By
From the * Laboratory of Mammalian Genes and Development, National Institute of Child Health
and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and the Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, Maryland 20892
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Abstract |
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Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of
T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5
surface expression during normal thymocyte ontogeny and in mice with developmental and/or
signal transduction defects. The results demonstrate that low level expression of CD5 on
CD4CD8
(double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the
pre-TCR and is dependent upon the activity of p56lck. At the CD4+CD8+ (double positive,
DP) stage, intermediate CD5 levels are maintained by low affinity TCR-major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the
surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and
CD4+ or CD8+ (single positive, SP) thymocytes is induced by engagement of the
/
-TCR
by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature
SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the
positively selecting TCR-MHC-ligand interaction. Taken together, these observations suggest
that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.
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Introduction |
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CD5 is a monomeric cell surface glycoprotein expressed
on thymocytes, all mature T cells, and a subset of B
cells, B-1 cells (1). Putative CD5 ligands include CD72,
a pan-B cell antigen, and CD5L, a recently described protein expressed on activated splenocytes, B cells, and activated murine T cell clones (5, 6), suggesting that CD5 may
be involved in regulating immune cell interactions. The
cytoplasmic domain of CD5 contains three potential tyrosine phosphorylation sites, including a putative ITAM
(immunoreceptor tyrosine-based activation motif)1 or
ITIM (immunoreceptor tyrosine-based inhibition motif) sequence (4, 7) and multiple potential Ser/Thr phosphorylation sites (4). After TCR engagement, CD5 is tyrosine
phosphorylated and becomes associated with a multimolecular complex that may include TCR-, CD2, CD4, CD8,
p56lck, p59fyn, PTPC1, and Zap70 (8). The physiological role of CD5 is still not clearly understood. Previous
studies have shown that treatment of T cells with anti-CD5
enhances TCR-mediated activation, proliferation, and IL-2
production (13). On the other hand, more recent data indicate that CD5 acts to negatively regulate signaling
through both the B and T cell antigen receptors (16, 17).
In the absence of CD5, peritoneal B-1 cells, which normally are triggered to undergo apoptosis in response to
mIgM cross-linking, develop resistance to apoptosis and
enter the cell cycle (17). Likewise, thymocytes from CD5
/
mice are hyperresponsive to stimulation through the TCR,
and the efficiency of thymocyte selection in CD5
/
,
/
-TCR transgenic mice is altered in a manner consistent with enhanced TCR signaling (16).
CD5 surface expression is tightly regulated throughout T
cell development. Low levels of CD5 are expressed on immature CD4CD8
(double negative, DN) thymocytes.
CD5 surface expression then increases at both the
CD4+CD8+ (double positive, DP) and CD4+ or CD8+
(single positive, SP) stages and relatively high levels of CD5 are maintained on circulating SP T cells (3, 18). In this study, we sought to identify the cellular mechanisms regulating CD5 expression during development. Our results
demonstrate that CD5 is upregulated at crucial points during thymocyte development by pre-TCR and TCR engagement and that the level of CD5 surface expression is
directly related to pre-TCR and TCR signaling intensity. Significantly, CD5 surface levels were found to vary considerably among mature SP thymocytes and T cells that
express distinct TCRs, and the level of CD5 expression
paralleled the avidity of the positively selecting TCR-MHC-ligand interaction. Together, these results suggest that the
ability to regulate CD5 surface expression in response to
TCR signaling is important for fine tuning the TCR signaling response and for selection of the mature TCR repertoire.
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Materials and Methods |
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Mice
C57 BL/6 (B6) mice were bred within our facility. Mutant
strains of mice used for this study included Rag2/
(19); MHC
class I
/
(
2M
/
; reference 20); MHC class II
/
(A
/
; reference 21); MHC class I × II
/
(
2M
/
× A
/
; reference
22); TCR-
/
(23); and lck
/
(24).
/
-TCR transgenic lines
included P14 (25), H-Y (26), AND (27), and DO11.10 (DO10;
28). TCR-
chain transgenic and TCR-
/
mice were generated as previously described (29, 30). For positive selection experiments, mice were bred to C57 BL/6, B10.D2 or B10.A(5R)
mating partners to change the selecting haplotype.
Antibodies
mAbs used for flow cytometric analysis were purchased from
PharMingen (San Diego, CA) and included fluorochrome-
(FITC or PE) or biotin-conjugated anti-Thy1.2 (53-2.1), anti-B220 (RA3-6B2), anti-CD4 (H129.19), anti-TCR- (H57-597), anti-CD8
(53-6.7), anti-CD3
(145-2C11), anti-CD5
(53-7.3), anti-CD69 (H1.2F3), anti-CD25 (7D4), anti-CD44
(IM7), anti-V
11 (RR8-1), and anti-V
2 (B20.1). Unconjugated anti-CD16/CD32 (2.4G2), and rat IgG2a,
(R35-95) were used to block nonspecific Fc receptor binding and as control antibody, respectively. The anti-H-Y clonotypic receptor mAb
(T3.70) and anti-DO10 clonotypic receptor mAb (KJ126) were
purified from cell culture supernatants and labeled with FITC in
our laboratory. Streptavidin Red 670 (GIBCO BRL, Gaithersburg, MD) was used in conjunction with biotinylated antibodies
for flow cytometry.
Flow Cytometric Analysis
Thymi and lymph nodes were excised from mice and single
cell suspensions were prepared. For multicolor flow cytometry
(FCM), thymocytes or lymph node cells were first incubated with
antibody to the Fc receptor (mAb 2.4G2) to prevent nonspecific
binding of antibodies. Background staining was measured using
fluorochrome-conjugated rat IgG2a, and designated as control
peaks. For two- and three-color FCM, cells were incubated with
FITC-conjugated, PE-conjugated, and biotinylated antibodies,
followed by the addition of Red 670 streptavidin. FCM was performed on a FACScan® using standard Cell Quest software (Becton Dickinson, San Jose, CA). Data were collected on 104 viable
cells as determined by forward and side light scatter. Cell numbers
among the various thymocyte populations were normalized by
gating and collecting on DN, DP, or SP thymocytes. CD5 levels on thymocyte subpopulations were measured by staining with
CD5, CD4, and CD8 antibodies, followed by a three-color
FACS® analysis and gating according to the CD4/CD8 profile.
Purification of DN Thymocyte Populations
Total thymocytes from B6 mice were stained with a mixture
of biotinylated anti-CD3, -CD4, -CD8, and -B220 mAb antibodies, washed, and incubated with Streptavidin microbeads
(Miltenyi Biotec, Auburn, CA). DN (CD3CD4
CD8
B220
)
thymocytes were then purified by magnetic separation according to the manufacturer's protocol (Miltenyi Biotec). Purity of
magnetically separated DN thymocytes was >95% as assessed
by FCM.
Thymocyte Stimulation
In Vitro CD3 Cross-linking.
24-well plates were coated with 1-50 µg of anti-CD3In Vivo CD3 Cross-linking.
Rag-2 ![]() |
Results |
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Examination of thymocyte subsets from B6 mice reveals a stepwise progression in CD5
surface expression that correlates with thymocyte maturity
(Fig. 1). Low levels of CD5 are expressed on immature
CD4CD8
(double negative, DN) thymocytes (CD5lo),
followed by an approximately sixfold increase in CD5 expression at the CD4+CD8+ (double positive, DP) stage
(CD5int), and a further three- to fivefold increase in CD5
expression as thymocytes reach the mature CD4+ or CD8+
(single positive, SP) stage (CD5hi). Relatively high levels of
CD5 are maintained on mature peripheral SP T cells (Fig.
1), with the highest expression on CD4+ T cells (Fig. 1).
To investigate how CD5 expression is controlled during
development, we began by examining thymocytes from
mutant strains of mice that exhibit defects in T cell development, signal transduction, or both.
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Thymocytes from mice deficient in expression of the
Rag-2 gene product (Rag-2/
) fail to develop beyond the
DN stage and fail to express either pre-TCR or
/
-TCR
surface complexes owing to a defect in the initiation of
V-(D)-J recombination (19, 31). Yet thymocytes from
Rag-2
/
mice express low levels of CD5 (Fig. 2). CD5
expression was also observed on TCR
DN thymocytes
from TCR-
/
mice (results not shown) demonstrating
that neither the pre-TCR nor the
/
-TCR is required for
low-level expression of CD5. DN thymocytes from mice in
which TCR-
rearrangement is inhibited (TCR-
/
)
mature to the DP stage, but are unable to progress further in development (23). Significantly, DP thymocytes from
TCR-
/
mice express higher levels of CD5 than do DN
thymocytes (Fig. 2). In fact, CD5 levels on the majority of DP
thymocytes from control (TCR-
+/+) and TCR-
/
mice
are similar, differing mainly in the absence of a small CD5hi
DP population in TCR-
/
mice (Fig. 2; reference 32).
|
Since in the absence of TCR- chain, thymocytes can
express pre-TCR but not
/
-TCR complexes, we next
investigated whether pre-TCR signals could upregulate
CD5 surface expression. Expression of the pre-TCR begins
at the DN CD44lo/
CD25+ stage of development and is
required for transition to the DN CD44
CD25
stage (33,
34). Examination of DN CD44
CD25
thymocytes revealed that these cells express three- to fourfold higher levels of surface CD5 than do their immediate precursors, DN
CD44lo/
CD25+ thymocytes (Fig. 3 A). Signals transduced by the pre-TCR induce cell cycle progression, resulting in the expansion of cells that have undergone productive
-rearrangement, a process termed "
selection"
(34). Consequently, in normal mice, the DN CD44lo/
CD25+ thymocyte compartment consists of two subsets
("E" and "L") that can be distinguished on the basis of cell
size (34). The E subset contains small resting cells that either have not yet undergone
selection or have failed to
productively rearrange their
chain genes, whereas the L
subset consists of larger cells that have received pre-TCR
signals and have entered the cell cycle (reference 34; Fig.
3 A). Examination of CD5 surface expression on DN
CD44lo/
CD25+ thymocyte subsets revealed that L cells
express approximately fourfold higher levels of CD5 than
do E cells (Fig. 3 A). Collectively, these results indicate that
signaling through the pre-TCR upregulates CD5 surface
expression on DN thymocytes. To test this hypothesis further, we next determined the effect of CD3 cross-linking
on CD5 expression. Injection of anti-CD3 antibodies into
Rag-2
/
mice has been shown to mimic pre-TCR signaling as it promotes the formation of DP thymocytes (35,
36). This response has been attributed to the low level expression of CD3 signaling subunits on the surface of Rag-2
/
thymocytes in the absence of
and pre-T
chains
(37). To ascertain whether direct engagement of these surface complexes could induce CD5 expression on DN thymocytes before their transition to the DP stage, Rag-2
/
mice were analyzed at multiple time points after intraperitoneal injection of anti-CD3 antibody. Significantly, as
early as 24 h after anti-CD3 injection, thymocyte cellularity was increased fourfold and Rag-2
/
thymocytes,
which still consisted entirely of DN cells, expressed high
levels of CD5 (Fig. 3 B). Induction of the early activation antigen, CD69, was also observed on DN thymocytes 24 h
after injection (Fig. 3 B). At 3 d after injection, CD5 levels
on DN thymocytes remained high and large numbers of
CD5int-hi DP thymocytes were present in the thymus of
Rag-2
/
mice (data not shown).
|
Because signaling by the pre-TCR has been shown to be
dependent upon the activity of the protein tyrosine kinase
p56lck (24, 38), we also examined the importance of Lck for
the induction of CD5 expression. DN, CD44lo/CD25+
thymocytes from lck
/
mice were found to express lower
levels of CD5 than DN, CD44lo/
CD25+ thymocytes from
lck+/+ mice (Fig. 3 C). In addition, although lck
/
mice
contained
/
-TCR+ DP thymocytes, these cells also expressed low levels of CD5 relative to DP thymocytes from
lck+/+ mice (Fig. 3 C).
To determine if induction of CD5 on DN thymocytes
correlates with pre-TCR signal intensity, Rag-2/
mice
were injected with varying amounts of stimulating (anti-CD3
) antibody. The results demonstrated a direct relationship between anti-CD3
dosage and CD5 surface expression (Fig. 3 B). The relationship between pre-TCR
signaling capacity and CD5 expression was also examined
by analyzing CD5 expression on DP thymocytes from
TCR-
/
×
/
mice, which express pre-TCR in the
absence of associated
chain. Previous work has shown
that the pre-TCR is expressed and can function, though
less efficiently, in the absence of
to promote the formation of DP thymocytes, indicating that
chain contributes quantitatively to the pre-TCR signaling response (29, 38). Indeed, DP thymocytes from TCR-
/
×
/
mice expressed significantly lower levels of CD5 than did DP thymocytes from TCR-
/
mice (Fig. 3 D). Taken together,
these results demonstrate that signals transduced by the pre-TCR induce the upregulation of CD5 on DN thymocytes,
resulting in the formation of DP CD5int thymocytes.
CD5 surface expression has been
shown previously to correlate with expression of the /
-TCR during T cell development (3, 18). To investigate
this relationship further, we used mice generated previously
that express different levels of TCR due to variable expression of
chain (30). Examination of DP thymocytes from
+/
and
+/+;
tg mice, which express low or high levels
of
/
-TCR relative to
+/+ mice, respectively, revealed a
direct relationship between TCR surface expression and
mean CD5 surface expression on DP thymocytes (Fig. 4 A).
To determine if TCR engagement was specifically required for CD5 expression on DP thymocytes, we then examined thymocytes from mice lacking MHC (class I × II)
/
in which development is arrested at the TCR+ DP
stage. Although most DP thymocytes from MHC class (I × II)
/
mice express higher levels of
/
-TCR than do DP
thymocytes from control mice, these cells express low levels of CD5 (Fig. 5 A). Indeed, the level of CD5 expressed
on DP thymocytes from MHC class (I × II)
/
mice is
similar to that expressed on DN thymocytes (Fig. 5 B). In
contrast, DP thymocytes from mice lacking expression of
either MHC class I (Fig. 5, A and B) or MHC class II alone
(data not shown) express normal (intermediate) levels of
CD5. Thus, interaction of TCR+ DP thymocytes with
self-MHC appears to be required to sustain CD5 surface
expression at levels above those observed on DN thymocytes. Consistent with this idea, anti-TCR cross-linking
resulted in induction of both CD5 and CD69 on DP
thymocytes from class (I × II)
/
mice to levels comparable
to those observed on DP thymocytes from control mice
(Fig. 5 C).
|
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The relationship between TCR signal intensity and
CD5 expression on DP thymocytes was examined by in
vitro cross-linking with varying amounts of anti-CD3.
The results of this experiment revealed a direct relationship
between the concentration of cross-linking antibody and
CD5 surface levels (Fig. 4 B). The correlation between TCR signaling and CD5 induction could also be assessed
using transgenic mice that express similar levels of TCR
but whose TCRs differ in their signaling potential due to
differences in the total number of ITAMs contained within
the
chain (30).
/
mice that have been reconstituted
with a transgene encoding the full-length
chain (
-3
ITAM) express surface TCRs that contain a full complement of ITAMs (10 ITAMs per TCR complex), whereas reconstitution of
/
mice with a truncated
chain transgene lacking all three ITAMS (
-0 ITAM) results in surface
expression of TCRs that contain only four ITAMs per
TCR complex (provided by the CD3 chains) (30). Although the TCR levels on DP thymocytes from these
transgenic mice are nearly identical, mean CD5 levels are
significantly lower on the majority of DP thymocytes from
-0 ITAM mice relative to the
-3 ITAM mice (Fig. 4 B).
Thus, the level of TCR surface expression and the intensity
of the TCR signal quantitatively influence CD5 surface expression on DP thymocytes.
The
observation that CD5 is upregulated to high levels on DP
thymocytes in vivo after engagement of the TCR by
ligands that promote either positive or negative selection,
or in vitro by cross-linking of the TCR (references 32, 39-
42; Fig. 4) suggested the possibility that CD5 surface expression might be quantitatively influenced by the affinity
of the TCR for selecting ligand, together with the concentration of selecting ligand (TCR avidity). To address this
question, we analyzed CD5 surface expression on thymocytes from two different MHC class I-restricted (H-Y,
reference 26, and P14, reference 25) and two different
MHC class II-restricted (DO10, reference 28, and AND,
reference 27) TCR transgenic lines, each expressing distinct clonotypic TCRs. Under conditions known to promote positive selection, CD5 levels on DP thymocytes from all of the transgenic mice were higher than those expressed on the majority of DP thymocytes from nontransgenic mice (Fig. 6, A and B). As the shift in CD5 surface
expression from CD5int to CD5hi on the bulk of DP thymocytes in transgenic mice is observed in either positively
or negatively selecting but not in nonselecting backgrounds
(data not shown), this result presumably reflects the fact
that most DP thymocytes in TCR transgenic mice, but
only a small percentage of DP thymocytes from non-TCR
transgenic mice express TCRs that are engaged by selecting
ligand (Fig. 6, A and B). However, gating on transgenic
TCRhi SP thymocytes revealed clear differences in the
level of CD5 expression among the various transgenic lines,
with P14 > H-Y on CD8 SP thymocytes for the MHC
class I-restricted TCRs, and AND > DO10 on the CD4
SP thymocytes for the MHC class II-restricted TCRs (Fig.
6, A and B). Significantly, the relative differences in CD5
surface expression were maintained on peripheral SP T
cells generated by positive selection (CD8+ T cells in the
case of P14 and H-Y TCR transgenic mice, and CD4+ T
cells in the case of AND and DO10 TCR transgenic mice;
Fig. 6, A and B). Since TCR levels on SP T cells from the
class I- or class II-restricted TCR transgenic mice were
similar as assessed by staining with anti-CD3 or anti-
TCR-
antibodies (data not shown), these results suggested that the differences in CD5 levels might reflect differences in the avidity of the positive selecting interaction
(i.e., P14 > H-Y and AND > DO10). Although the natural positively selecting ligands for these TCRs are unknown, their relative avidity can be inferred by the efficiency of positive selection (43). Experimental data support
the idea that within the "window" of TCR-ligand-MHC
interactions that promote positive selection, higher avidity
interactions increase the efficiency of positive selection, resulting in the generation of increased numbers of TCRhi SP
thymocytes (43). In the case of the H-Y and P14 transgenic TCRs, several observations indicate that the efficiency of
positive selection is greater in P14 than in H-Y (female)
TCR transgenic mice (25, 26). For example, both the total
number of transgenic TCRhi thymocytes and the number
of transgenic TCRhiCD8+ thymocytes and T cells in P14
TCR transgenic mice exceeds that observed in H-Y transgenic female mice (Fig. 6 A). These observations suggest,
although by no means prove, that positive selection is mediated by higher avidity TCR-ligand-MHC interactions in
P14 transgenic mice than in H-Y (female) transgenic mice.
Applying similar criteria, positive selection also appears to
be mediated by a higher avidity TCR-ligand-MHC interaction in AND transgenic mice than in DO10 transgenic
mice (Fig. 6 B).
|
To further investigate the possible relationship between
CD5 expression and TCR avidity, we analyzed positive selection in the same transgenic line (DO10) under conditions that have been shown to alter the avidity of the positively selecting TCR-MHC-ligand interaction (44, 45).
Thymocytes with DO10 TCR are positively selected in
both the H-2d and H-2b backgrounds; however, the small
thymus size and reduction in DP thymocytes observed in
DO10, H-2b mice is consistent with the induction of partial clonal deletion as a result of increased TCR-MHC-ligand avidity interactions (44, 45). Significantly, the CD4+
transgenic TCRhi (KJ-126hi) thymocytes and T cells generated in DO10 H-2bxd mice were found to express higher
surface levels of CD5 than do CD4+ KJ-126hi thymocytes
and T cells from DO10 H-2dxd mice (Fig. 7 A). Notably,
the KJ-126hiCD4+ T cells from DO10 H-2bxd and DO10
H-2dxd express similar levels of /
-TCR and CD4 (Fig.
7 A). Thus, the difference in CD5 levels could not be attributed to differences in TCR or CD4 coreceptor surface
expression. In addition, since the identical transgenic TCR
was used in these experiments, the differences observed
could not reflect variation in the timing or onset of transgene expression during thymocyte development. Rather,
these results suggest that the level of CD5 expression on
mature T cells is determined during development in the
thymus by the avidity of the TCR for selecting ligand.
|
A prediction of this hypothesis is that the avidity of the
positively selecting TCR-MHC-ligand interaction may
regulate the level of CD5 surface expression by influencing
the intensity of the TCR signaling response. To examine
this relationship, we analyzed the effect on CD5 expression
produced by lowering the TCR signaling potential from
10 to 4 ITAMs per TCR complex. For these experiments, we chose the P14 TCR transgene because prior experiments had revealed that significant numbers of P14 TCRhi
(V2hi) CD8 SP T cells are generated in both
-3 ITAM
and
-0 ITAM P14-TCR transgenic mice (Fig. 7 B). We
found that lowering the TCR signaling capacity by removal of
chain ITAMs (
-0 ITAM tg versus
-3 ITAM tg) resulted in the generation of V
2hi CD8-SP cells that
express lower levels of CD5 (Fig. 7 B). Taken together,
these results indicate that CD5 surface expression is developmentally regulated in response to the avidity of the TCR for positively selecting ligands.
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Discussion |
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Interest in the role of CD5 in lymphocyte development has grown considerably in light of recent data suggesting that CD5 functions to negatively regulate signaling through the B and T cell antigen receptors (16, 17). In this study, we examined CD5 expression during normal thymocyte development and in mice with defects in thymocyte maturation. Our results demonstrate a critical role for pre-TCR and TCR signals, and notably TCR avidity, in regulating CD5 surface expression.
Early DN thymocytes express very low levels of CD5
independent of their ability to undergo TCR gene rearrangement, and therefore independent of their ability to
express pre-TCR and/or TCR complexes. That the pre-TCR is not required for initial CD5 expression on early
DN thymocytes is not surprising, as previous studies have documented very low levels of CD5 on pro-T cells in the
thymus (46). Of significance, however, is that signals transduced by the pre-TCR upregulate CD5, and that the level
of CD5 surface expression reflects the extent or intensity of
pre-TCR signaling (Fig. 3 B; reference 47). Consistent
with this observation, both p56lck and chain, which appear to act primarily by quantitatively influencing the pre-TCR signaling response (24, 29, 38, 47, 48), are required
for full CD5 induction by the pre-TCR (29, 49; Fig. 3 C).
Although these results suggest a possible role for CD5 at
the DN-DP transition, perhaps through modulation of
pre-TCR signaling threshold, the fact that thymocyte maturation appears unaffected in CD5
/
mice argues that this
function is not critical for T cell development (50).
Interestingly, most DP thymocytes from TCR-/
mice (which can express pre-TCR complexes but are
/
-TCR
) express nearly normal (intermediate) levels of
CD5, whereas CD5 levels on DP thymocytes from MHC
(class I × II)
/
mice (which are
/
-TCRint-hi) express
low levels of CD5. We interpret these observations as indicating that continued signaling, either through the pre-TCR or
/
-TCR, is required to maintain CD5 expression at intermediate levels on DP thymocytes. Thus in
TCR-
/
mice, continued expression of the pre-TCR
provides this signaling function, whereas in MHC (class I × II)
/
mice, replacement of the pre-TCR by TCR complexes that are incapable of generating signals by interacting
with MHC leads to the downregulation of CD5. Results
consistent with our own have also been reported by Dutz
et al., who noted a specific requirement for MHC class I
for the generation of CD5int DP thymocytes in class
I-restricted H-Y TCR transgenic mice (51). The observation that most DP thymocytes from transgenic mice express intermediate levels of CD5 in a nonselecting background
(51) further indicates that extremely low avidity or "noncognate" TCR-MHC interactions are sufficient to maintain intermediate levels of CD5 expression on DP thymocytes.
CD5 is upregulated to high levels on a small subset of
DP thymocytes, before TCR upregulation or CD4 /CD8
lineage commitment as assessed by coreceptor downregulation (32, 39, 51). Data from our own and previous
work indicate that this response is mediated by higher avidity TCR-MHC-ligand interactions than those that are required to maintain intermediate levels of CD5 expression on DP thymocytes (32, 39, 51). In agreement with this
model, CD5hi DP thymocytes are absent both in TCR-/
mice that lack
/
-TCR and in MHC (class I × II)
/
mice that express
/
-TCR but are incapable of engaging
ligand. Conversely, in TCR transgenic mice in which a
defined TCR is expressed on a high percentage of DP thymocytes, a large cohort of CD5hi DP thymocytes is generated under conditions that promote either positive or negative selection but not in a nonselecting background (51).
Thus, only those DP thymocytes that express TCRs that can interact with MHC-ligand with sufficiently high affinity to promote either positive or negative selection upregulate CD5 surface expression to high levels.
Although CD5 upregulation on DP thymocytes upon TCR engagement has been previously shown, the current study reveals that CD5 surface expression on SP thymocytes and T cells differs depending upon the clonotypic TCR that they express. Transgenic TCRhi SP T cells were found to express similar (P14), lower (H-Y, AND, D011.10/H-2d), or higher (D011.10/H-2bxd) levels of CD5 compared with mean CD5 expression on corresponding SP T cell populations from nontransgenic mice (Figs. 6 and 7). Our data also suggest that the level of CD5 expressed on SP thymocytes and T cells generated during positive selection corresponds to the avidity (signal intensity) of the positively selecting TCR-MHC-ligand interaction. When the avidity of the positively selecting TCR-MHC-ligand interaction was varied for the same TCR by changing the MHC haplotype or the signal intensity was varied by removal of TCR ITAMs, the results also supported this hypothesis. Because the antigen specificity of the transgenic TCRs used in this study should result in the generation of "naive" SP thymocytes and T cells that should not encounter their native antigen in vivo, our results also argue that CD5 levels are "fixed" during positive selection on the basis of TCR specificity and these relative levels are subsequently maintained on circulating mature SP T cells independent of engagement of the TCR by antigen. The mechanism by which CD5 expression levels are established during thymocyte development could involve modulation of CD5 in response to TCR signal intensity followed by maintenance of this relative level of expression on mature T cells. Alternatively, it is possible that only those thymocytes that constitutively express the appropriate level of CD5 with respect to their clonotypic TCR are selected to mature to the SP stage. Since these mechanisms are not mutually exclusive, both may also be operative.
Since CD5 acts to attenuate signaling by the TCR (16), the capacity of cells to regulate CD5 surface levels could serve several important functions during development. For example, induction of CD5 on thymocytes and mature T cells after TCR engagement may serve to dampen the TCR signaling response, especially after restimulation. At the DP stage, upregulation of CD5 represents an early response to engagement of the TCR by selecting ligand as it precedes upregulation of the TCR (32, 40, 51). The rapid increase in CD5 levels after initial TCR engagement may act to inhibit subsequent TCR-mediated signaling responses. Increased CD5 expression in response to TCR engagement could also provide an off signal that participates in terminating the activation response. Another role suggested by our data is that CD5 levels may influence the triggering threshold for T cell activation. This function might be particularly important during thymocyte selection when the TCR signaling response dictates the fate of DP thymocytes (i.e., either positive or negative selection). Indeed, analysis of CD5-deficient mice has shown that CD5 does participate in thymocyte selection through its ability to negatively regulate the TCR signaling response (16). Interestingly, the effect of CD5 deletion on thymocyte selection varied among different TCR transgenic mice, resulting in enhanced positive selection in H-Y TCR transgenic mice and a shift from positive selection to partial negative selection in P14 TCR transgenic mice (16).
The ability of thymocytes to regulate CD5 surface expression also suggests that the relative impact of CD5 on TCR signal transduction can be varied in relation to the avidity of the TCR for selecting ligand, providing a mechanism for fine tuning of the TCR signaling response. Surface levels of both CD4 and CD8, which unlike CD5 provide costimulatory signals, also have been shown to vary among individual SP T cells that express distinct TCRs (52). An advantage of using multiple surface structures such as CD4, CD8, and CD5 to influence the overall signaling response delivered to thymocytes and T cells could be to enable a much broader range of TCRs to transduce signals appropriate for positive selection than would otherwise be possible if the affinity of the TCR for selecting ligand alone dictated the outcome. Thus, the potential for TCR signal modulation by CD5 may be particularly useful for generating the maximum possible diversity in the mature T cell repertoire.
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Footnotes |
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Address correspondence to Paul E. Love, Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. Phone: 301-402-4946; Fax: 301-480-3223; E-mail: pel{at}helix.nih.gov
Received for publication 30 June 1998 and in revised form 2 October 1998.
We thank Drs. Fred Alt, Rudolph Jaenisch, Laurie Glimcher, Susumo Tonegawa, Dennis Loh, Tak Mak, Roger Perlmutter, Harald von Boehmer, Rolf Zinkernagel, and Steve Hedrick for making mice available for these experiments. We are also grateful to B.J. Fowlkes and C.L. Sommers for reading the manuscript and for helpful discussions.
Abbreviations used in this paper
DN, CD4CD8
double negative;
DP, CD4+CD8+ double positive;
SP, CD4+ or CD8+ single positive;
FCM, multicolor flow cytometry;
ITAM, immunoreceptor tyrosine-based activation motif.
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