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Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
phosphorylation and down-regulation of CD5, both of which are also MHC
contact dependent, indicating that the Src family kinases are critical
for transducing a TCR-MHC survival signal. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The question of whether MHC-TCR interactions are required for prolonged T cell survival is one that has been addressed by a number of studies in recent years. While many of these evoke a role for MHC-TCR interactions in T cell maintenance (1, 5, 6, 7, 8, 9), a significant number of others have suggested that survival is not linked to self-MHC recognition (10, 11, 12). It has been argued that since MHC ligands also induce T cell expansion under conditions of lymphopenia, in the absence of MHC it is difficult to differentiate between a defect in expansion vs one in survival. Conversely, studies that failed to find a requirement for MHC ligands in promoting T cell survival have involved T cell transfers to MHC-deficient environments under conditions where it is difficult to absolutely exclude the possibility of cotransferring MHC-expressing APCs or their precursors. The situation is further confused in that cytokines also play an essential role in promoting the survival of naive T cells (13, 14, 15), and their impact on TCR-mediated survival signals has, in general, not been addressed in these studies.
Previous studies in our laboratory have attempted to characterize the signals involved in peripheral T cell homeostasis by directly manipulating the intracellular signaling molecules important in transducing TCR signals. Using mice bearing a tetracycline-inducible transgene for the Src family kinase, p56lck (Lck1ind), we showed that expression of Lck was essential for transducing the TCR signals that mediate homeostatic proliferation, but surprisingly Lck-mediated signals were not required for long term survival of naive T cells (16). Given that Lck plays such a central role in initiating and propagating TCR signals (17, 18), the dissociation of T cell survival from Lck expression might argue against the existence of a TCR-mediated survival signal. However, p59fyn is another Src family kinase expressed in T cells that has been shown to be able to substitute for some of the functions of Lck (19). In the present study we generated mice with the inducible lck transgene that lacked both endogenous Lck and Fyn and show that Src family kinases are crucial for the prolonged survival of naive T cells and, therefore, that survival is indeed TCR signal dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of inducible Lck-transgenic Lck1ind mice was described previously (20). Briefly, mice expressing the rtTA trans-activator domain under control of the human CD2 promotor on an endogenous Lckneg background (rtTA-C/Lckneg) were intercrossed with mice bearing the mouse lck transgene under control of a tetO/CMVmin promotor, also on an endogenous Lckneg background (Lck1/Lckneg). Mice were fed doxycycline (dox)3 in food (1 mg/g) throughout pregnancy and before weaning. After weaning, Lck1hetrtTA-ChetLckneg (Lck1ind) offspring were fed dox-containing food to maintain lck transgene expression. Lck1indFynneg mice were similarly derived by intercrossing rtTA-C LcknegFynneg and Lck1 LcknegFynneg strains. C57BL/10 wild-type, Lckneg, Fynneg, and LcknegFynneg mice were bred in a conventional colony free of pathogens at National Institute for Medical Research (London, U.K.). All lines used were the H-2b haplotype.
Flow cytometry and cellularity determinations
Flow cytometry was conducted using 2–5 x
106 thymocytes, lymph node cells, or spleen
cells. Lymph node cell suspensions were prepared from superficial
cervical, brachial, axilary, inguinal, and mesenteric lymph nodes of
individual mice. Cell concentrations were determined using a Casy
Counter (Scharf Instruments, Reutlingen, Germany). Cells were
incubated with saturating concentrations of Abs in 100 µl PBS/BSA
(0.1%)/azide (1 mM) for 1 h at 4°C, followed by three washes in
PBS/BSA/azide. Biotin-conjugated Ab labeling was developed with
streptavidin-PE-Cy7 (Caltag, San Francisco, CA). The mAb used in this
study were as follows: PE-CD4 (GK1.5; BD PharMingen, San Diego, CA),
PerCP-CD8
(53-6.7; BD PharMingen), allophycocyanin-CD8 (53-6.7;
BD PharMingen), FITC-TCR (H57-597; BD PharMingen), allophycocyanin-CD44
(Leinco Technologies), and IL-7R (A7R34) and FITC-CD5 (BD PharMingen).
Four-color cytometry staining was analyzed on a FACSCalibur (BD
Biosciences, Mountain View, CA), and data analysis was performed with
CellQuest software (BD Biosciences). Statistical significance of
reductions in cell recoveries compared with controls was tested using
one-tailed Student’s t test (unpaired).
Western blot analysis
Cell suspensions from thymus, lymph node, and spleen were
recovered by centrifugation and resuspended in ice-cold lysis buffer
(50 mM Tris-HCl (pH 8.0); 150 mM NaCl; 1% Triton X-100; 5 µg/ml each
of chymostatin, leupeptin, pepstatin A, and antipain (Sigma-Aldrich,
St. Louis, MO); 1 mg/ml iodoacetamide (Sigma-Aldrich); and 0.2 mg/ml
Pefabloc (Roche, Indianapolis, IN)) for 30 min, and postnuclear
supernatants were obtained by centrifugation. Immunoprecipitation,
SDS-PAGE, and Western blotting were conducted as reported previously
(21). Total cell lysates for Lck immunoblotting were
loaded at 2 x 106 T cell
equivalents/lane, while anti-CD3 immunoprecipitates were from
the equivalent of 5 x 106 T cells using
H146-968 mAb coupled to protein A-Sepharose beads (Sigma-Aldrich).
Lysates and immunoprecipitates were separated on 12.5% SDS-PAGE under
reducing conditions, blotted onto Immobilon-P membranes
(Millipore, Bedford, MA), and probed using anti-phosphotyrosine
RC20-HRP (Transduction Laboratories, Lexington, KY), rabbit
anti-Lck sera, rabbit anti-Fyn (Santa Cruz Biotechnology, Santa
Cruz, CA), anti-CD3 (H146-968), and rabbit anti-actin sera
(Sigma-Aldrich). Unconjugated Igs were detected using protein A-HRP
(Amersham, Arlington Heights, IL). Membranes were washed using
PBS/Tween (0.5%) between sequential probing steps. Rabbit anti-Lck
serum and the H146-968 anti-CD3-secreting hybridoma were gifts
from A. Magee and R. Kubo, respectively.
Treatment of mice with anti-IL-7R mAb
A7R34, a gift from Prof. S. Nishikawa (Department of Molecular Genetics, Kyoto University, Kyoto, Japan), was purified from hybridoma tissue culture supernatants with Mono-S Sepharose (Pharmacia, Piscataway, NJ) and dialyzed into PBS. Treatment of mice with A7R34 in PBS was performed on a 7-day cycle of i.p. injections of 300 µg/mouse of mAb on days 1, 3, and 5.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the requirement of both Fyn and Lck for naive T cell survival, mice with an inducible lck transgene (20) were back-crossed to a Fynneg background. These mice have an lck transgene under a tetracycline-sensitive promotor (Lck1) and a second tet-on trans-activator domain (rtTA-C) transgene under the control of human CD2 regulatory elements, targeting expression to T cells. Lck-inducible (Lck1ind) mice lack endogenous Lck and express the lck transgene in response to administration of the tetracycline-derivative dox.
Thymic development appears normal in Fynneg mice
(22, 23). To confirm this was also true of
Lck1indFynneg mice, thymic
phenotypes were analyzed from mice fed since gestation with dox. As
described previously, we were able to overcome the block in
thymic development found in Lckneg mice to
near-normal levels by expression of an inducible lck
transgene (20) (Fig. 1
A).
LcknegFynneg mice exhibit a
complete block in thymocyte development and have virtually no
double-positive or mature single positive thymocytes (19, 24) (Fig. 1, A and B). Introduction of the
lck1ind transgene was able to restore
thymic differentiation in
LcknegFynneg mice to levels
comparable with those observed on
LcknegFyn+ backgrounds.
Consistent with earlier studies, we found that the loss of Fyn had
little impact on either the frequency or number of thymocytes,
providing Lck is present in the wild-type (WT) or inducible form (Fig. 1, A and B). Furthermore, expression of TCR by
single-positive and double-positive thymocytes from WT or
Lck1indFyn+ mice was
comparable to that expressed by the corresponding
Fynneg strain mice (Fig. 1C).
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Analysis of thymi from
Lck1indFynneg mice
suggested that thymic development and production of mature
single-positive T cells occur comparably to those in
Lck1indFyn+ controls, so we
examined their peripheral naive T cell numbers. In the presence of
endogenous Lck, the frequencies of TCRhigh cells
and CD4+ and CD8+ subsets
found in lymph node from Fynneg mice were similar
to those in WT mice (Fig. 2A).
Interestingly, this was not found to be the case when lymph node cells
from Lck1indFynneg mice
were compared with those from
Lck1indFyn+ controls. The
overall frequency of TCRhigh lymph node cells was
consistently lower in
Lck1indFynneg mice than in
Lck1indFyn+ controls (Fig. 2A, histograms), although the ratios of
CD4+ to CD8+ cells were
similar (Fig. 2A, dot plots). Analysis of the absolute
numbers of naive phenotype
CD4+CD44low and
CD8+CD44low cells in
defined lymph nodes and spleens confirmed this observation. The numbers
of naive T cells in lymph nodes and spleen from
Fynneg mice were similar to those in WT mice
(Fig. 2B). However, there was a reproducible and
statistically significant reduction (
2.5-fold) in the numbers of
naive CD4+CD44low and
CD8+CD44low cells found in
lymph node and spleen of
Lck1indFynneg mice compared
with Lck1indFyn+ controls
(Fig. 2B). Examination of the frequency of
CD44high T cells in lymph node and spleen
revealed no significant difference between FynWT
strain mice and their Fynneg counterparts (Fig. 2C).
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Naive peripheral T cells in Fynneg mice are
found in comparable numbers to those in WT mice, so it was surprising
to find such a significant reduction in the numbers of naive T cells in
the periphery of
Lck1indFynneg mice compared
with Lck1indFyn+ controls.
We have previously reported that there are differences in
expression of the induced lck1 transgene between thymocytes
and peripheral cells (16). Therefore, we compared
expression of lck and fyn in the thymus and
periphery of Lck1ind and WT mice on
Fyn+ and Fynneg
backgrounds; lck expression in the thymus of
Lck1indFyn+ mice was
comparable to that seen in WT and Fynneg mice
(Fig. 3). However, while
LckWT strains express equivalent levels of Lck
protein in thymus and periphery, expression of the lck1
transgene in the periphery of both
Lck1indFyn+ and
Lck1indFynneg mice was
considerably lower. No change was observed in the level of Fyn
expressed in the periphery of WT and
Lck1indFyn+ T cells (Fig. 3), and consistent with other studies, Fyn expression was much lower in
thymus than in the periphery (25).
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20% of WT levels of lck
in the periphery; this, it seems, is insufficient to maintain survival
of peripheral T cells. Naive T cells do not survive the absence of both lck and fyn
To test whether there is a requirement for Src family kinases for the prolonged survival of peripheral T cells, lck1 transgene expression was switched off. Dox was removed from the diets of cohorts of Lck1indFynneg mice (Lck1OFFFynneg) and Lck1ind mice (Lck1OFFFyn+) to switch off lck expression, while groups of control littermate Lck1indFynneg mice (Lck1ONFynneg) and Lckind mice (LckONFyn+) were maintained on dox food. The numbers of T cells in lymph nodes and spleen were determined in 6- to 8-wk-old mice at the start of the experiment, and then in additional groups of mice 4, 8, and 12 wk later.
As we have shown previously, absolute numbers of naive
CD4+CD44low and
CD8+CD44low T cells did not
decline notably over the 12-wk period following removal of dox and
switching off the lck transgene in the presence of WT Fyn
(Fig. 4A). Both
CD4+ and CD8+
cells from Lck1OFFFyn+ mice
had half-lives in excess of 90 days in these experiments. Similarly, in
dox-fed Lck1ONFynneg mice, the
number of CD8+CD44low cells
was maintained at constant levels, while
CD4+CD44low T cell numbers
showed a slight increase over time (Fig. 4A). Interestingly,
there was a marked difference in the behavior of naive T cells from
Lck1OFFFynneg mice that
lack both lck and fyn expression. After removal
of dox, CD4+CD44low and
CD8+CD44low naive T cells
disappeared with half-lives of 26 and 18 days, respectively,
indicating that the presence of a Src family kinase member is essential
for long term survival. It is important to note that this experimental
model allows us to monitor the survival of a cohort of peripheral T
cells in the absence of either additional thymic export or homeostatic
expansion. We have shown previously (16) that cessation of
dox feeding and loss of lck induce rapid thymic atrophy,
resulting in a functionally thymectomized animal. In addition, we
showed that TCR-mediated peripheral homeostatic proliferative responses
are completely dependent on the expression of lck. Thus,
removal of dox from the diet of these animals prohibits either newly
exported naive T cells or expansion of existing T cells from
influencing our analysis of the survival of the naive T cell
pool.
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B). In the absence of both Lck and Fyn,
Lck1OFFFynneg mice, there
was an initial drop in the numbers of both
CD4+CD44high and
CD8+CD44high cells during
the first 4 wk, but subsequently numbers remained constant (Fig. 4B) over the same time period in which naive T cells
continued to disappear.
Survival of naive T cells correlates with CD3-chain
phosphorylation
The disappearance of T cells from Lck1OFF
Fynneg mice suggests that survival of these cells
was dependent on signals through a Src family kinase member.
Unstimulated T cells have partially phosphorylated CD3 chains ex
vivo (29, 30), and it has been suggested that this is a
consequence of survival signals delivered through engagement of the TCR
with MHC. We examined the phosphorylation state of CD3 chains in T
cells from Lck1OFFFynneg
and control mice. Cell lysates were prepared from T cells
of WT,Fynneg,
Lck1ONFyn+,
Lck1OFFFyn+,
Lck1ONFynneg, and
Lck1OFFFynneg mice, and the
presence of phosphorylated (Phos-) was determined by Western
blot analysis of immunoblots. Analysis of total cell lysates from
the same mice for the presence of Lck protein confirmed its loss of
expression in Lck1OFF mice. Fig. 5A shows data from one
representative experiment, in which Phos- was visible in cells
from both Lck1ONFyn+ and
Lck1OFFFyn+ mice. In
contrast, while Phos- could be detected readily in T cells from
Lck1ONFynneg mice, there
was a complete absence of Phos- in T cells from
Lck1OFFFynneg mice.
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-chains that were phosphorylated in
Lck1ind strain mice appeared to be less than that
in either WT or Fynneg mice. Quantitation by
densitometry confirmed this observation, as shown in Fig. 5B, which contains data pooled from three independent
experiments. T cells from WT and Fynneg mice had
similar levels of Phos-, while those of T cells from
Lck1ONFyn+,
Lck1OFFFyn+, and
Lck1ONFynneg mice were all
approximately half the WT levels. As previously reported, switching off
lck in the presence of WT fyn did not reduce the
detectable Phos- compared with that in mice maintained on dox.
However in the absence of Fyn, there was total loss of
phosphorylation within 1 wk of cessation of dox feeding (Fig. 5B). Fyn and Lck regulate CD5 expression in peripheral naive T cells
Recent studies have shown that levels of CD5 expressed by
peripheral T cells are modulated by TCR interactions with MHC in a
manner that reflects the avidity of a specific TCR for self MHC-peptide
complexes, and that, certainly for CD4+ cells,
this is in part dependent on Lck expression (31). To
determine whether this is also true of CD8+ cells
and whether Fyn can influence the adaptation of CD5, we examined T
cells from Lck1indFyn+ and
Lck1indFynneg mice for
their expression of CD5. Fig. 6 shows
representative histograms of TCR and CD5 expression by
CD4+CD44low (Fig. 6A) and
CD8+CD44low (Fig. 6B) cells from WT,
Lck1OFFFyn+, and
Lck1OFFFynneg mice. Levels
of TCR expression were constant between T cells from the different
mouse strains (Fig. 6, A and B, left
histogram). However, both Lck and Fyn were found to influence the
levels of CD5 expressed in CD4+ and
CD8+ cell subsets, since CD5 levels were reduced
in Lck-deficient cells compared with those in WT cells and were lowest
in T cells lacking both Lck and Fyn. Fig. 6C summarizes CD5
expression, expressed as a percentage of WT levels, by
CD4+CD44low and
CD8+CD44low cells from
Fynneg,
Lck1ONFyn+,
Lck1OFFFyn+, and
Lck1OFFFynneg mice.
Interestingly, while Lck and Fyn mediated the adaptation of CD5 levels
in both CD4+ and CD8+
cells, the influence of these two kinases was not the same for both
subsets. CD5 expression on CD4+ cells was more
sensitive to the presence of Lck, since its reduction in
Lck1indFyn+ ON mice, which
express low levels of Lck, and in
Lck1indFyn+ OFF mice which
have no Lck, was much more marked in the CD4+ vs
the CD8+ subset (Fig. 6C). In
contrast, CD5 expression on CD8+ cells was more
sensitive to the presence of Fyn, shown firstly by the reduced
expression of CD5 on CD8+ cells compared with
CD4+ cells in Fynneg
(LckWT) mice. Secondly, in the absence of Lck (or
in the presence of reduced levels of Lck, in
Lck1ON mice; data not shown) there was a 35%
drop in CD5 mean fluorescence intensity when Fyn was also removed from
CD8+ cells compared with only a 20% drop in
CD4+ cells (Fig. 6C). These data
demonstrate that Fyn contributes to the sensory adaptation of CD5 on
peripheral T cells and reinforce the view that naive T cells are
continually making contact with MHC-peptide complexes, as deprivation
of this contact has been demonstrated to reduce CD5 levels (31, 32).
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As the data in Fig. 4 show, failure of naive T cells to express
both Lck and Fyn resulted in their disappearance. However, although the
TCR-mediated survival signal, as visualized by CD3-chain
phosphorylation and CD5 expression, was lost within 1 wk of switching
off Lck expression (Fig. 5), disappearance of T cells was considerably
slower. Studies of T cell survival in the absence of either MHC or TCR
expression report a similarly slow rate of T cell decay (9, 32, 33). In contrast, T cells in vitro die rapidly without
stimulation, suggesting that factors other than TCR signals contribute
to T cell survival. The importance of cytokines whose receptors use the
common 
-chain in the survival of naive T cells has been well
documented (13), in particular IL-7 (14, 15, 34). To determine whether the slow decline in naive T cell
numbers in the absence of continued TCR signals was because cells were
still receiving survival signals from IL-7, the fate of T cells were
followed after treatment of mice with an IL-7R-specific mAb
(IL-7R), that blocks the biological activity of IL-7.
At time zero the numbers of naive T cells in the lymph nodes of groups
of 6- to 8-wk-old
Lck1indFyn+ and
Lck1indFynneg mice were
determined. The remaining littermates were taken off dox and treated
with saturating levels of IL-7R Ab or PBS as a control. As shown
previously, the loss of Lck
(Lck1OFFFyn+ mice) had
little effect on the survival of CD44low naive
cells (Fig. 7). The removal of either the
IL-7R-mediated survival signals by treatment of
Lck1OFFFyn+ mice with
IL-7R Ab or TCR-mediated survival signals by the absence of both Lck
and Fyn (Lck1OFFFynneg)
resulted in similar reductions of naive T cell survival over the 28
days. However, the combined loss of both these survival signals
(Lck1OFFFynneg mice treated
with IL-7R) precipitated a severe decline in naive T cell numbers.
Interestingly, CD8+ cells seemed to be even more
sensitive to the loss of these survival signals than
CD4+ cells. Loss of naive T cells in
IL-7R-treated mice was not a cytolytic effect of the coating mAb,
since the numbers of memory CD8+ cells, whose
survival is not dependent on IL-7 (34), were unaffected in
treated hosts compared with controls even though they express higher
levels of IL-7R than naive T cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The essential role of Src family kinases in the prolonged survival of
naive T cells was apparent by examining
Lck1indFynneg mice even
when expression of the lck1 transgene was maintained.
Lck1indFynneg mice have
reduced numbers of both naive CD4+ and
CD8+ cells compared with
Lck1ind controls (Fig. 2), even though thymic
development and mature single-positive cell production are normal in
both strains (Fig. 1). The defect in peripheral T cell survival was not
seen in Lck1indFyn+ mice
expressing WT Fyn, which was more abundant in peripheral T cells than
Lck (25). Similarly, no defect in survival was seen in
Fynneg mice expressing WT Lck, probably because
WT levels of Lck are 5 times greater than those found in resting
peripheral T cells from mice expressing the induced lck1
transgene. However, in
Lck1indFynneg mice, T cells
can rely only on the low level of lck1 transgene expression
for their continued survival, and this appears to be insufficient.
The implication that Src family kinases are required for naive T cell
survival was confirmed when Lck1 expression was switched off in
Lck1indFynneg mice (Fig. 4). T cell numbers declined with kinetics similar to those described
previously following ablation of either MHC expression (9)
or expression of the TCR itself (33). However, both Lck
and Fyn have been implicated in the signaling of other cell surface
molecules as diverse as CD28 (35), CD44 (36),
L-selectin (37), and cytokine receptors (38, 39). In principle, therefore, the requirement of Lck and Fyn for
T cell survival may not be specifically for transducing TCR signals.
While involvement of other surface receptors is a possibility that
cannot be excluded, we could show that phosphorylation of CD3 in ex
vivo unstimulated T cells was entirely Src kinase dependent (Fig. 5),
and its absence correlated with a failure in prolonged T cell survival.
Furthermore, there is good evidence that phosphorylation of TCR is
dependent on interaction of TCR with MHC molecules both in vitro
(31) and in vivo (9, 11).
Quantitative analysis revealed that the extent of TCR-chain
phosphorylation broadly correlates with expression levels of Lck and
Fyn. TCR-chain phosphorylations in WT and
Fynneg mice are similar, suggesting that Lck at
WT levels is sufficient to maintain normal phosphorylation. When the
levels of Lck are greatly reduced
(Lck1ONFyn+ mice) or when
Lck is absent (Lck1OFFFyn+
mice), the extent of TCR phosphorylation is 50% that in WT mice.
This phosphorylation must be mediated by Fyn, as all Phos-
disappears when Fyn is also absent. It will be interesting to determine
whether Fyn and Lck preferentially phosphorylate different
immunoreceptor tyrosine-based activation motifs on , because
while those phosphorylated by Fyn are entirely sufficient to promote T
cell survival, they are not sufficient to transduce the TCR signals
that drive homeostatic proliferation of naive T cells under conditions
of lymphopenia (16). Furthermore, in another study we
could find no evidence that Fyn played any role in mediating either TCR
or IL-7R signals that induce homeostatic proliferation by T cells under
conditions of lymphopenia (42) strongly suggesting that
promotion of cell survival and induction of homeostatic proliferation
involve discrete signaling pathways.
Further evidence suggesting that Src kinase-dependent survival signals
were TCR dependent came from analysis of CD5 expression. Previous
studies have shown that the maintenance of CD5 expression was MHC
dependent, was correlated with the degree of TCR-chain
phosphorylation, and was influenced by the expression of Lck, all of
which evoke an MHC-dependent TCR signaling mechanism (31).
Here we show that adaptation of CD5 expression is regulated by Fyn as
well as Lck (Fig. 6). Interestingly, we found evidence that these two
Src family kinases have different influences for
CD4+ and CD8+ subsets. This
may reflect more general preferences of CD4+ and
CD8+ subsets for the use of either Lck or Fyn for
TCR signaling, and it is interesting to note that in
Lckneg mice, the presence of Fyn seems to favor
the selection of CD8+ cells over
CD4+ cells (40). In addition, CD5
expression was found to correlate well with cell survival. T cells from
Lck1indFynneg and
Lck1OFFFynneg mice that
exhibited defects in naive T cell survival also had the lowest levels
of CD5.
The role of IL-7 as a survival factor for naive T cells is well
documented (13, 14, 15, 34). However, the interaction between
IL-7 and TCR survival signals has not previously been addressed. In the
present study we found that blocking one or another of these signals
compromised T cell survival to a broadly similar extent. Significantly,
when both IL-7R and TCR signals were blocked in IL-7R-treated
Lck1OFFFynneg mice, nearly
all naive T cells were lost within 2 wk (Fig. 7). Therefore, it appears
that TCR and IL-7R signals have a limited capacity to sustain naive T
cells independently of one another, and it is not until both signals
are blocked that a rapid attrition of T cells is observed. While in a
"full" host, these signals are individually insufficient to sustain
survival for any longer than a few weeks, combined they can maintain
naive T cells in the periphery for many months. A similar synergy
between TCR and IL-7R signals occurs when homeostatic proliferation is
induced in T cells under conditions of lymphopenia (42).
In this way the size of the T cell pool is regulated by both stromal
cell IL-7 production and access to MHC expressing APCs, and is
ultimately limited by whichever of the two is lesser.
In the light of these findings, therefore, the differences in T cell half-lives previously reported (9, 32, 33) in the absence of TCR or its ligand are more likely a reflection of the host cytokine milieu than a function of TCR signaling itself. It is clear that even in a full host, IL-7 can prolong T cell survival in the absence of TCR signaling for a limited period. IL-7 can induce homeostatic proliferation of naive T cells independently of TCR signals, but depends on the extent of lymphopenia within a host (42). Therefore, under lymphopenic conditions, greater availability of IL-7 may increase its capacity to maintain T cell survival in the absence of TCR signals than is observed in full hosts, and this may explain reports of naive T cell survival upon adoptive transfer to MHC-deficient hosts (10, 11). Finally, we found that CD8+ cells were more reliant on both TCR and IL-7R signals than were CD4+ cells, exhibiting a more rapid loss in the absence of either or both signals. This greater reliance may reflect a greater sensitivity to such signals, and it is interesting to note that CD8+ cells survive better than CD4+ cells in an intact host (41).
In conclusion, it is clear from the present study that Src kinase
expression is essential for prolonged survival of naive T cells. We
cannot exclude the possibility that Lck and Fyn mediate survival
signals via surface receptors other than the TCR; however, evidence in
favor of the latter is very strong. CD3 chain phosphorylation, which
has been shown to be MHC dependent (9, 11), is absolutely
Src kinase dependent; sensory adaptation of CD5 expression, also
dependent on TCR-MHC interactions (31), correlates with
both Src kinase expression and T cell survival. Therefore, the most
direct interpretation of these data is that a TCR survival signal for
naive T cells is delivered by MHC and is mediated by Src family
kinases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rose Zamoyska, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K. NW7 1AA. E-mail address: rzamoys{at}nimr.mrc.ac.uk
3 Abbreviations used in this paper: dox, doxycycline; Phos-, phosphorylated ; WT, wild type.
Received for publication May 1, 2002. Accepted for publication July 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain required for naive CD4+ T cell survival but not for antigen proliferation. Nat. Immunol. 1:54.[Medline]
chain. J. Immunol. 152:397.[Abstract]
T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429.[Medline]
subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR . Mol. Cell. Biol. 13:5771. in murine thymocytes and lymph node T cells. Immunity 1:675.[Medline]
T cells deal with induced TCR alpha ablation. Proc. Natl. Acad. Sci. USA 98:8744. chain is critical for the IL-2-induced activation of p56lck. EMBO J. 12:759.[Medline]
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M. Takase, E. M. Kanagawa, and O. Kanagawa Age-Dependent TCR Revision Mediated by Interaction between {alpha}beta TCR and Self-Antigens J. Immunol., August 15, 2007; 179(4): 2163 - 2169. [Abstract] [Full Text] [PDF]
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 M. Lovatt, A. Filby, V. Parravicini, G. Werlen, E. Palmer, and R. Zamoyska Lck Regulates the Threshold of Activation in Primary T Cells, While both Lck and Fyn Contribute to the Magnitude of the Extracellular Signal-Related Kinase Response Mol. Cell. Biol., November 15, 2006; 26(22): 8655 - 8665. [Abstract] [Full Text] [PDF]
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 E. Buentke, A. Mathiot, M. Tolaini, J. Di Santo, R. Zamoyska, and B. Seddon Do CD8 effector cells need IL-7R expression to become resting memory cells? Blood, September 15, 2006; 108(6): 1949 - 1956. [Abstract] [Full Text] [PDF]
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D. K. Taylor, P. T. Walsh, D. F. LaRosa, J. Zhang, M. A. Burchill, M. A. Farrar, and L. A. Turka Constitutive Activation of STAT5 Supersedes the Requirement for Cytokine and TCR Engagement of CD4+ T Cells in Steady-State Homeostasis J. Immunol., August 15, 2006; 177(4): 2216 - 2223. [Abstract] [Full Text] [PDF]
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B. Martin, C. Becourt, B. Bienvenu, and B. Lucas Self-recognition is crucial for maintaining the peripheral CD4+ T-cell pool in a nonlymphopenic environment Blood, July 1, 2006; 108(1): 270 - 277. [Abstract] [Full Text] [PDF]
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M.-W. Dobenecker, C. Schmedt, M. Okada, and A. Tarakhovsky The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function Mol. Cell. Biol., December 1, 2005; 25(23): 10533 - 10542. [Abstract] [Full Text] [PDF]
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 R. J. Ingham, J. Raaijmakers, C. S. H. Lim, G. Mbamalu, G. Gish, F. Chen, L. Matskova, I. Ernberg, G. Winberg, and T. Pawson The Epstein-Barr Virus Protein, Latent Membrane Protein 2A, Co-opts Tyrosine Kinases Used by the T Cell Receptor J. Biol. Chem., October 7, 2005; 280(40): 34133 - 34142. [Abstract] [Full Text] [PDF]
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F. J. Schnell and G. J. Kersh Control of Recent Thymic Emigrant Survival by Positive Selection Signals and Early Growth Response Gene 1 J. Immunol., August 15, 2005; 175(4): 2270 - 2277. [Abstract] [Full Text] [PDF]
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) or Fynneg (
)
backgrounds. Histograms (C) are of TCR expression by
CD4+CD8+ double-positive (DP), CD4 SP, and CD8
SP subsets from LckWT (C, upper
row) and Lck1ind mice (C,
lower row). Dotted lines are TCR expression by the
corresponding FynWT strain (
and 
, Cell numbers
from