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Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T
cells are
derived from bone marrow precursor cells. These precursor cells migrate
to the thymus, where they differentiate and are subject to selection
(reviewed in 1 . To complete this process, double negative
(DN)3
(CD4-CD8-) precursor cells must successfully
rearrange their TCR ß-chain to allow for expression of the pre-TCR
(2, 3) and progression to the double positive (DP)
(CD4+CD8+) stage. A significant expansion of
this population occurs before these cells rearrange their TCR -chain
and begin to express their TCRs (4). It is this population of immature
DP TCR+ cells that are subject to thymic selection (1, 5).
These processes entail both the selection of T cells bearing receptors
able to recognize peptides in the context of self-MHC (positive
selection) and the deletion of autoreactive T cells (negative
selection). Several hypotheses have been put forward to explain how the
TCR can propagate signals for both positive and negative selection. In
a qualitative model, the final outcome depends on signaling events that
somehow differ from one another qualitatively (6, 7). In a quantitative
model, the strength of the signals propagated by the TCR results in
either deletion or selection (8, 9). Recent results in fetal thymic
organ cultures have provided support for both the qualitative (10, 11)
as well as the quantitative models (12, 13). However, despite
differences in observations between these studies, the data are
consistent with an affinity/avidity model for thymic positive and
negative selection (1). Thus, increasing the overall avidity during the
selection process by either increasing peptide concentrations (13) or
by increasing the surface expression levels of CD8 (14, 15, 16) led to the
conversion of a positively selecting thymus to a negatively
selecting one.
Recent work has suggested that the efficiency of signaling from the TCR
during development can influence the outcome of the positive and
negative selection processes (17). In this study, Yamazaki et al. (17)
demonstrated that with TCR signaling compromised by the deletion of the
TCR 
-chain, thymocytes expressing a transgenic male-specific TCR
were able to develop in the male background, which would normally have
been deleting (18). If deletion of the TCR -chain allows for
survival of autoreactive T cells in such a strong deleting background,
then it is likely that signaling molecules associated with the TCR
-chain play a role in the signaling events during selection.
The Fyn protein tyrosine kinase (PTK) has been shown to be
constitutively associated with the TCR -chain (19). Despite its
close link to the TCR -chain, however, initial work with
Fyn-/- mice showed that thymocytes in these
mice appear to undergo normal development (20, 21). Thymocytes derived
from these mice revealed no differences in CD4/CD8 staining profiles
compared with control mice, and thymocyte yields were unaffected.
Several lines of evidence do, however, indicate that Fyn
plays a role in thymocyte development. Recent studies in
Lck-/-Fyn-/- mice
demonstrate that ß T cell development was completely halted by the
concurrent disruption of the lck and fyn genes
(22, 23). As mice lacking a functional lck gene (24) or
overexpressing a catalytically inactive form of Lck (25)
display a substantial but not absolute reduction in single positive
(SP) thymocyte numbers, these results suggest that Fyn is at
least able to perform some of the signaling functions required for T
cell development.
Examinations of SP thymocytes from Fyn-/- mice
showed that although T cell development was apparently normal, SP
thymocytes from Fyn-/- mice demonstrated
functional defects in TCR- (20, 21) and Thy-1- (26) mediated responses.
Proliferation responses, calcium ion fluxes, and IL-2 production were
significantly depressed in Fyn-/- thymocytes.
Studies of peripheral T cell responses have provided confusing results.
Stein et al. (20) found that proliferation was largely normal but IL-2
secretion and calcium flux were decreased. In contrast, Appleby et al.
(21) found that the proliferative response was decreased, as was
calcium flux. Both groups found decreases in tyrosine-phosphorylated
substrates in peripheral Fyn-/- T cells. It
has been demonstrated that Fyn phosphorylates tyrosine
residues within immunoreceptor tyrosine-based activation motifs on the
TCR/CD3 chains upon TCR stimulation (27). Fyn has also been
shown to phosphorylate Zap-70, leading to activation of this kinase
(27). More recent studies have also shown that
Fyn-/- thymocytes have a block in their
ability to up-regulate CD25 (IL-2R) but are able to secrete normal
levels of IL-2 (28).
Whether the role of Fyn is as a redundant partner to
Lck or as a kinase able to perform a unique and essential
role in TCR signaling has not been satisfactorily answered. To address
the question of whether Fyn is required for the development
of T cells bearing specific subsets of TCRs, we mated mice with the
Fyn-/- mutation with mice transgenic for TCRs
with differing affinities for their Ag ligands. The 2C TCR, whose
ligand has been identified as the naturally processed peptide p2Ca
(LSPFPFDL) presented by Ld MHC class 1 molecules (29) was
chosen as an example of a high affinity TCR. The affinity of the 2C TCR
for the p2Ca/Ld ligand is 
2 x 106
M-1 (29). We also studied the H-Y TCR (18) as a
representative of a low affinity TCR (30). Our results demonstrate that
Fyn plays a role both in signaling during thymocyte
development and during the response of thymic and peripheral T
lymphocytes to stimulation by specific Ags. The function of
Fyn is most evident in the response of T cells to low
affinity ligands.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeders for the H-2b 2C TCR transgenic mice (31, 32) were kindly provided by Dr. Denis Loh (Nippon Roche Research Center, Kamakura, Kanagawa, Japan). The H-2b 2C TCR mice were in the seventh to eighth generation of backcross to C57BL/6 (H-2b) mice. The 2C TCR is specific for the naturally processed peptide, p2Ca (LSPFPFDL), presented by Ld MHC class 1 molecules (29). The p2Ca peptide is derived from the mitochondrial protein 2-oxoglutarate dehydrogenase (33). H-2b H-Y TCR transgenic mice were produced as previously described (34). The H-Y TCR transgenic mice have been backcrossed onto a C57BL/6 background. Fyn-/- mice, on a mixed 129 (H-2b) and C57BL/6 background, were kindly provided by Dr. R. Perlmutter (Howard Hughes Medical Institute, University of Washington, Seattle, WA). TCR transgenic mice and Fyn-/- mice were mated to produce transgenic animals with the Fyn-/- mutation. C57BL/6 (B6), DBA/2(H-2d), and B10.BR (H-2k) mice were obtained from The Jackson Laboratory, Bar Harbor, ME. BDF1 mice were F1 mice obtained by mating B6 mice with DBA/2 mice. All animals were maintained in the animal facility at the University of British Columbia in the Department of Microbiology and Immunology.
Flow cytometric analysis of lymphocytes
Single cell suspensions of thymocytes and lymph node cells were
prepared. Cells (1 x 105) were stained with mAb
for 15 min on ice in 50 µl of FACS buffer (PBS with 2% FCS). Cells
were washed and then incubated with secondary Ab for 15 min on ice in
50 µl of FACS buffer. Cells were then washed and resuspended in FACS
buffer for analysis on a FACScan IV flow cytometer using LYSIS II
software (Becton Dickinson, Mountain View, CA). Data were collected for
1 x 104 viable cells, as determined by forward and
side light scatter analysis. mAbs used were T3.70 (anti-V3,
specific for the -chain of the H-Y TCR) (35, 36); F23.1 (37)
(anti-Vß8; specific for the ß-chain of the H-Y or 2C TCR); 1B2
(anti-2C TCR Id) (38); 53.67 (anti-CD8) FITC conjugate;
anti-CD4 (GK1.5) phycoerythrin (PE) conjugate; and M1/69
(anti-mouse heat-stable Ag (HSA)). mAbs were biotinylated except
where indicated. The hybridoma lines producing mAbs specific for CD4,
CD8, and HSA were obtained from the American Type Culture
Collection, Manassas, VA.
Proliferation assays
Thymocytes and lymph node cells were harvested from transgenic
mice and were used as responder cells in standard proliferation assays.
Purification of CD8 SP cells was as previously described (39).
Thymocytes were first depleted of CD4+ cells by
incubating with anti-CD4 (GK1.5) mAb followed by depletion of
CD4+ cells by anti-mouse Ig-coated Dyna beads (Dynal,
Oslo, Norway). The nonadherent cells after this treatment contained
CD4-CD8- and
CD4-CD8+ thymocytes. These nonadherent cells
were then incubated with biotinylated anti-CD8ß mAb and the
CD8+ cells positively selected with streptavidin-conjugated
MicroBeads (Miltenyi Biotec, Auburn, CA). The purity of the positively
selected cells was determined by staining the positively selected cells
with fluoresceinated goat F(ab')2 anti-mouse Ig Abs
(Southern Biotechnology Associates, Birmingham, AL), which reacted with
the anti-CD8ß mAb, and phycoerythrin-conjugated anti-CD4 mAb.
Thymocytes purified in this manner were >99%
CD4-CD8+. Highly purified CD8 SP lymph node
cells were isolated by the same method. Anti-CD3
(2C11) mAb
stimulations were done using 1 µg/ml of purified Ab and 20 U/ml of
exogenous IL-2. Stimulator cells (5 x 105 cells/well)
were irradiated splenocytes (2000 rad) from female or male B6 mice for
H-Y TCR transgenic cells and BDF1, B6, or B10.BR mice in
the case of 2C TCR transgenic mice. The peptide transporter-deficient
cell lines T2-Ld and T2-Kb (40) were derived by
transfecting the human (T x B) hybridoma T2 with Ld
or Kb. Cells were cultured in Iscove’s modified
Dulbecco’s medium (Life Technologies, Burlington, Canada) supplemented
with 5 x 10-5 M ß-mercaptoethanol, 10% FCS (Life
Technologies), and antibiotics. Cells were cultured in 96-well
round-bottom plates. The stimulation period was 3 days for 2C TCR and 4
days for H-Y TCR thymocyte and lymph node cultures. A total of 1 µCi
of [3H]TdR was added to each culture well 16 h
before harvest.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous analyses of Fyn-/- mice have revealed these mice to have similar thymocyte subset distribution as their control littermates (20, 21). However, these analyses were done on normal mice with heterogeneous TCR repertoires. We sought to determine the role of Fyn in T cell development by following the developmental fate of Fyn+/+ and Fyn-/- T cells that express a defined TCR. Using this approach, we hoped to identify conditions in Fyn-/- mice in which TCR signaling was not sustained by the activities of other Src kinases. We chose to study two TCRs with differing affinities for their Ag ligands: the 2C TCR, with high affinity for its Ag ligand, and the H-Y TCR, representative of a low affinity TCR. The H-Y TCR is specific for an undefined male (H-Y) peptide presented by the H-2Db MHC class I molecule; this TCR is positively selected in female H-2b mice and is negatively selected in male H-2b mice (18, 41).
The CD4/CD8 phenotype of thymocytes from female H-2b H-Y
TCR transgenic mice with or without the Fyn-/-
mutation is shown in Figure 1
. The
Fyn-/- mutation had several effects on
thymocyte development in female H-2b H-Y TCR transgenic
mice. In Fyn-/- mice, the proportion of DP
thymocytes was slightly increased. A corresponding reduction in the
proportion of DN thymocytes compensated for this increase. The
expression of the CD4 and CD8 coreceptor molecules was slightly
down-regulated on DP thymocytes from Fyn-/-
mice. Furthermore, the number of Fyn-/- DP
thymocytes that expressed higher levels of the transgenic TCR ß-chain
(detected by the F23.1 mAb) and the transgenic TCR -chain (detected
by the T3.70 mAb) was also increased. Previous studies have shown that
DP thymocytes that have been positively selected by thymic MHC ligands
up-regulate TCR expression levels (42, 43). If the lack of
Fyn facilitated positive selection, this would have been
accompanied by an increase in the production of CD8 SP thymocytes in
Fyn-/- mice. However, this was not observed
(Fig. 1). One explanation for this observation is that while
Fyn is not required for the up-regulation of TCR levels on
positively selected DP thymocytes, it may be required for the efficient
differentiation of positively selected DP thymocytes into SP
thymocytes. Thus, the lack of Fyn may result in the
accumulation of DP thymocytes that expressed higher levels of the
H-Y TCR.
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, Table I). This increase in DP thymocytes in
Fyn-/- mice was associated with an increase in
the number of DP thymocytes that expressed high levels of CD8 in 2C
Fyn-/- mice (Fig. 1). This result can be
explained by assuming that Fyn is involved in the negative
selection of thymocytes. In the absence of Fyn, thymocytes
that would normally have been deleted will be able to survive. In
H-2b Fyn-/- 2C TCR transgenic
mice, this translates into a higher proportion of DP thymocytes that
retained high expression of the CD8 coreceptor molecule. This effect of
Fyn is specific for H-2b 2C TCR transgenic mice
since the Fyn-/- mutation did not lead to an
increase in CD8 expression in female H-2b H-Y TCR
transgenic mice (Fig. 1).
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2 x 107 cells from either
Fyn+/+ or Fyn-/- mice),
and the surviving population in Fyn+/+ or
Fyn-/- mice was predominantly of the DN
phenotype (Fig. 2). These results
indicate that Fyn is not essential for efficient negative
selection in these mice, though Fyn did have a subtle effect
in male H-Y transgenic mice. We found that the surviving thymocytes in
Fyn-/- mice differed from
Fyn+/+ mice in the expression levels of CD8 and
TCR (Fig. 2). In Fyn-/- male mice, there were
more thymocytes that expressed higher levels of the CD8 coreceptor and
the H-Y TCR (Fig. 2). Thus, the lack of Fyn enables the
survival of thymocytes that expressed slightly higher levels of CD8 and
the H-Y TCR.
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), it did affect the
expression of HSA in CD8 SP thymocytes from these mice (Fig. 3). In Fyn+/+ H-Y
TCR transgenic mice, the majority of CD8 SP thymocytes maintained high
HSA expression, and a minority of these cells had down-regulated HSA.
However, almost all Fyn-/- CD8 SP thymocytes
from these mice maintained high expression of HSA. Similarly, in
Fyn+/+ 2C TCR transgenic mice, the majority of
CD8 SP thymocytes had down-regulated HSA. However, the majority of CD8
SP thymocytes in 2C Fyn-/- mice maintained
high expression of HSA. This delayed down-regulation of the HSA
maturity marker may reflect a role for Fyn in the
development of CD8 SP thymocytes. No significant differences in
transgenic TCR - or ß-chain expression were detected in CD8 SP
thymocytes of H-2b 2C and female H-2b H-Y TCR
transgenic mice with the Fyn-/- mutation
(Fig. 3).
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). By contrast, CD8 SP cells from the
lymph nodes of female H-2b H-Y TCR transgenic mice showed a
very heterogeneous expression of the TCR -chain. Only a minority of
these CD8 SP cells continued to express high levels of the transgenic
TCR -chain (Fig. 4). The predominant expression of endogenous TCR
-chains in peripheral CD8 SP cells has previously been reported and
attributed to the preferential expansion of CD8 SP cells that express
endogenous TCR -chains in these mice (48). Significantly, the
Fyn-/- mutation did not alter the TCR
repertoire of peripheral CD8 SP cells in H-Y TCR transgenic mice. This
result suggests that the positively selected CD8 SP cells in
Fyn+/+ or Fyn-/- mice,
regardless of whether they expressed transgenic or endogenous TCR
-chains, were expanded in a similar fashion in peripheral lymphoid
organs. In contrast to CD8 SP thymocytes, the vast majority of CD8 SP
lymph node cells expressed low levels of HSA (Fig. 4). This likely
reflects the more functionally mature status of CD8 SP lymph node cells
and suggests that Fyn is not essential for the functional
maturation of peripheral CD8 SP cells.
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We next sought to determine the responsiveness of cells with or
without the Fyn-/- mutation to stimulation by
anti-CD3 mAb. To ensure that any responses we observed were
strictly those of CD8 SP cells, we first depleted cell populations of
CD4 SP, DP, and DN cells as previously described (39). Once highly
purified (>99% CD8 SP) cell populations were obtained, the
responsiveness of CD8 SP thymocytes and lymph node cells to
anti-CD3 (2C11) mAb stimulation in the presence of 20 U/ml of
exogenous IL-2 was determined. As seen in Figure 5, thymocytes from female
H-2b H-Y TCR transgenic mice with the
Fyn-/- mutation were hyporesponsive to
stimulation by anti-CD3 mAb. This functional defect was not
observed in CD8 SP lymph node cells, since no significant difference in
proliferative response was observed between the
Fyn+/+ and Fyn-/-
populations. This result agrees with the previously published data (20, 21) in that only Fyn-/- thymocytes, but not
Fyn-/- lymph node cells, were refractive to
stimulation by anti-TCR mAb. Similar experiments with CD8 SP
thymocytes and lymph cells expressing the 2C TCR provided different
results, however. 2C11 stimulation of 2C CD8 SP thymocytes and lymph
node cells (Fig. 5) revealed comparable proliferation in both the
Fyn-/- and Fyn+/+
populations. This difference in responsiveness between H-Y and 2C CD8
SP thymocytes to stimulation by 2C11 likely reflects differences in the
functional maturity of these thymocytes.
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-chain. Therefore, this approach provides a
more critical evaluation of the requirement of Fyn for the
activation of T cells by their cognate ligands. Purified CD8 SP
thymocytes and lymph node cells were stimulated with irradiated
splenocytes in a standard proliferation assay. Cells transgenic for the
H-Y TCR were stimulated with syngeneic (C57/BL6) male splenocytes,
whereas 2C TCR cells were stimulated with BDF1
(C57/BL6 x DBA/2) splenocytes. As seen in Figure 6, both Fyn-/-
CD8 SP thymocytes and lymph node cells transgenic for the H-Y TCR were
hyporesponsive to the male Ag. The response of this TCR to its cognate
ligand was 10-fold lower than the Fyn+/+
cells. Neither the Fyn+/+ nor the
Fyn-/- cells were able to respond to female B6
splenocytes, indicating that the observed response was indeed male
specific.
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). These CD8 SP cells did not
respond to B6 or B10.BR splenocytes, indicating that the response was
indeed specific for H-2d. Thus, depending on the transgenic
TCR, CD8 SP thymocytes or lymph node cells display differential
requirement for Fyn in optimal proliferative response to
stimulation by their specific Ag.
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2 x
106 M-1 and 3 x 103
M-1, respectively (29). This approach therefore enabled us
to determine whether CD8 2C TCR+ SP thymocytes or lymph
node cells can display differential requirements for Fyn in
their response to a high affinity (Ld/p2Ca) or a low
affinity (Kb/p2Ca) ligand. As shown in Figure 8, 2C Fyn-/-
thymocytes required 100- to 1000-fold more p2Ca peptide to respond to
the same extent as the Fyn+/+ thymocytes when
stimulated with the T2-Ld cells and exogenous p2Ca peptide.
This response was p2Ca specific since no proliferative response was
observed in the presence of another Ld-binding peptide
(YPHFMPTNL) (29) (data not shown). Thus, even for a high affinity
ligand, CD8 SP thymocytes required a relatively high ligand density for
optimal proliferation to the specific Ag. These results indicate that
the lowering of the avidity of the T cell/APC interaction revealed a
requirement for Fyn by CD8 SP thymocytes expressing a high
affinity TCR for its specific Ag. By contrast, only minor differences
were evident between CD8 SP Fyn-/- and
Fyn+/+ lymph node cells in their response to the
Ld/p2Ca ligand, even at the lowest peptide concentrations
studied (Fig. 8). This independence of Fyn by CD8 SP lymph
node cells even at low ligand density likely reflects the more
functionally mature status of the lymph node cells relative to
thymocytes.
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. As a result of such a low affinity
interaction between T cells and APCs, it was necessary to add exogenous
IL-2 to these cultures to detect a proliferative response to the
Kb/p2Ca ligand. As seen in Figure 9, even in the presence
of exogenously added IL-2, proliferative responses were only observed
at higher peptide concentrations. Significantly, both
Fyn-/- CD8 SP thymocytes and lymph node cells
were hyporesponsive to stimulation by the Kb/p2Ca ligand
relative to their Fyn+/+ counterparts. Not only
were the proliferative responses by Fyn-/-
cells substantially reduced, the responses attained by the
Fyn-/- cells did not approach that of
Fyn+/+ cells, even at high ligand density. These
observations emphasized the importance of Fyn in optimizing
the responses of T cells expressing low affinity TCRs to stimulation by
their specific Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A previous study suggests that the positive selection of CD8 SP
thymocytes expressing high levels of the H-Y TCR occurs normally even
in the absence of Fyn expression (49). Our results are
consistent with this report in that we also observed normal production
of CD8 SP thymocytes expressing high levels of the H-Y TCR in female
H-2b Fyn-/- H-Y TCR transgenic
mice. Our study extended this finding by demonstrating that
Fyn does have subtle effects on positive selection. Our
results suggest that although Fyn is not essential for the
positive selection of the H-Y TCR, it may facilitate the transition of
positively selected DP thymocytes into SP thymocytes. Furthermore, we
found that Fyn facilitated the down-regulation of HSA on
positively selected CD8 SP thymocytes. Previous reports have provided
evidence for the 2C TCR being strongly selected in H-2b 2C
mice (31), with increases in CD8 expression leading to deletion (14, 15). With a recent report indicating that the efficiency of TCR
signaling during T cell development is greatly reduced by the
abrogation of TCR--derived signals (17), it is reasonable to
hypothesize that other molecules that contribute to TCR signaling may
also influence these processes.
Deletion of Fyn is an ideal means to study partial
disruption of TCR- signaling. The Fyn PTK is
constitutively associated with the TCR -chain and has been shown to
phosphorylate it upon TCR stimulation, allowing for the association of
Zap-70 with the cytoplasmic tail (reviewed in 50 . Murine
Zap-70-/- thymocytes arrest at the DP stage (51),
suggesting that Zap-70 signaling is essential for T cell development.
As Fyn phosphorylates and activates Zap-70 (27), it may play
a role during thymocyte development. However, this situation is
complicated by the finding that another PTK, namely Lck, can
also phosphorylate Zap-70 (27). Thus, the question of whether
Fyn is a redundant kinase for T cell development and
function has not been resolved.
In this study, we found that although Fyn is not essential
for negative selection, it alters the cell surface phenotype of
surviving thymocytes in either a very strongly positively selecting MHC
background (H-2b 2C mice) or a negatively selecting MHC
background (male H-2b H-Y mice). In H-2b 2C TCR
transgenic mice, introduction of the Fyn-/-
mutation led to a twofold increase in the number of DP thymocytes. In
male H-2b H-Y TCR transgenic mice, the
Fyn-/- mutation enables the survival of
thymocytes that expressed higher levels of the CD8 coreceptor and the
H-Y TCR. These observations are consistent with the hypothesis that the
Fyn-/- mutation may reduce the signaling
efficiency of the TCR/CD3 complex during the negative selection
process. This hypothesis agrees with previous findings showing that
lowering the TCR signaling efficiency by altering the number of
-chains or by the deletion of the -chain can allow for the
survival of thymocytes that would have been expected to have been
deleted (17, 52).
If the Fyn-/- mutation lowers the effective signaling capabilities of the TCR/CD3 complex and allows for higher avidity interactions before deletion, then one would anticipate higher levels of coreceptor expression on Fyn-/- cells. This was indeed the case in the 2C Fyn-/- thymocyte population, where CD8 expression was higher than that of Fyn+/+ thymocytes. The reduced deletion and increased CD8 expression in the Fyn-/- 2C system demonstrate that Fyn does play a role in TCR signaling during thymocyte development. This effect of Fyn was not observed in female H-2b H-Y TCR transgenic mice. This can be explained by postulating that the H-Y TCR is positively selected by relatively low affinity/avidity thymocyte/selecting cell interactions in female transgenic mice. This level of interaction leading to the positive selection of H-Y thymocytes in female mice is presumed to be well below the deletion threshold, but above the minimum positive selection threshold. Under these conditions, we do not expect Fyn to have a discernible effect on the survival of DP thymocytes, and this is observed.
We have shown previously that high cell surface expression of the HSA molecule on CD8 SP thymocytes correlates with positive selection by low affinity/avidity ligands and that these cells are functionally immature (39). Here, we have observed that HSA levels in CD8 SP thymocytes from both H-Y Fyn-/- and 2C Fyn-/- thymocytes are elevated relative to those that expressed Fyn. This observation also argues for the selection of these thymocytes by weaker selecting signals and further implicates a role for Fyn in the positive selection process. However, we noted that although Fyn-/- thymocytes from both TCR transgenic backgrounds had increased HSA expression, only the H-Y Fyn-/- thymocytes were hyporesponsive to stimulation by anti-TCR Abs. This observation may be explained by the hypothesis that the H-Y TCR is positively selected with low efficiency in the first place. The further lowering of this efficiency by introducing the Fyn-/- mutation will lead to the production of CD8 SP thymocytes that are functionally less mature. We have recently shown that the 2C TCR, when selected by very weak selecting ligands in an H-2k thymus, also led to the production of CD8 SP HSAhigh thymocytes that were hyporesponsive to stimulation by anti-TCR Abs (39). That only minimal differences can be observed in the 2C thymocytes can be explained by the same hypothesis. In this situation, the selecting ligand for these cells is of sufficiently high affinity/avidity that the Fyn-/- mutation does not lower the effective signal enough to slow the development of these cells as significantly as in the H-Y system. Hence, these cells are able to proliferate when stimulated with 2C11 and IL-2.
Stimulation of CD8 SP Fyn-/- cells with their physiologic ligands provided evidence that Fyn is indeed involved in the proliferation responses of both thymocytes and lymph node cells. As these stimulation protocols do not circumvent the natural affinity of the TCR for its ligand, these results provide clear evidence for a role for Fyn in signaling from low affinity TCRs. Using purified CD8 SP cell populations, we saw that both the thymocyte and lymph node cell populations from H-Y Fyn-/- mice were hyporesponsive in an anti-male response. This observation suggested that the low affinity H-Y response was compromised by the Fyn mutation. However, the response of CD8 SP 2C thymocytes or lymph node cells to naturally processed ligands on BDF1 splenocytes did not reveal a role for Fyn in this response. We attribute this to the high affinity of the 2C TCR for the Ld/p2Ca ligand. Thus, Fyn is not essential for an optimal response by CD8 SP thymocytes or lymph node cells, which expressed a high affinity TCR for its cognate ligand. Importantly, we found that we can reveal a role for Fyn in 2C TCR signaling by lowering the affinity/avidity of the 2C/APC interaction by using T2-Kb or T2-Ld cells as APCs and varying the concentrations of the p2Ca peptide. Under these conditions, we observed a role for Fyn in the response of CD8 SP 2C thymocytes to the high affinity Ld/p2Ca ligand at low ligand density. In the case of the low affinity Kb/p2Ca ligand, we were able to demonstrate a role for Fyn even at high ligand density. These observations underscore the importance of Fyn in optimizing responses to low affinity ligands and provide independent evidence supporting the conclusion that the H-Y TCR is indeed a low affinity TCR.
The results presented here provide evidence of a role for Fyn in TCR signaling during both thymocyte development and the activation of positively selected thymocytes and peripheral T cells. That the role of Fyn was not as evident in previous reports can be explained by the heterogeneous TCR backgrounds studied. We propose that Fyn is an important player in the positive selection of T cells by low affinity/avidity ligands and in the activation of positively selected T cells by low affinity/avidity ligands. This function of Fyn appears not to be compensated for by other PTKs and argues against Fyn being a redundant kinase in T cell development and T cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, 6174 University Boulevard, Vancouver, B.C., Canada V6T 1Z3. E-mail address:
3 Abbreviations used in this paper: DN, double negative (CD4-CD8-); DP, double positive (CD4+CD8+); Fyn, p59fyn; H-Y TCR, transgenic TCR specific for the male Ag presented by H-2Db; Lck, p56lck; PTK, protein tyrosine kinase; SP, single-positive (CD4-CD8+ or CD4+CD8-); 2C TCR, transgenic TCR specific for the p2Ca peptide presented by H-2Ld; PE, phycoerythrin; HSA, heat-stable Ag.
Received for publication December 1, 1997. Accepted for publication February 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  A. A. Mamchak, B. M. Sullivan, B. Hou, L. M. Lee, J. K. Gilden, M. F. Krummel, R. M. Locksley, and A. L. DeFranco Normal Development and Activation but Altered Cytokine Production of Fyn-Deficient CD4+ T Cells J. Immunol., October 15, 2008; 181(8): 5374 - 5385. [Abstract] [Full Text] [PDF]
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A. Filby, B. Seddon, J. Kleczkowska, R. Salmond, P. Tomlinson, M. Smida, J. A. Lindquist, B. Schraven, and R. Zamoyska Fyn Regulates the Duration of TCR Engagement Needed for Commitment to Effector Function J. Immunol., October 1, 2007; 179(7): 4635 - 4644. [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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N. B. Martin-Cofreces, D. Sancho, E. Fernandez, M. Vicente-Manzanares, M. Gordon-Alonso, M. C. Montoya, F. Michel, O. Acuto, B. Alarcon, and F. Sanchez-Madrid Role of Fyn in the Rearrangement of Tubulin Cytoskeleton Induced through TCR J. Immunol., April 1, 2006; 176(7): 4201 - 4207. [Abstract] [Full Text] [PDF]
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 K. Sugie, M.-S. Jeon, and H. M. Grey Activation of naive CD4 T cells by anti-CD3 reveals an important role for Fyn in Lck-mediated signaling PNAS, October 12, 2004; 101(41): 14859 - 14864. [Abstract] [Full Text] [PDF]
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P. Gadue, L. Yin, S. Jain, and P. L. Stein Restoration of NK T Cell Development in fyn-Mutant Mice by a TCR Reveals a Requirement for Fyn During Early NK T Cell Ontogeny J. Immunol., May 15, 2004; 172(10): 6093 - 6100. [Abstract] [Full Text] [PDF]
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 K. L. Puls, K. A. Hogquist, N. Reilly, and M. D. Wright CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics Int. Immunol., March 1, 2002; 14(3): 249 - 258. [Abstract] [Full Text] [PDF]
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O. Utting, J. J. Priatel, S.-J. Teh, and H.-S. Teh p59fyn (Fyn) Promotes the Survival of Anergic CD4-CD8- {{alpha}}{{beta}} TCR+ Cells but Negatively Regulates Their Proliferative Response to Antigen Stimulation J. Immunol., February 1, 2001; 166(3): 1540 - 1546. [Abstract] [Full Text] [PDF]
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J. Huang, D. Tilly, A. Altman, K. Sugie, and H. M. Grey Inaugural Article: T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase PNAS, September 26, 2000; 97(20): 10923 - 10929. [Abstract] [Full Text] [PDF]
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 P. Gadue, N. Morton, and P. L. Stein The Src Family Tyrosine Kinase Fyn Regulates Natural Killer T Cell Development J. Exp. Med., October 18, 1999; 190(8): 1189 - 1196. [Abstract] [Full Text] [PDF]
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G. Eberl, B. Lowin-Kropf, and H. R. MacDonald Cutting Edge: NKT Cell Development Is Selectively Impaired in Fyn- Deficient Mice J. Immunol., October 15, 1999; 163(8): 4091 - 4094. [Abstract] [Full Text] [PDF]
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S. Miscia, A. Di Baldassarre, G. Sabatino, E. Bonvini, R. A. Rana, M. Vitale, V. Di Valerio, and F. A. Manzoli Inefficient Phospholipase C Activation and Reduced Lck Expression Characterize the Signaling Defect of Umbilical Cord T Lymphocytes J. Immunol., September 1, 1999; 163(5): 2416 - 2424. [Abstract] [Full Text] [PDF]
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