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*
Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and
Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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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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Surface receptors other than the TCR also participate in thymocyte
selection by directly or indirectly influencing the TCR signaling
response. CD4 and CD8, which also bind to MHC, potentiate TCR signaling
by increasing the avidity of TCR/MHC/ligand interaction and by
recruitment of the Src family protein tyrosine kinase, Lck, to the
proximity of the TCR complex (4, 5). CD45 also functions
to augment TCR signaling by positively regulating the activity of both
Lck and Fyn (4, 5). CD5, a monomeric cell surface
glycoprotein expressed on thymocytes, T cells, and a subset of B cells,
has been shown to negatively regulate signaling through both the B cell
and T cell Ag receptors (6, 7). In the absence of CD5,
peritoneal B-1 cells that normally are triggered to undergo apoptosis
in response to IgM cross-linking develop resistance to apoptosis
and enter the cell cycle (7). Moreover, thymocytes from
CD5-/- mice are hyperresponsive to stimulation
through the TCR (6). The cytoplasmic domain of CD5
contains four potential tyrosine phosphorylation sites, including an
imperfect immunoreceptor tyrosine-based activation motif
(ITAM),2 an
immunoreceptor tyrosine-based inhibition motif (ITIM), and multiple
potential Ser/Thr phosphorylation sites (8, 9, 10). The
precise mechanism by which CD5 inhibits TCR signaling has not been
elucidated. However, CD5 is tyrosine-phosphorylated after TCR
engagement and associates with several effector molecules that could
potentially influence the TCR signaling response, including Cbl,
rasGAP, SHP-1, casein kinase 2, and TCR-
/ZAP-70
(11, 12, 13, 14, 15).
The function of CD5 in development, particularly during thymocyte
selection, remains unclear. CD5-/- mice exhibit
no obvious defects in T cell development (16). However,
when cohorts of thymocytes expressing a defined (transgenic) 
TCR
were analyzed, thymocyte selection was found to be dramatically altered
in mice lacking CD5 (6, 17). In positively selecting MHC
backgrounds, CD4+CD8+
(double-positive, or DP) thymocyte numbers are reduced in
TCR-transgenic (tg)/CD5-/- mice relative to
TCR-tg; CD5+/+ mice suggesting a shift in cell
fate from positive selection toward negative selection (6, 17). However, the impact of CD5 deficiency on positive
selection varies depending on the specificity of the TCR transgene. For
example, in P14 TCR-tg/CD5-/- mice and
(Abd) DO10.10
(DO10)TCR-tg/CD5-/- mice, few thymocytes
express high levels of the clonotypic TCR, and few single-positive (SP)
clonotype-TCRhigh cells are present in the
periphery, suggesting that in the absence of CD5, most
clonotype-TCRhigh thymocytes undergo negative
selection (6, 17). In contrast, large numbers of
clonotype-TCRhigh thymocytes and SP T cells are
generated in H-Y TCR-tg/CD5-/- and
(Ad) DO10 TCR-tg/CD5-/-
mice, indicating that thymocytes expressing these TCRs can still be
positively selected in the absence of CD5 (6, 17).
A possible explanation for the variable effects of CD5 deletion on thymocyte selection is suggested by the recent observation that CD5 surface expression is regulated by the intensity of the TCR signal and by the avidity of the TCR/ligand interaction during selection (18). Relatively high-avidity positively selecting interactions result in high surface expression of CD5 on DP and SP thymocytes, whereas lower avidity interactions induce lower surface expression of CD5 (18). Consequently, the impact of CD5 deletion (or CD5 overexpression) on thymocyte selection might be predicted to be different depending on the avidity of the TCR for its selecting ligand.
To test this idea, we generated CD5-tg mice and then compared the effect of CD5 overexpression or CD5 deletion on thymocyte selection in different TCR-tg backgrounds. Significantly, the effect on thymocyte selection of altering CD5 surface levels critically depended on the avidity of the TCR for its selecting ligand. Moreover, the cytoplasmic domain of CD5 was required for its inhibitory function, as substitution of endogenous CD5 with a transgene encoding a mutant form of the protein lacking the imperfect ITAM and distal sequences failed to rescue the CD5-/- phenotype. Together, these results demonstrate that regulation of CD5 surface expression by TCR signaling serves as a mechanism for fine tuning the TCR signaling response during thymocyte selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human (hu) CD2-CD5 transgene (CD5-tg) was generated by
substituting murine CD5 coding sequences for the cDNA sequences in
construct -CT108 (19). The huCD2-mutant CD5-tg
(mCD5-tg) was generated in a similar fashion, except that the murine
CD5 coding sequence was first mutated in vitro with a synthetic primer:
5'-CAT-GTG-GAC-AAT-*TAA-TAC-AGC-CAG-CC-3'
that substitutes a stop codon (TAA) for Glu428
(GAA; see Fig. 5A
). The CD5 and mCD5 cDNAs were sequenced before
insertion into the hCD2 transgenic cassette. Four CD5-tg founder lines
and four mCD5-tg founder lines were generated by zygote injection. CD5
expression was quantitated by Northern blotting, Western blotting, and
flow cytometric analysis. CD5-tg and mCD5-tg founder lines that
expressed similar levels of surface protein that fell within the
high-normal range (see Figs. 1 and 6) were selected for use in the
current experiments. CD5-/- mice
(16) were obtained from Dr. Ron Schwartz, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health (Bethesda, MD). TCR-tg mice used in these studies included the
MHC class I-restricted TCRs P14 (20) and H-Y
(21) and the MHC class II-restricted TCRs AND
(22) and DO10 (23) Mice were maintained in
the H-2Db background by mating with C57BL/6
partners and were moved into or maintained in the
H-2Dd background by mating with B10.D2
partners.
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mAbs used for flow cytometric analysis were purchased from BD
PharMingen (San Diego, CA), unless noted otherwise, and included:
FITC-, PE-, or CyChrome-conjugated anti-CD4 (H129.19),
anti-TCR(H57-597), anti-CD8 (53-6.7), anti-CD3
(145-2C11), anti-CD5 (53-7.3), anti-V11 (RR8-1), and
anti-V2 (B20.1). Unconjugated anti-CD16/CD32 (2.4G2) was
used to block nonspecific Fc receptor binding. 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. In some experiments, Quantum
red-cÓonjugated anti-CD4 (H129.19) or anti-CD8 (53-6.7)
(Sigma, St. Louis, MO) were used for flow cytometry. Abs for
immunoprecipitation and Western blotting included anti-CD5 (53-7.3)
(BD PharMingen), Q-20 (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-phosphotyrosine-HRP (4G10) (Upstate Biotechnology, Lake
Placid, NY), and polyclonal anti-goat-HRP (Santa Cruz
Biotechnology).
Flow cytometric analysis and measurement of calcium flux
Thymi and lymph nodes were excised from mice and single-cell suspensions were prepared in FACS buffer (HBSS (Life Technologies, Rockville, MD) plus 0.1% BSA). For multicolor flow cytometry, thymocytes or lymph node cells first were incubated with unlabeled Ab to the Fc receptor (mAb 2.4G2) to prevent nonspecific binding of Abs. Background staining was measured with fluorochrome-conjugated rat IgG2a (BD PharMingen) and designated as control. For two- and three-color multicolor flow cytometry, cells were incubated with fluorochrome-conjugated Abs for 1 h at 4°C, washed, and resuspended in FACS buffer. Analysis was performed on a Becton Dickinson Immunocytometry Systems (Mountain View, CA) FACScan with standard CellQuest software. Data were collected on 1 x 104 viable cells as determined by forward and side light scatter. Calcium flux measurements were performed as described (24).
Western blot analysis
Thymocytes were enumerated, washed twice in ice-cold PBS, and resuspended in PBS at a concentration of 1 x 108/ml. Thymocyte stimulations, immunoprecipitations, Western blotting, and PAGE were performed as described (24, 25). Briefly, thymocytes were incubated for 5 min in medium lacking (-) or containing (+) pervanadate (1 mM; Sigma). Thymocyte lysates were prepared and incubated at 4°C for 1 h in the presence of protein G beads plus anti-CD5 (53-7.3). Beads were washed and eluted proteins were resolved by 10% SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membranes and blotted with antiphosphotyrosine-HRP and detected by ECL (Amersham, Arlington Heights, IL). For detection of CD5, blots were stripped and reprobed with polyclonal goat anti-mouse CD5 followed by polyclonal anti-goat-HRP (Santa Cruz Biotechnology).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we used two MHC class I-restricted TCR-tg lines
(H-Y and P14) and two MHC class II-restricted TCR-tg lines (DO10 and
AND) to test the effect on thymocyte selection of altering the level of
CD5 surface expression. In a previous report (Ref. 18 ;
also shown in Fig. 1
A), we
observed that mean CD5 surface expression was higher on DP thymocytes
and CD8-SP T cells from P14 TCR-tg mice than on equivalent populations
of cells from H-Y TCR-tg mice. Likewise, CD5 surface levels were higher
on DP thymocytes and CD4-SP T cells from AND TCR-tg mice than on
similar cells from (Ad) DO10 TCR-tg mice (Ref.
18 and Fig. 1A). Significantly, these
differences in CD5 surface expression correlated with the presumed
affinity/avidity of the individual TCRs for their positively selecting
ligands in the thymus (i.e., P14 > H-Y and AND >
(Ad) DO10; Ref. 18).
To examine the effect of loss of CD5 surface expression on thymocyte
selection, each of the TCR transgenes was mated into the
CD5-/- background (16).
Alternatively, CD5 surface expression was augmented by generating
transgenic mice that express CD5 under the control of the huCD2
promoter/enhancer and then mating the CD5 transgene into the different
TCR-tg backgrounds. For these studies, a transgenic founder line was
selected that increased CD5 expression on thymocytes to levels within
the high-normal range observed on total thymocytes from non-tg mice
(Fig. 1B). As previously noted for
non-TCR-tg/CD5-/- mice (16), no
obvious alterations in T cell development were detected in
non-TCR-tg/CD5-tg mice (Fig. 1B and data not shown).
Results from representative experiments performed with MHC class
I-restricted TCR transgenes are shown in Fig. 2. The effect of the CD5 transgene on
thymocyte selection was different in mice expressing the H-Y TCR or the
P14 TCR transgenes (Fig. 2). Augmentation of CD5 surface expression
markedly inhibited positive selection in H-Y TCR-tg mice as assessed by
the selective reduction of clonotype-TCRhigh
(T3.70high) thymocytes and
T3.70high CD8-SP T cells in H-Y TCR-tg/CD5-tg
mice (Fig. 2A). In contrast, applying the same criteria,
positive selection was only slightly impaired in P14 TCR-tg/CD5-tg mice
(Fig. 2B). In this respect, it is important to note that
although introduction of the CD5 transgene increased the level of CD5
surface expression by the same increment in both H-Y and P14 TCR-tg
mice, the level of baseline CD5 expression (i.e., in non-CD5-tg mice)
was 
50% greater on thymocytes from P14 TCR-tg mice than on
thymocytes from H-Y TCR-tg mice (Ref. 18 and Figs. 1A and 2).
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, total thymocyte numbers are reduced 2- to
3-fold in both H-Y TCR-tg/CD5-/- and P14
TCR-tg/CD5-/- mice. Moreover, the reduction in
thymocyte cellularity occurs predominantly in the DP compartment,
suggesting that in both cases, there is a shift from positive toward
negative selection in the absence of CD5 (6). However, the
number of T3.70high thymocytes and
T3.70high CD8-SP T cells was not reduced in H-Y
TCR-tg/CD5-/- mice relative to H-Y
TCR-tg/CD5+/+ mice (Fig. 2A). In
contrast, clonotype-TCRhigh
(V2high) thymocytes were nearly absent in P14
TCR-tg/CD5-/- mice and few
V2high CD8-SP T cells were present in the
peripheral lymphoid organs of these mice (Ref. 6 and Fig. 2B). Thus, the relative impact of CD5 deletion was much more
severe in P14 TCR-tg mice than in H-Y TCR-tg mice, resulting in marked
reduction in the number of clonotype-TCRhigh
thymocytes and CD8-SP T cells in the former but not the latter.
To determine whether the effect on thymocyte selection of altering CD5
expression also varied according to TCR specificity in mice expressing
MHC class II-restricted TCRs, we next examined
(Ad) DO10 TCR-tg and AND TCR-tg mice (Fig. 3). Previous data argue that the avidity
of the positively selecting interaction is greater for AND TCR than for
(Ad) DO10 TCR (18). Significantly,
the results obtained with DO10 TCR-tg mice were similar to those
observed with H-Y TCR-tg mice. Positive selection was inhibited in
(Ad) DO10 TCR-tg/CD5-tg mice, as assessed by the
selective reduction in clonotype-TCRhigh
(KJ126high) CD4-SP thymocytes and peripheral T
cells (Fig. 3A). In contrast, the results obtained with AND
TCR-tg mice were similar to those obtained with P14 TCR-tg mice in that
the CD5 transgene had minimal effect on the generation of clonotype
TCRhigh (V11high)
thymocytes and V11high CD4-SP T cells (Fig. 3B). Again, it is important to note that although
introduction of the CD5 transgene increased CD5 surface levels by
roughly the same increment in both cases, mean CD5 surface expression
was 50% greater on thymocytes from AND
TCR-tg/CD5+/+ mice than on thymocytes from
(Ad) DO10 TCR-tg/CD5+/+mice
(Ref. 18 and Figs. 1 and 3). As in H-Y TCR-tg mice (Fig. 2A), loss of CD5 expression in (Ad)
DO10 TCR-tg mice led to a reduction in DP thymocytes, but did not lead
to a reduction in the number of clonotype-TCRhigh
(KJ126high) CD4-SP thymocytes or peripheral
KJ126high CD4-SP T cells (Fig. 3A).
However, in AND TCR-tg/CD5-/- mice, DP
thyomocytes and V11high thymocytes and T cells
also were markedly reduced (Fig. 3B).
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B), events that have been
associated with strongly activating signals (26). In
contrast, the presence of normal numbers of
clonotype-TCRhigh thymocytes and T cells in H-Y
TCR-tg/CD5-/- and (Ad)
DO10 TCR-tg/CD5-/- mice demonstrates that loss
of CD5 does not result in deletion of most thymocytes that express
putative low-avidity TCRs. Conversely, forced overexpression of CD5
markedly attenuated positive selection of thymocytes expressing
putative low-avidity TCRs (H-Y and (Ad) DO10) but
did not impair positive selection of thymocytes expressing putative
high-avidity TCRs (P14 and AND).
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).
Significantly, CD5 surface expression is increased nearly 2-fold on
thymocytes from (Abd) DO10 TCR-tg mice relative
to (Ad) DO10 TCR-tg mice (Ref. 18
and Figs. 1 and 4). Accordingly, the effects of CD5 deletion or
overexpression on selection of thymocytes in
(Abd) DO10 TCR-tg mice were found to more closely
resemble those obtained with the putative higher-avidity TCRs (AND and
P14) than those observed for the lower-avidity TCRs
((Ad) DO10 and H-Y; Fig. 4). Specifically,
deletion of CD5 in (Abd) DO10 TCR-tg mice
resulted in a reduction in DP thymocyte numbers and loss of
KJ126high thymocytes and
KJ126high CD4-SP T cells (Fig. 4). In contrast,
expression of the CD5 transgene did not impair, and in fact appeared to
slightly improve, positive selection in (Abd)
D010 TCR-tg mice as assessed by the increase in
KJ126high thymocytes and
KJ126high CD4-SP T cells in
(Abd) DO10 TCR-tg/CD5-tg mice relative to
(Abd) DO10 TCR-tg mice (Fig. 4). The cytoplasmic domain of CD5 is required for negative regulation of TCR signaling during selection
To determine whether signal transduction by CD5 is required for
its inhibitory activity in thymocytes, we generated a transgene that
expresses a mutant form of the protein (mCD5) lacking a large portion
of the cytoplasmic tail, including the imperfect
ITAM(Tyr429-Tyr441)
sequences (Fig. 5A). The
wild-type and mCD5 transgenes then were used to reconstitute CD5
surface expression in CD5-/- mice by mating.
Analysis of CD5 expression in thymocytes from in
CD5-/-/CD5-tg and
CD5-/-/mCD5-tg mice revealed
proteins of the predicted mobilities (67 and 62 kDa for CD5 and mCD5,
respectively; Fig. 5B). Moreover, although wild-type CD5 was
tyrosine phosphorylated after stimulation with pervanadate, tyrosine
phosphorylation of mCD5 was undetectable (Fig. 5B).
Thymocyte development appeared grossly normal in non-TCR-tg mice where
the mCD5 transgene was substituted for endogenous CD5 (Fig. 6). Moreover, thymocytes from
CD5-/-/CD5-tg and
CD5-/-/mCD5-tg mice expressed surface levels of
CD5 that were within the high-normal range (Fig. 6). The narrow single
peak of CD5 expression in CD5-/-/CD5-tg and
CD5-/-/mCD5-tg mice indicated further that the
relatively broad range of CD5 surface expression on thymocytes from
CD5+/+ mice is attributable to regulation by
endogenous CD5 regulatory sequences (Fig. 6). CD5 surface expression on
all peripheral T cells (both CD4-SP and CD8-SP) from
CD5-/-/CD5-tg and
CD5-/-/mCD5-tg mice was similar to the levels
normally expressed on CD8-SP T cells from CD5+/+
mice (i.e., 2-fold lower than normally present on the majority of
CD4-SP T cells).
Thymocytes from CD5-/- mice have been shown to
be hyperresponsive to TCR stimulation relative to
CD5+/+ mice as assessed by several criteria,
including the TCR-mediated calcium mobilization response
(6). To assess whether the cytoplasmic tail of CD5 is
required for its inhibitory activity, we compared the TCR-mediated
calcium responses elicited by thymocytes from
CD5-/- mice and CD5-/-
mice reconstituted with either the CD5 or mCD5 transgenes. DP
thymocytes from CD5-/- and
CD5-/-/mCD5-tg mice were equally
(hyper)-responsive to TCR cross-linking, indicating that the sequences
deleted from the mCD5 protein are required for TCR signal inhibition
(Fig. 7). Interestingly, neither CD4-SP
thymocytes (Fig. 7) nor CD8-SP thymocytes or peripheral T cells (data
not shown) from CD5-/- and
CD5-/-/mCD5-tg mice were hyperresponsive to TCR
cross-linking, indicating that DP thymocytes are selectively sensitive
to the inhibitory effects of CD5. To determine whether the mCD5 protein
also was functionally inert with respect to its effect on thymocyte
selection, we introduced the H-Y and (Ad) DO10
TCR transgenes into CD5-/-/CD5-tg and
CD5-/-/mCD5-tg mice. In both TCR-tg
backgrounds, we observed that the phenotype of
CD5-/-/mCD5-tg mice was nearly identical with
that of CD5-/-mice, whereas the phenotype of
CD5-/-/CD5-tg mice more closely resembled
CD5+/+ or CD5-tg mice (Fig. 8) confirming that the CD5 cytoplasmic
domain is required for its inhibitory activity during thymocyte
selection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a recent study, we reported that the level of CD5 surface expression on developing thymocytes and mature T cells parallels the avidity of the positively selecting TCR/MHC-ligand interaction (18). In view of these findings, we proposed that CD5 surface expression is titrated by the strength of the TCR signal (18). We also speculated that the requirement for CD5-mediated signal inhibition during thymocyte selection increases when the avidity of the positively selecting TCR/ligand interaction (and the TCR signal strength) is relatively high. Conversely, the requirement for CD5-mediated signal inhibition decreases when the avidity of the positively selecting TCR/ligand interaction (and the TCR signal strength) is relatively low. This model predicts that the impact on thymocyte selection of either CD5 deletion or CD5 overexpression will depend on the avidity of the selecting interaction, which in turn is reflected by the level of endogenous CD5 surface expression.
To test this model, we generated CD5-tg mice and examined the effect of
augmented CD5 expression or CD5 deletion (using
CD5-/- mice) on the selection of thymocytes
that express the identical (transgenic) TCR. The results of these
experiments (summarized in Fig. 9) were
entirely consistent with the proposed model for CD5 function. In TCR-tg
systems where the avidity of the selecting interaction is thought to be
relatively high (P14, AND and (Abd) DO10) and,
consequently, where the level of endogenous CD5 surface expression also
was relatively high (Fig. 1A), loss of CD5 converted
positive selection to strong negative selection as assessed by the
reduction in DP thymocytes, the reduction in mature
clonotype-TCRhigh thymocytes and T cells, and the
down-modulation of the TCR on DP thymocytes (
Figs. 2–4). In contrast,
in TCR-tg systems where the avidity of the positively selecting
interaction is thought to be relatively low (H-Y and
(Ad) DO10) and the level of endogenous CD5
surface expression is lower (Fig. 1A), deletion of CD5 did
not fully convert positive selection to negative selection, as normal
numbers of clonotype TCR+ SP thymocytes and T
cells are still generated in these mice (Figs. 2A and
3A). The reduction in DP thymocytes in H-Y
TCR-tg/CD5-/- and (Ad)
DO10 TCR-tg/CD5-/- mice suggests that even in
these cases, large numbers of thymocytes are deleted in the absence of
CD5. The reason why some thymocytes are deleted and others escape
negative selection and are positively selected in H-Y
TCR-tg/CD5-/- and (Ad)
DO10 TCR-tg/CD5-/- mice remains unclear. This
could be attributed to other compensating factors similar to CD5 that
function to adjust TCR signal intensity or to variable expression of
signaling molecules in the pool of developing thymocytes, making some
cells more or less susceptible to negative selection. These
compensatory mechanisms are presumably sufficient to partially offset
the loss of CD5 if the thymocytes express relatively low-avidity TCRs
but not if they express high-avidity TCRs and are therefore closer to
the signaling threshold for negative selection.
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B
and 3B). The selective decrease in
clonotype-TCRhigh SP thymocytes and T cells but
relatively unchanged total thymus cellularity in H-Y TCR-tg/CD5-tg and
(Ad) DO10 TCR-tg/CD5-tg mice suggests that
increased expression of CD5 converted positive selection to
"nonselection" in these mice (Fig. 9). It is important to note that
the distinct effects of the CD5 transgene on positive selection in H-Y
TCR-tg vs P14 TCR-tg mice or AND TCR-tg vs (Ad)
DO10 TCR-tg mice cannot be attributed to variable CD5 transgene
expression, as surface expression of CD5 on thymocytes was increased by
the same increment (Figs. 2 and 3). Thus, thymocytes that normally
express low levels of CD5 are much more susceptible to a forced
increase in CD5 surface expression than are thymocytes that normally
express relatively high levels of CD5. This could indicate that the
inhibitory effect of CD5 is already at or near maximal in P14 TCR-tg
and AND TCR-tg mice such that the increase in CD5 surface expression
has little or no additional effect on TCR signaling. Alternatively, the
CD5 transgene could attenuate TCR signaling to the same degree in all
TCR-tg/CD5-tg mice; however, the decrease in TCR signal intensity is
sufficient to convert positive selection to nonselection in the case of
thymocytes expressing lower-avidity TCRs (H-Y and
(Ad) DO10 TCR-tg mice), whereas a similar
decrease in TCR signal intensity is not sufficient to alter cell fate
in the case of cells expressing higher-avidity TCRs (P14 and AND TCR-tg
mice; Fig. 9).
A difficulty that arises when interpreting these and other studies
concerning thymocyte selection is that the relative affinities of the
transgenic TCRs for their selecting ligands and the concentration of
these ligands in the thymus are currently unknown. In the case of the
MHC class I-restricted TCRs, several observations lead us to infer that
the relative avidity of the positively selecting interaction is
P14-TCR > H-Y-TCR. First, the "efficiency" of positive
selection, reflected by the percentage of CD8-SP T cells that express
high levels of the clonotopic TCR, and the ratio of CD8 to CD4 SP T
cells in the periphery is greater in P14-TCR-tg mice than in H-Y-TCR-tg
mice (Fig. 2). Second, reduction of the TCR signaling potential by
substitution of endogenous -chain (which contains three ITAMs) with
a signaling-deficient protein (-0 ITAM) markedly inhibits positive
selection in H-Y TCR-tg mice but not in P14 TCR-tg mice
(29, 30, 31). The relative avidities of the positively
selecting interactions in the MHC class II-restricted TCR-tg systems
are more difficult to discern with the first set of criteria. However,
the results of experiments similar to those described above in which
signaling deficient -chain variant transgenes were substituted for
endogenous suggest that AND > (Ad)
DO10, as positive selection is less dependent on the presence of
-chain ITAMs in AND TCR-tg mice (29, 31). We also
compared the effect of CD5 deletion or introduction of the CD5
transgene on selection of thymocytes that express the same MHC class
II-restricted transgenic TCR (DO10) under conditions that have been
shown to alter the avidity of the positively selecting TCR/MHC-ligand
interaction. When the effects of CD5 deletion or forced CD5
overexpression on thymocyte selection were examined in
(Abd) DO10 TCR-tg mice, the outcome was
consistent with that predicted for a relatively high-avidity
selecting interaction (i.e., resembling the results obtained with P14
TCR-tg and AND TCR-tg mice rather than (Ad) DO10
TCR-tg or H-Y TCR-tg mice;
Figs. 2–4).
To determine whether the CD5 cytoplasmic domain (and by
extension, CD5-mediated signaling) is required for its inhibitory
effect, we generated transgenic mice that express a mutant form of CD5
lacking most of the cytoplasmic tail (mCD5-tg). The mCD5 protein was
expressed on the cell surface but, unlike the intact CD5 transgene,
failed to reconstitute inhibitory function in
CD5-/- mice. These results demonstrate that the
sequences deleted in mCD5 (which include the imperfect ITAM and the
distal tyrosine residue, Y463) are required for
its inhibitory activity in thymocytes. Deletion mutants structurally
similar to mCD5 also were found to lack inhibitory activity when
expressed in mature T cells, T cell hybridomas, or B lymphoma cell
lines (17, 32, 33). However, results reported in another
study performed with T lymphoma lines indicate that
Y378 is required for CD5 inhibitory activity
(34). Y378 is contained
within a membrane-proximal sequence that matches the consensus for an
ITIM (10), and this motif has been shown to bind to the
protein tyrosine phosphatase SHP-1 (14, 34). Our results
do not rule out a role for the putative ITIM in mediating CD5
inhibition, as the deletion in mCD5 could render the ITIM sequences
nonfunctional, perhaps because of suboptimal tyrosine phosphorylation
(Fig. 5). Thus, the precise mechanism of CD5 inhibition of TCR
signaling in vivo remains to be established. Comparison of the proteins
that associate with CD5 and mCD5 before and after activation should
assist in identifying the signaling components responsible for CD5
inhibitory function in thymocytes.
Our results indicate that inhibition of TCR signaling by CD5 does not
require its coligation with the TCR (Fig. 7), suggesting that CD5 may
spontaneously coaggregate with the TCR after TCR cross-linking. The
present data also reveal that DP thymocytes are especially sensitive to
the inhibitory effects of CD5 relative to mature SP thymocytes, even
when SP thymocytes are suboptimally stimulated by TCR cross-linking
(Fig. 7). Indeed, an inhibitory effect of CD5 has been difficult to
demonstrate in mature T cells (6, 34, 35, 36, 37). However, SP T
cells are not refractory to CD5 inhibition, as transfection of
full-length CD5 into mature T cells from CD5-/-
mice inhibits Ag-induced IL-2 production (17). These
results are not necessarily contradictory. Preselected thymocytes might
be especially sensitive to fluctuations in CD5 surface expression.
Because these cells have not been subjected to the process of thymocyte
selection, their TCR signaling response directly reflects the input of
the TCR plus costimulatory and inhibitory molecules. In contrast,
postselected cells (SP thymocytes or peripheral T cells) presumably
survive selection only if the integrated signal delivered by the TCR
plus costimulatory and inhibitory molecules is appropriate to allow
them to mature to the SP stage. Thus, in CD5-/-
mice, only those thymocytes that can generate the appropriate signaling
response for positive selection in the absence of CD5 emerge as SP T
cells.
A potential role for negative regulators of TCR signaling such as CD5 could be to influence the repertoire of TCRs that undergo positive selection in the thymus. The constitutive low-level surface expression of CD5 on all DP thymocytes could function to prevent thymocytes expressing TCRs that fail to bind to self-MHC plus self-ligand or do so with extremely low affinity from inadvertently receiving survival signals through nonspecific cell surface interactions. Most T cells expressing these TCRs would presumably not be activated by foreign Ag presented by self-MHC and would therefore not be useful to the mature T cell repertoire. In contrast, the inducible regulation of CD5 surface expression in response to TCR signal strength could enable thymocytes that express TCRs that bind with relatively high affinity to self-MHC plus self-ligand to transduce signals in the range appropriate for positive selection. As shown here and in previous studies (6, 17), thymocytes expressing these TCRs undergo negative selection in the absence of CD5, presumably because the intensity of the signals transduced by these TCRs is too strong for positive selection. Biasing the repertoire toward relatively high-avidity TCRs may be important for mature T cell survival and/or subsequent activation by Ag. Thus, "fine tuning" of TCR signaling by CD5 may help to optimize the repertoire of TCRs expressed on mature T cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine based inhibition motif; DP, double positive (CD4+CD8+); SP, single positive (CD4+CD8- or CD4-CD8+); tg, transgenic; DO10, DO10.10; hu, human; m, mutant.
Received for publication November 22, 2000. Accepted for publication February 23, 2001.
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molecules in human CD3+ thymocytes. J. Immunol. 159:3739.[Abstract]
and 
transgenes on early / T cell development. J. Exp. Med. 179:1485. chain signaling motifs. J. Immunol. 160:163. chain signaling motifs in selection of the T cell repertoire. J. Exp. Med. 185:893. ITAMs does not impair T cell receptor signaling. Immunity. 10:409.[Medline]
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,
CD5+/+; 
, CD5-/-; 
,
CD5-/-/CD5-tg; •, CD5-/-/mCD5-tg.
