+CD8+ double positive (DP)
cells. The process of allelic exclusion arrests further rearrangement at the TCR-
locus and ensures the generation
of DP thymocytes with a single productive TCR-
chain. DP cells then undergo TCR-
chain rearrangements and
upon expression of a functional
/
-TCR complex are
subjected to the processes of positive and negative selection
that ensures the generation of CD4+CD8
or CD4
CD8+
single positive (SP) thymocytes that exit to the periphery.
The transition of cells from the CD44
CD25+ to the
CD44
CD25
compartment is a critical checkpoint for
thymocyte differentiation and is controlled by signals generated by the pre-TCR complex. Mice lacking expression
of any of the components of this complex such as the
CD3
subunit (3), the TCR-
chain (4), or the pT
subunit (5) show a developmental block at the CD44
CD25+
stage.
The signal transduction pathways used by the pre-TCR
to control T cell differentiation involve the activation of
protein tyrosine kinases. Mouse strains with null mutations
in the tyrosine kinases p56lck (6), ZAP70, and Syk (7), or
their regulatory molecules such as CD45 (8) show defects
at the pre-T cell stage of development. Several other studies in particular highlight the importance of the tyrosine kinase p56lck in the transmission of pre-TCR-mediated signals. Mice lacking p56lck or overexpressing a dominant
inhibitory p56lck mutant (6, 9) show a defect in early thymopoiesis similar to that seen in pT or TCR-
chain-deficient mice. Additionally, transgenic expression of a constitutively active form of p56lck can induce the transition of
thymocyte progenitors from the CD44
CD25+ to the
CD44
CD25
compartment and induce the generation of
DP thymocytes in mice lacking expression of the pre-TCR
complex, e.g., in RAG
/
or pT
/
mice (10, 11). This
indicates that activated p56lck is sufficient to initiate all the
critical events that occur as CD44
CD25+ early pre-T cells
develop into CD44
CD25
late pre-T cells: the onset of
rapid cell cycle progression, allelic exclusion at the TCR-
chain locus and ultimately expression of CD4 and CD8.
We recently generated mice that lack function of the GTPase Rho in thymocytes due to tissue-specific transgenic expression of a Rho inhibitor, C3 transferase, which selectively
ADP-ribosylates Rho and thereby abolishes its biological
activity (12). The phenotype of these mice indicated that Rho
might also be important for pre-TCR function (13). Mice
lacking Rho function in the thymus were able to generate
DP and SP thymocytes, but at severely reduced levels. Loss
of Rho function resulted in decreased cell proliferation in
CD44CD25
late pre-T cells due to a partial block in G1/S
cell cycle progression. This stage of thymocyte development
is regulated by p56lck which raises questions about the positioning of Rho in the context of the p56lck signals that
control the proliferation of early thymocyte progenitors. In
fact, very little is known about the signaling pathways downstream of p56lck that control pre-T cell differentiation. Given the involvement of both p56lck and the GTPase Rho
in the regulation of proliferation in late pre-T cells we considered it possible that Rho might function in the signaling
pathways used by p56lck to regulate the checkpoints for
proliferation and differentiation at the pre-T cells stage of
thymocyte development. To explore these questions, we generated mice double transgenic for active p56lck (p56lckF505;
reference 14) and C3 transferase (12) and examined the
ability of active p56lck to drive thymocyte proliferation and
differentiation in the absence of Rho function. Mice expressing active p56lck show accelerated pre-T cell proliferation
and generate CD4+CD8+ DP thymocytes that fail to express
/
-TCR complexes via a process that mimics allelic exclusion. The present study reveals that the proliferative signals
generated by p56lck are suppressed in mice lacking thymic
Rho function. In contrast the ability of active p56lck to
suppress expression of
/
-TCR complexes is unimpaired. p56lck thus uses either qualitatively or quantitatively different signals to control thymocyte development at the pre-T
cell stage, p56lck-mediated signaling pathways for proliferation are dependent on the Rho GTPase whereas signals
for regulation of antigen receptor expression are not.
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Materials and Methods |
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Transgenic Mice.
Transgenic mice expressing the C3 transferase transgene under the control of the proximal p56lck promoter were generated as previously described (12). Mice transgenic for an activated form of the tyrosine kinase p56lck (p56lckF505; strain pLGF-2964, 10 copies of p56lckF505 transgene) were provided by Dr. Roger M. Perlmutter (Merck Research Labs.; 14). Expression of this transgene is also driven by the proximal p56lck promoter, restricting expression of the transgene to thymocytes. Mice were kept under SPF conditions and bred to C57BL/6/J mice to maintain the transgenic lines. Mice double transgenic for C3 transferase and p56lckF505 (pLGF/C3) were generated by cross-breeding of the parental strains.PCR.
Offspring were genotyped for the presence of both transgenes by PCR analysis of genomic mouse DNA using transgene specific primer pairs: b-actin (actin 1: 5'-GTGGCCATCTC-CTGCTCGAAGTC-3' and actin 2: 5'-GTTTGAGACCTTCAACACCCC-3'); C3 transferase (G9112: 5'-GCCACCATGGAGCAGAAGCTGATCTCCG-3' and C3NT: 5'-CTGATTTGCTTAGTCCATAC-3'), and p56lckF505 (pLGF-Fwd: 5'-ATGACTTCTTCACAGCCACAGAGGG-3' and pLGF-Rev: 5'-TTTTATTAGGACAAGGCTGGTGGGC-3'). PCR reactions were performed in 20 mM ammonium sulphate, 75 mM Tris-HCl, pH 9.0, 0.01% Tween 20, 1.2 mM MgCl2, 200 µM dNTP, 0.5 U Taq DNA polymerase, and 1 µM of each primer. Genomic DNA was purified from mouse ear clips and used as template for 30 cycles of polymerase chain reaction (57°C annealing, 72°C elongation, 95°C denaturation). PCR products were separated in 2% agarose gels and stained with ethidium bromide.Thymocyte Preparation.
Young adult mice of ~4-6 wk of age were typically analyzed. Thymocytes were obtained by carefully mincing the thymus with forceps and filtering through a fine mesh filter to obtain a single cell suspension. Total cell numbers were determined by counting of trypan blue stained thymocytes within a representative volume of cell suspension using a Neubauer hemocytometer.Flow Cytometric Analysis.
Freshly isolated thymocytes were stained with saturating concentrations of antibody in 100 µl cold PBS supplemented with 1% BSA on ice for 30 min in a 96-well V-bottom shaped microtiter plate. Cells were washed twice in between incubations with this buffer. In a first step cells were incubated with anti-FcgRII mAb in order to block any unspecific staining. All mAb used were conjugated to either FITC, PE, or biotin. Biotinylated mAb were revealed using streptavidin-Tricolor (Caltag Laboratories, South San Francisco, CA). The following mAb were used (all PharMingen, San Diego, CA): CD8 (53-5.8), CD4 (RM4-5), CD25 (IL2RCell Cycle Analysis.
Cellular DNA content was assayed by standard techniques using staining with propidium iodide. In brief, thymocytes were stained with biotinylated mAb against CD4, CD8, CD3, B220, NK, Mac-1, Gr-1, and CD44 and surface staining revealed with FITC conjugated Avidin (Sigma Chemical Co., St. Louis, MO). Cells were fixed in 70% ethanol for 30 min on ice, washed free of ethanol, RNase (1 mg/ml) treated for 15 min at room temperature, and then resuspended in propidium iodide (50 ug/ml in PBS) in order to stain DNA. Maintaining a low flow rate events were collected and propidium iodide fluorescence was measured >600 nm on a FACS®Calibur (Becton Dickinson & Co.). DNA content of FITC-negative (CD44 ![]() |
Results |
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The developmental maturation of pre-T cells is associated with downregulation of the CD25 surface marker
and the acquisition of CD4 and CD8 molecules (15). At
this stage, only thymocytes that productively rearranged
their TCR- chain and express a complete pre-TCR complex are selected for further development and transition into
the DP compartment, a process known as
-selection (16, 17). Current models propose that the pre-TCR complex
triggers this maturation program via intracellular signaling
pathways that involve activation of the tyrosine kinase
p56lck (18).
-selection is bypassed in thymocytes that express a constitutive activated form of p56lck (19), resulting
in differentiation and proliferation of all CD44
CD25+
pre-T cells, regardless of a productive or unproductive
chain rearrangement. Accordingly, pLGF mice expressing a
constitutively active p56lck (p56lckF505) transgene in the
thymus show enhanced generation of CD44
CD25
pre-T
cells compared with normal control mice (10). Comparison of CD25 expression on CD44
triple negative (TN) thymocytes (CD44
CD4
CD8
CD3
) from pLGF mice with
normal control mice shows how expression of activated
p56lck results in an increased frequency of CD44
CD25
late pre-T cells and a concomitant low frequency of
CD44
CD25+ early pre-T cells (Fig. 1 a).
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Analysis of the cellular DNA content of pre-T cells from
pLGF mice compared with normal control mice is shown
in Fig. 1 b. We observed a marked increase of cells in the
proliferative S and G2 phases of the cell cycle in CD44 TN
thymocytes from pLGF mice when compared with cells of
the same phenotype from normal control mice. Thymocyte-specific transgenic expression of activated p56lck
thus accelerates proliferative responses in pre-T cells. The
dramatic enhancement of the production of late pre-T cells
by active p56lck is illustrated by a comparison of total cell
numbers for this population in pLGF mice compared with
control mice. A normal thymus contains an average number of CD25
CD44
late pre-T cells of 1.2 × 106, whereas
in pLGF mice their numbers are typically increased to ~6.0 × 106. There is thus a fivefold increase in total numbers of CD44
CD25
late pre-T cells in thymi from pLGF
mice compared with thymi from normal control mice.
Proliferative responses in late pre-T cells are reduced in
thymi lacking endogenous Rho function as a consequence
of transgenic expression of C3 transferase (C3). These cells
show a severe decrease in the production of late pre-T cells
(12). To explore whether p56lck-mediated responses in
early T cell development required endogenous Rho function
we crossed pLGF mice (14) to transgenic mice with inactivated thymic Rho function (C3). We then used CD44 and
CD25 markers to analyze early thymocyte progenitors in
TN thymocytes of pLGF/C3 double transgenic mice. Total cell numbers and the cell cycle status of the CD4425
pre-T cell compartment were monitored. The data in Fig.
2 a show that the increases in the percentage of cycling
pre-T cells seen in pLGF mice are lost in pLGF/C3 double
transgenic mice, i.e., loss of Rho function antagonizes the
action of p56lck in driving cell cycle progression in pre-T
cells. There are still some cycling cells in pre-T cells from
pLGF/C3 double transgenic mice but the loss of Rho
function appears to prevent the ability of activated p56lck
to induce a hyperproliferative response in CD44
CD25
late pre-T cells.
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The most striking consequence of the decreased proliferative capacity of pre-T cells in pLGF/C3 double transgenic
mice is presented in Fig. 2 b, which shows absolute numbers of CD44CD25
late pre-T cells in pLGF and pLGF/C3
mice. The data reveal that in the absence of endogenous
Rho function p56lck is unable to enhance the production
of CD44
CD25
late pre-T cells. Thus, pLGF/C3 double
transgenic mice do not show the skewed distribution of
CD44
CD25
and CD44
CD25+ subpopulations seen in
pLGF mice. These results indicate that Rho function is required for active p56lck to accelerate the transition of early
thymocytes from the CD44
CD25+ to the CD44
CD25
compartment.
The consequence of a productive rearrangement
of the TCR- locus in thymocytes is expression of the pre-TCR complex and the initiation of the process of allelic
exclusion that ensures that mature T cells express a TCR
complex with only one
chain (20). p56lck function is required for this pre-TCR initiated response. Moreover,
transgenic expression of activated p56lck inhibits V-DJ
-rearrangements and expression of the TCR-
chain in thymocytes. Consequently, DP thymocytes that develop in
pLGF mice show defective expression of the TCR-CD3
complex (19). The data in the left panel of Fig. 3 a show
/
-TCR staining profiles of thymocytes from pLGF mice in
comparison with thymocytes from control littermate mice.
Normal thymocytes contain a large subpopulation of cells expressing intermediate levels of the TCR-CD3 complex
and a smaller subset of cells with upregulated, higher levels
of TCR expression. In contrast, thymocytes derived from
pLGF mice show an abnormal
/
-TCR staining pattern
with few intermediate or high
/
-TCR positive cells.
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If Rho function was required for p56lck-mediated suppression of TCR- chain rearrangements then expression
of activated p56lck in Rho
thymocytes would not prevent expression of the
/
-TCR complex. Thymocytes
from pLGF/C3 double transgenic mice should then express a mature
/
-TCR complex, undergo positive selection,
and generate mature SP thymocytes in the normal ratio.
Conversely, if Rho function was not required for p56lck-mediated regulation of
/
-TCR expression then pLGF/
C3 thymocytes should show a pattern of TCR expression and CD4/CD8 differentiation like that seen in pLGF thymocytes that express active p56lck alone. Therefore, we
analyzed expression of the TCR-CD3 complex and the
development of CD4 and CD8 mature SP cells in thymocytes from pLGF/C3 double transgenic mice. As shown
in the right panel of Fig. 3 a, pLGF/C3 thymocytes that express active p56lck on a Rho
background fail to normally express a mature TCR-CD3 antigen receptor complex.
The absence of a mature TCR-CD3 complex interrupts
the normal processes of positive and negative selection and
inhibits CD4 and CD8 SP development in the thymus of
pLGF mice. As shown in the upper right panel of Fig. 3 b,
thymocytes expressing active p56lck comprise CD4CD8
DN and CD4+CD8+DP cells but abnormally low levels of
CD4+ or CD8+ SP cells. We have shown previously that
positive and negative selection processes of DP thymocytes
occur undisturbed in the absence of Rho function (12).
Adult thymi which lack endogenous Rho function show
severe hypocellularity of only ~5-10% of normal cell
numbers but do contain DP and mature SP thymocytes
with normal expression levels of the TCR-CD3 complex
(Fig. 3 b, lower left). The lower right panel in Fig. 3 b shows
the result of a representative analysis of CD4/CD8 subpopulations in pLGF/C3 thymocytes. The frequencies of DP
and mature SP cells of thymocytes from pLGF/C3 double
transgenic mice are identical to that of thymocytes from pLGF mice. The data in Fig. 3 thus show that introduction
of an activated p56lck transgene into thymocytes lacking
endogenous Rho function prevents the expression of the
/
-TCR complex and consequently, because positive selection can not occur, the generation of mature CD4 and
CD8 SP thymocytes. In this context, the outcome of expression of activated p56lck in terms of inhibition of
/
-TCR complexes and suppression of the differentiation of
CD4 and CD8 SP cells is the same in Rho
and normal
thymocytes.
Immature CD8 single positive cells (ISP), distinguished by
low levels of CD8 antigen expression but no expression of the
TCR-CD3 complex, represent an intermediate stage in the
transition from the CD4CD8
DN to the CD4+CD8+
DP compartment (21, 22). These cells are not easy to discern in normal thymocytes but can accumulate in mice
where there is a partial block or a delay in the DN to DP
transition. For example, an accumulation of CD8lowCD3
ISPs was seen in mice lacking expression of the transcription factors Tcf-1 and Lef-1 (23). A consistent and reproducible observation was that thymocytes from pLGF/C3
double transgenic mice contained higher frequencies of
CD8 low ISPs when compared with pLGF single transgenic
mice (Fig. 3 c).
In normal thymocyte development pre-TCR-mediated downregulation of CD25 expression is accompanied by upregulation of the expression of the CD2 antigen (24, 25). The data in Fig. 4 a show CD2 expression of thymocytes isolated from a normal young C57/BL6 mouse. CD2 expression is found on CD4+CD8+ DP and SP thymocyte populations. There is a subtle but clear regulation of CD2 expression levels during normal thymocyte development in that SP thymocytes express about threefold higher levels of the CD2 antigen than DP thymocytes.
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Fig. 4 b shows a comparison of CD2 antigen expression on thymocytes from normal mice, pLGF transgenic and pLGF/C3 double transgenic mice. The representative profiles depicted here show that expression of active p56lck results in an about fourfold reduction in CD2 antigen expression levels compared with normal control thymocytes. Expression profiles of other T cell markers such as Thy-1.2 are normal in pLGF thymocytes (Fig. 4 b, lower panel). Similarly, levels of CD4 and CD8 on DP thymocytes from pLGF thymocytes are comparable to levels seen in normal control mice (Fig. 3 b). Expression of active p56lck thus selectively downregulates expression of CD2 antigen. In contrast, loss of Rho function during thymocyte development does not alter CD2 expression levels in adult thymocytes (13). To explore whether the downregulation of CD2 expression in pLGF thymocytes was a Rho dependent phenomenon we analyzed CD2 antigen levels on thymocytes from LGF/C3 double transgenic mice (Fig. 4 b, upper right panel). These experiments reveal normal levels of CD2 antigens on pLGF/C3 thymocytes. Thus, transgenic expression of active p56lck in the thymus interferes with the generation of normal levels of CD2. However, induced reduction of CD2 antigens occurs only in normal thymocytes but not in thymocytes lacking endogenous Rho function, p56lck downregulation of CD2 is thus a Rho-dependent response.
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Discussion |
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The differentiation and proliferative expansion of pre-T
cells is mediated via signaling pathways which involve activation of the tyrosine kinase p56lck (10, 11). However, nothing is known about other signaling molecules that operate
with p56lck to mediate the complex molecular changes that
occur at this stage of thymocyte development. To examine
the role of the GTPase Rho in p56lck-mediated signals in
the thymus, we generated mice double transgenic for active
p56lck and the Rho inhibitor C3 transferase. This enabled us
to compare thymocyte development in mice expressing active p56lck on a wild-type or Rho background.
Mice with thymocyte-specific expression of a transgene
encoding active p56lck show strong potentiation of proliferative responses in pre-T cells that results in a fivefold increase in numbers of CD44CD25
late pre-T cells. This
p56lck-mediated response is abrogated when the active
p56lck transgene is expressed in thymocytes which lack endogenous Rho function. Rho function is thus required for p56lck to drive the differentiation of pre-T cells from the
CD44
CD25+ into the CD44
CD25
compartment and
to accelerate proliferative responses in this population. It
has been shown that expression of active p56lck inhibits
TCR-
chain rearrangements; DP thymocytes that develop in pLGF thymocytes fail to normally express
/
-TCR complexes (19). We now show that active p56lck
can prevent expression of
/
-TCR complexes on DP
thymocytes in the absence of endogenous Rho function.
Interestingly, active p56lck also generates DP thymocytes that express downregulated levels of CD2 antigens when
compared with normal thymocytes. However, our data
show that p56lck does not prevent expression of CD2
when expressed on a Rho
background. p56lck thus regulates thymocyte development at the pre-T cell stage by
both Rho-dependent and Rho-independent responses.
It has been suggested that one critical role for p56lck at
the pre-T cell stage of thymocyte development is to mediate allelic exclusion at the TCR- chain locus (19, 26, 27).
Expression of a constitutive active form of p56lck can initiate thymocyte differentiation in RAG
/
or pT
/
mice (10, 11) and switch off rearrangements at the TCR-
locus. Further evidence for the role of p56lck in preventing
rearrangement at the TCR-
locus stems from observations that DP thymocytes that develop in the presence of
active p56lck fail to express TCR-
subunits. They do not
show rearrangements at the TCR-
locus, do not express a
functional
/
-TCR complex and fail to develop into mature CD4+ or CD8+ SP thymocytes (19, 26).
The current model to explain this phenomenon is that
p56lck acts as a sensor for the expression of a functional
pre-TCR complex. Successful expression of a functional
pre-TCR complex would normally activate p56lck and
initiate the feedback mechanism that prevents further rearrangements of TCR- chains, i.e., p56lck mediates allelic
exclusion at the pre-T cell stage. The failure of TCR-
chain rearrangements in cells expressing active p56lck is
thus thought to reflect the fact that such cells initiate the allelic exclusion mechanism before they have produced a
competent TCR-
subunit. Once such cells differentiate
into DP thymocytes there is no TCR-
chain to partner
newly synthesized TCR-
subunits and consequently expression of a mature TCR-CD3 antigen receptor complex
does not occur. This results in failed positive and negative selection and impaired development of mature SP thymocytes. The present data show that loss of Rho function
has no influence on the ability of active p56lck to prevent
expression of
/
-TCR complexes. Since the failure to express
/
-TCR complexes in thymocytes expressing active
p56lck is a model for allelic exclusion, the simplest interpretation of the present data is that Rho function is not required for allelic exclusion at the TCR-
locus.
In this study, we demonstrate that expression of active
p56lck in thymocytes not only prevents expression of mature /
-TCR complexes but also regulates expression of the
accessory molecule CD2. The downregulation of CD25
expression on pre-T cells is normally accompanied by upregulation of the expression of the CD2 antigen (24, 25).
Active p56lck can push the differentiation processes that
downregulates expression of CD25, but the resultant cells do not express normal levels of CD2. The negative regulatory effect of active p56lck on CD2 expression can be reversed by a concomitant loss of Rho function. This was a
particularly striking result because loss of Rho function did
not restore expression of the
/
-TCR complex on pLGF
thymocytes. The ability of active p56lck to prevent CD2
antigen expression on DP thymocytes is thus dependent on
Rho function whereas the ability of p56lck to inhibit expression of the
/
-TCR complex is not.
In pLGF/C3 double transgenic mice we observed a consistent increase in frequencies of CD4CD8low cells when
compared with pLGF single transgenic mice. These ISP represent an intermediate stage in the transition from the
DN to the DP compartment (21, 22). This result suggests
that the transition of late pre-T cells into DP cells is partially suppressed in thymocytes lacking endogenous Rho
function. However, loss of Rho function does not completely suppress pre-T cell differentiation. Moreover, although it strikingly decreases proliferative responses, it does
not completely abrogate proliferation in late pre-T cells. The present results thus demonstrate that Rho is a regulator
of pre-T cell development but they equally illustrate that
compensatory, Rho-independent proliferative and differentiation signals must operate in early thymic progenitors.
There is increasing awareness that pre-T cell differentiation
is controlled by overlapping and compensatory signaling
pathways. For example, the tyrosine kinases ZAP70 and
Syk have overlapping and compensatory roles in pre-T cells (7) as do the transcription factors Tcf and Lef (23). The present data show that Rho has an important function
in p56lck-mediated signals for thymocyte development.
The fact that pre-T cell differentiation is not absolutely dependent on Rho function is consistent with the fact that
the thymic developmental block in mice deficient for
p56lck is not absolute. In p56lck-deficient mice, the related kinase p59fyn can partially compensate for p56lck-mediated signals. Thymocytes that are deficient for both p56lck
and p59fyn show a complete block in thymocyte development at the pre-T cell stage analogous to the developmental block seen in RAG
/
thymocytes (28). Therefore, one
speculation is that loss of Rho function causes defects in
p56lck-mediated function in pre-T cells but does not impact compensatory p59fyn signaling pathways. In this respect, the strategy used in this study to inactivate Rho
function is extremely selective. C3 transferase has no inhibitory effects on the function of other Rho family GTPases
such as Rac-1, Rac-2, or Cdc42 (29). It will be intriguing
to know if loss of Rho function in the thymus can be compensated by the actions of these other Rho family GTPases.
In summary, the tyrosine kinase p56lck regulates pre-T
cell proliferation and differentiation and controls expression
of /
-TCR complexes and CD2 antigens. This study
shows that these p56lck-mediated responses can be distinguished by their requirement for Rho function. This requirement of Rho function for p56lck-mediated regulation of pre-T cell development is consistent with a model
where Rho is part of signaling pathways that emerge from
p56lck and regulate cell cycle progression and CD2 expression at this developmental stage. The signaling pathways
emerging from p56lck that prevent expression of
/
-TCR complexes by suppressing TCR gene rearrangement at the
-locus are apparently independent of the function
of Rho. One interpretation of these results is that qualitatively different signaling pathways, some involving Rho
and some not, are being used to control these different
p56lck responses (Fig. 5 a). However, an alternative possibility is that Rho is regulating p56lck-signaling responses
quantitatively. In this model, the same p56lck-induced signaling pathways controls thymocyte proliferation,
/
-TCR and CD2 expression but these responses require
quantitatively different signaling thresholds (Fig. 5 b). Rho
could thus regulate the strength of p56lck-induced signals.
This model predicts that regulation of TCR-
chain rearrangements has a low signaling threshold compared with
the thresholds required for proliferation and control of CD2 antigen expression. Future studies will determine
whether Rho is positioned directly downstream of p56lck
at the pre-T cell stage or whether Rho is part of a parallel
signaling pathway required for pre-T cell development.
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Footnotes |
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Address correspondence to Doreen A. Cantrell, Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK. Phone: 44-171-269-3307; Fax: 44-171-269-3479; E-mail: cantrell{at}icrf.icnet.uk
Received for publication 13 May 1998 and in revised form 16 June 1998.
We are indebted to Ian Rosewell from the Imperial Cancer Research Fund Transgenic Unit for microinjection of the C3 transferase transgenic construct. The p56lckF505-expressing pLGF-2964 mice were kindly provided by Dr. Roger M. Perlmutter. We also wish to thank Tracy Crafton, Gary Martin and Barbara Turner at the transgenic mouse breeding unit for excellent assistance in animal care and Derek Davies for helpful advice on flow cytometry. Drs. L. Bruno and M.J. Owen are acknowledged for helpful discussion and critical reading of the manuscript.
This work was supported by the Imperial Cancer Research Fund and the European Human Capital and Mobility Program (CHRX-CT94-0537). S.W. Henning was a fellow of the European Training and Mobility of Researchers Program (ERBFMBICT 960518).
Abbreviations used in this paper DN, double negative; DP, double positive; ISP, immature CD8 single positive cells; SP, single positive; TN, triple negative.
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References |
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