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-Independent CD28 Signaling and Costimulation Require Non-CD4-Associated Lck1
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Department of Immunology, University of Toronto, and Arthritis and Immune Disorder Research Centre, Toronto, Ontario, Canada;
McGill Cancer Centre, Departments of Biochemistry, Medicine, and Oncology, McGill University, Montreal, Quebec, Canada;
Oncology Gene Therapy Program, The Toronto Hospital, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and
Ontario Cancer Institute, Toronto, Ontario, 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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The mechanisms underlying CD28 signal transduction remain controversial. Two lines of evidence support a role for src family protein tyrosine kinases (PTKs)5 in CD28 signaling. Both p59fyn and p56lck have been reported to associate with CD28 upon its aggregation (11, 12, 13). Furthermore, aggregation of CD28 on the Lck- cell line, JCAM1, failed to induce an increase in the phosphotyrosyl content of cytosolic proteins, including that of CD28, until it was reconstituted with exogenous Lck (14). However, both of these lines of evidence are contentious. A number of reports demonstrate an absence of a stable association between CD28 and Lck, and fail to detect reproducible changes in the tyrosine phosphorylation and/or kinase activity of Lck following mAb-mediated CD28 aggregation (15, 16). In addition, CD28 aggregation on JCAM1 has been demonstrated to induce levels of IL-2 comparable with those observed in Lck-sufficient Jurkat cells (17). Furthermore, these analyses do not correspond well with those analyzing CD28-mediated costimulation, as assays used to assess its physiological consequences do not involve the deliberate coaggregation of CD28 with either CD4/Lck complexes or TCR/CD3, which may provide a source of Fyn (18). Thus, invoking a role for src family PTK in CD28-mediated costimulation is conceptually problematic.
Toward addressing this issue, we have taken advantage of the recent demonstrations that mAb-mediated CD28 aggregation on primary resting T cells from either rat or human results in IL-2 production and ensuing cellular growth (13, 19, 20). This treatment was shown to correlate with increased CD28-associated kinase activity and an increase in the phosphotyrosyl content of cellular substrates, including that of CD28 itself (11, 14, 21, 22). In the present study, these observations are extended to a series of murine T cell clonal variants that are particularly amenable to analyses of Lck-dependent signaling processes (23, 24).
Specifically, a CD4-, but not a
CD4+ T cell variant is shown to respond to CD28
aggregation with both early and late indicators of cell activation.
Forced expression of wild-type CD4 (WT CD4), but not A418/A420 CD4 (DC
CD4), unable to associate with Lck, in the CD4-
variant ablated CD28 responsiveness, thus implicating the requirement
for the non-CD4-associated pool of Lck in the signaling process. The
obligate role of this pool of Lck was formally demonstrated by
generating variants of the CD4+ clone in which
either S20/S23 Lck (DC Lck), unable to associate with CD4, or wild-type
Lck (WT Lck) was overexpressed. Despite achieving comparable levels of
expression, only the DC Lck variant responded to
TCR
-independent CD28 aggregation, and further, responded to
CD28-mediated costimulation more robustly than WT Lck variants. The
results support the conclusion that CD28 signaling utilizes
non-CD4-associated Lck exclusively, and that in physiological
circumstances of T cell activation, it is this pool that functionally
limits essential Lck-dependent signals emanating from CD28.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old C57BL/6 male mice were obtained from the National Cancer Institute animal facility. CD28-/- C57BL/6 mice (25) were a kind gift from Dr. T. Mak (Amgen Institute, Toronto, Canada).
Antibodies
Polyclonal rabbit anti-murine Lck was prepared as described
(26), and rabbit anti-murine CD28 was generated by
immunization with peptide 201–218, conjugated to keyhole limpet
hemocyanin. Murine-specific mAbs for CD28, 37.51 (27),
TCRC, H57.597 (28), and rabbit anti-CD28 were
purified on protein A-conjugated Sepharose 4B (Pharmacia, Piscataway,
NJ). The murine IL-2-specific mAb S4B6.34.1 (29), and
CD4-specific mAb H129 (30) were purified on mouse
anti-rat Ig
-conjugated Sepharose 4B (Pharmacia).
Phosphotyrosine-specific mAb, 4G10 (31), was purchased
from Upstate Biotechnology (Lake Placid, NY). Normal hamster IgG,
rabbit anti-hamster IgG, FITC F(ab')2 goat
anti-hamster IgG, and biotinylated F(ab')2
goat anti-hamster IgG were purchased from Bio/Can Scientific
(Mississauga, Ontario, Canada). PE-conjugated anti-CD4 and
streptavidin were purchased from Cedarlane (Hornby, Ontario, Canada)
and Southern Biotechnology Associates (Birmingham, AL), respectively.
Goat anti-mouse IgG-HRP and anti-actin were purchased from Sigma,
and protein A-HRP was purchased from ICN Pharmaceuticals (Costa
Mesa, CA).
Primary T cells, T cell clones, and cell lines
The CD4- and CD4+ T cell clones, as well as wild-type CD4 and A418/A420 (DC)-mutated CD4 infectants of the CD4- variant have been described previously (23). Clonal variants were maintained in serum-free IMDM containing 3 U/ml rIL-2 supernatant (32) and 1% soybean lecithin supplemented with 600 µg/ml active G418 (Life Technologies, Gaithersburg, MD). The packaging cell lines BOSC (33) and GP+E (34) were propagated in IMDM supplemented with 5% FCS. The IL-2-dependent CTLL-2 cell line (35) was propagated in serum-free IMDM supplemented with 20 U/ml rIL-2. Purification of primary CD4+ lymph node (LN) T cells was as described previously (26).
CD28-mediated proliferation assays
One of two protocols was employed. Soluble mAb was added at
various concentrations to cultures containing 3 x
104 cloned T cells and 5 x
105 irradiated (2000 rad) syngeneic splenocytes.
Alternatively, wells were coated directly or indirectly with varying
concentrations of CD28-specific mAb for 1 h at 37°C. In the
latter case, wells were precoated with 50 µg/ml of anti-hamster
IgG for 1 h, washed, blocked with a 5% solution of BSA
(Boehringer Mannheim, Indianapolis, IN) for 1 h, and then washed
again. Following the addition of anti-CD28, wells were washed
again. For costimulation assays, both TCRC- and CD28-specific mAbs
were indirectly coimmobilized at the indicated concentrations. In all
assays, 3 x 104 cloned T cells or 5 x
104 primary LN T cells were added to each well,
respectively. Cultures were pulsed with 1 µCi
[3H]TdR (Amersham, Arlington Heights, IL) at
20–40 h and harvested onto Unifilter plates (Canberra Packard,
Mississauga, Ontario, Canada) 6 h later. Thymidine uptake was
assessed by liquid scintillation spectroscopy.
IL-2 assay
Twenty-hour supernatants from cultures containing 3 x 104 cloned T cells stimulated with 1 µg/ml soluble anti-CD28, or not, were diluted 10-fold and incubated with 2 µg of IL-2-specific mAb (S4B6.34.1), an isotype control, mAb H129, or medium, for 1 h at 37°C. Five thousand CTLL-2 cells were then added to each well and cultured for an additional 24 h (36). Cultures were pulsed with 1 µCi [3H]TdR and harvested onto Unifilter plates 4 h later, and thymidine uptake was assessed.
CD28 stimulation, cell lysis, immunoprecipitations, and immunoblotting
Cells (20 x 106/ml) were precoated with either 10 µg/ml anti-CD28 or normal hamster IgG for 45 min on ice, pelleted, resuspended to 40 x 106 cells/ml, and incubated at 37°C for 5 min. Rabbit anti-hamster IgG was added to a final concentration of 40 µg/ml, and the cells were incubated at 37°C for the indicated times. Cells were lysed at 50 x 106/ml in buffer containing: 150 mM NaCl, 10 mM Tris, 1% Nonidet P-40, 2 mM Na3VO4, and 1 mM PMSF, adjusted to pH 7.8. CD28 was immunoprecipitated from 7–10 x 106 cell equivalents, as indicated. Immunoprecipitates were washed three times, resolved by 10% SDS-PAGE, and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH), and the filters were blocked as described below.
4G10 immunoblots were blocked in gelatin and revealed with HRP-conjugated polyclonal goat anti-mouse IgG. Filters were stripped, then reblocked in 5% milk containing either 15 µg/ml anti-CD28 or anti-Lck serum, and revealed using protein A-HRP. Anti-actin blots were also blocked in milk and developed using HRP-conjugated goat anti-mouse IgG. Immunoblots were developed using Amersham ECL reagents.
To determine Lck localization, CD4 and Lck were sequentially and quantitatively precipitated from 100 µg of protein derived from postnuclear fractions of cell lysate, using mAb specific for CD4, and purified polyclonal anti-Lck covalently coupled to Sepharose 4B, respectively.
MIEV vector, transfections, and infections
The murine stem cell virus (MSCV)-based MIEV retroviral vector was constructed by replacing the neomycin phosphotransferase (neo) gene in the MSCV-based IRES-neo virus retroviral vector (37) with a 725-bp NcoI-NotI fragment containing the enhanced green fluorescent protein gene (EGFP) from the pEGFP-1 plasmid (Clontech Laboratories, Palo Alto, CA) (38). The cDNAs encoding for WT Lck and S20/S23-mutated Lck (DC Lck) (39) were cloned into MIEV. BOSC cells were transfected with 10 µg of empty MIEV, MIEV-DC Lck, or MIEV-WT Lck constructs using lipofectin (Life Technologies). GP+E cells were infected with 48-h BOSC cell supernatant and GFP+ cells sorted for high expression, using a FACStarPlus. Sorted GP+E cells were cocultured with the CD4+ T cell clonal variants in the presence of 8 µg/ml polybrene for 48 h, followed by sorting of GFP+ T cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immobilized anti-CD28, over a broad concentration range,
induces robust DNA synthesis in murine CD4+ T
cells (Fig. 1
A). In contrast,
immobilized mAbs specific for CD4, CD8, CD45, and MHC class I, did not
(not shown). The specificity of CD28-mediated T cell growth was further
assessed by comparing the responses of CD4+ T
cells derived from either CD28 sufficient or deficient mice
(25). As illustrated in Fig. 1B,
CD28-/- T cells failed to respond to
immobilized anti-CD28, while their responses to soluble
anti-CD3
and Con A were comparable with those of
CD28+/+ T cells (not shown). These results
confirm previous reports demonstrating TCR/CD3-independent
CD28-mediated T cell activation (19, 20), and extend the
observation to primary murine T cells.
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The CD4- but not the CD4+ clonal variant responds to anti-CD28
As illustrated in Fig. 2A, the level of CD28
expression on the CD4- and
CD4+ clonal variants is comparable. However, in
the presence of irradiated syngeneic splenocytes, the
CD4- clonal variant proliferates in response to
soluble anti-CD28, while the CD4+ clonal
variant does not (Fig. 2B). The differential signaling
observed in the clonal variants was not mAb specific, as the same
response pattern was observed using another murine CD28-specific mAb,
PV-1 (not shown). However, PV-1 was reported to enhance autologous MLR
(40), suggesting that the responses observed in the
present study might reflect a CD28-mediated enhancement of the response
of self-reactive T cells. To exclude this possibility, the capacity of
the clonal variants to respond to immobilized anti-CD28, in the
absence of filler cells, was assessed. This mode of stimulation did not
alter the differential response patterns observed of the clonal
variants to CD28-specific mAb (Fig. 2C).
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A,
anti-CD28-mediated growth correlated with the production and
secretion of IL-2. Thus, only supernatant from the
anti-CD28-responsive CD4- clonal variant
supported the proliferation of the IL-2-sensitive indicator cell line,
CTLL-2, and this growth-promoting activity was neutralized by mAb
specific for IL-2, but not an isotype control. However, the production
of IL-2 is a late indicator of activated T cells and is preceded by the
induction of PTK activity and the phosphorylation of specific cellular
substrates (41). As illustrated in Fig. 3B,
these early events were also observed upon CD28 aggregation of the
CD4- clonal variant, exclusively, and therefore
correlate well with the induction of IL-2 and ensuing cell growth.
Aggregation of CD28 on the CD4- clonal variant
resulted in an increase in the phosphotyrosyl content of proteins with
apparent molecular masses of 135, 100, 65, 43, and 33 kDa, all of which
coprecipitated with CD28. Analogous phosphoproteins were either below
the level of detection or present in lower amounts in CD28 precipitates
derived from the stimulated CD4+ clonal variant
(Fig. 3B). The lower panel in Fig. 3B
demonstrates comparable loading of CD28 within each series of
precipitations. The kinetics of appearance of the phosphoproteins
associated with CD28 demonstrates that aggregation of CD28 was
required, but whether these proteins were constitutively associated
with CD28, or recruited upon its aggregation is not known. Importantly,
further immunoblotting analysis revealed that Lck was not detectable in
anti-CD28 precipitates (not shown).
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) contrasts with
results obtained using primary CD4+ T cells (Fig. 1). To determine whether CD4-mediated sequestration of Lck, shown
previously to affect the responsiveness of this IL-2-dependent clonal
variant to TCRC-specific mAbs (23, 24), could also be
inhibiting its response to CD28-specific mAb, we assessed the
responsiveness of variants of the CD4- clone in
which the expression of either A418/A420-mutated CD4 (DC CD4), unable
to associate with Lck (23, 42, 43, 44), or WT CD4, was forced.
Despite comparable levels of CD4 expression in these two infectants
(23), forced expression of DC CD4 did not alter the
capacity to respond to soluble anti-CD28, whereas forced expression
of WT CD4 ablated the response (Fig. 4).
Thus, CD4 expression per se does not impede anti-CD28-induced
growth; rather, lack of responsiveness correlates with the capacity of
CD4 to bind Lck.
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Forced expression of DC Lck rescues direct CD28 signaling and costimulation in the CD4+ clonal variant
The cDNAs for WT and DC Lck were cloned into the polycistronic
vector MIEV (Fig. 5A), 5' to
the cDNA encoding for the EGFP. The Lck and GFP cDNAs are transcribed
as one transcript, separated by the encephalomyocarditis virus internal
ribosome entry site (45). The CD4+
clonal variant was infected with these MIEV constructs and GFP-positive
cells were enriched such that each population exhibited comparable
levels of fluorescence (Fig. 5B). Levels of expression of
both CD4 (Fig. 5C) and CD28 (Fig. 5D) were also
comparable. Densitometric analysis of Lck immunoblots derived from
lysates revealed that levels of Lck in each of the WT and DC Lck
infectants were on average 2.5-fold higher than that observed in empty
MIEV infectants (Fig. 6A).
Protein loading was normalized to actin levels (Fig. 6A).
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B, the
CD4-associated pool of Lck is roughly 4.5-fold higher in the WT Lck
infectants in comparison with DC Lck infectants or those carrying the
empty MIEV vector. The amount of non-CD4-associated Lck in variants
expressing WT Lck and DC Lck is 13-fold, and 37-fold higher,
respectively, in comparison with the variant expressing the empty MIEV
(Fig. 6B). These results indicate that the level of
overexpression of WT Lck achieved saturates the available CD4 binding
sites. Cell fractionation studies revealed that >95% of Lck was
associated with the plasma membrane in each of the three MIEV
infectants. Importantly and consistent with this observation, of the
membrane pool of Lck, the proportion associated or not with CD4 was the
same as that observed in total cell lysates (not shown).
To determine whether a generalized increase in the basal levels of
tyrosine phosphorylation resulted from the forced expression of either
WT or DC Lck, cell lysate from each of these clonal variants was
examined. As illustrated in Fig. 6C, forced expression of
either form of Lck resulted in a comparable increase of basal levels of
tyrosine phosphorylation. In fact, the level of phosphorylation of some
proteins was higher in the WT Lck variant (Fig. 6C).
However, the observed increases were not global. As will be presented
below, the basal level of CD28 phosphorylation is unaffected in either
of these variants. The key question is whether overexpression of either
form of Lck altered the anti-CD28 response pattern of the
CD4+ clonal variant.
As illustrated in Fig. 7A, DC
Lck infectants, but not WT Lck infectants, respond to immobilized
anti-CD28. Furthermore, the responses of the empty MIEV or WT Lck
infectants were comparable, and not significantly different to that of
uninfected CD4+ clonal variants (cf Figs. 7A and 2), despite the significant overexpression of
non-CD4-associated Lck in the WT Lck infectant (Fig. 6B).
Similar response patterns of these infectants to soluble anti-CD28
were observed (not shown). As illustrated in Fig. 7B, CD28
aggregation in DC Lck, but not WT Lck infectants revealed the same
array of phosphoproteins associated with CD28 as was observed in the
CD4- clonal variant (cf. Figs. 3B and
7B). Of note is that the level of tyrosine phosphorylation
of the 44-kDa band, consistent with the molecular mass of CD28 itself,
increases more robustly following aggregation of CD28 in DC Lck
infectants compared with WT Lck infectants. In addition, the forced
expression of DC Lck resulted in the association of pp80, pp65, and
pp33 with CD28 that was not dependent upon CD28 aggregation, indicated
by arrows in Fig. 7B. Furthermore, while not correlating
with anti-CD28-induced growth, exceeding the binding capacity of
CD4 through the forced expression of WT Lck did result in an increased
level of CD28-associated phosphoproteins observed after CD28
aggregation (Fig. 7B).
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-independent CD28 signaling could be extended to CD28-mediated
costimulation, the three categories of infectants were stimulated with
submitogenic concentrations of immobilized mAbs specific for CD28 and
TCRC, separately, and in combination. Two concentrations of
TCRC-specific mAb were tested in combination with 100 ng/ml of
coimmobilized anti-CD28. Nonmitogenic combinations of CD28 and
either of the two concentrations of anti-TCRC did not stimulate
significant DNA synthesis in the empty MIEV variant (Fig. 7C). In contrast, DC Lck infectants responded to
submitogenic concentrations of TCRC-specific mAbs in the presence of
coimmobilized anti-CD28, with synergies ranging from 5–30-fold
(Fig. 7C). The WT Lck infectant responded with roughly a
5-fold synergy at either concentration of anti-TCRC (Fig. 7C). Thus, while direct CD28 signaling appears to be more
sensitive to levels of non-CD4-associated Lck than CD28-mediated
costimulation, this compartment of Lck appears to limit the delivery of
CD28-mediated signals in both circumstances. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B); the
demonstration that immobilized mAbs specific for CD4, CD8, CD45, or MHC
class I did not induce T cell proliferation; and finally, the
demonstration that soluble anti-CD28 stimulates
CD4- clonal variants, and
CD4+ variants overexpressing DC Lck.
The implicated role of non-CD4-associated Lck in CD28 signaling was
derived from analyses of the collection of CD4-
and CD4+ clonal variants. As illustrated by the
DC CD4 and WT CD4 infectants, the presence of CD4 that is able to bind
Lck correlates with the inability to induce growth through CD28. In
contrast, the cellular localization of Lck in the absence of CD4, or in
the presence of DC CD4, permits the signaling sequelae observed upon
CD28 aggregation (Fig. 4).
In apparent contradiction to the hypothesis forwarded is the differential responsiveness of CD4+ LN T cells and the IL-2-dependent CD4+ clonal variants to CD28 stimulation. The confusion arises due to the fact that the distribution of CD4-associated and nonassociated Lck is comparable in both populations. Thus, based on the model forwarded, one would have predicted that the CD4+ clonal variant would respond to anti-CD28 stimulation. However, more recent work has characterized the biochemical basis that accounts for the distinct signaling phenotypes observed in primary CD4+ T cells and these IL-2-dependent CD4+ clonal variants. Specifically, the exogenous IL-2 that is required for propagating the clonal variants before assay down-regulates the kinase activity of membrane-associated Lck, which in turn prohibits responsiveness (24). Therefore, comparisons of the levels of non-CD4-associated Lck among unrelated cell populations, for example IL-2-dependent clones and primary T cells, are not a good predictor of responsiveness. It is the combination of IL-2-mediated affects on the kinase activity of Lck, in conjunction with its distribution, that determines net availability of Lck in the system. Pertinent to the present study is the fact that the MIEV infectants are internally controlled for the effects of exogenous IL-2 on Lck activity. This enables the direct assessment of the affects of the cellular distribution of the kinase in regard to its delivery of function.
The formal demonstration of the involvement of non-CD4-associated Lck
came from the analysis of CD4+ clonal variants
overexpressing either WT Lck or DC Lck. Despite the comparable level of
overexpression of these two forms of Lck within the membrane fraction
of the cell, in the CD4+ clonal variant (Fig. 6, not shown), only those expressing DC Lck responded to anti-CD28
(Fig. 7). Thus, when able to bind Lck, CD4 expression results in the
redistribution of Lck such that CD28-mediated growth is ablated.
However, and consistent with this model, if the pool of
non-CD4-associated Lck is artificially increased, as it is in DC Lck
infectants, the effects of CD4-mediated Lck sequestration are
counteracted, and responsiveness to CD28 aggregation is restored.
The amount of non-CD4-associated Lck in variants expressing WT Lck and
DC Lck is 13-fold, and 37-fold higher, respectively, in comparison with
that observed in variants expressing the empty MIEV (Fig. 6B). These fold increments in CD4 and non-CD4-associated Lck
compartments, given their relative contributions to total Lck, are
consistent with the 2.5-fold increase in total Lck observed in WT Lck
and DC Lck infectants (Fig. 6A). It is remarkable that a
13-fold increment in non-CD4-associated Lck, generated as a consequence
of saturating CD4, did not rescue anti-CD28-induced growth (Fig. 7A). However, it did rescue an increase in phosphoproteins
associated with CD28 (Fig. 7B), as well as some
CD28-mediated costimulation (Fig. 7C). These results suggest
that a threshold of activation and/or recruitment of signaling elements
must be achieved before the biochemical alterations induced upon CD28
aggregation translate into the induction of growth. Furthermore, this
threshold is different when complemented by TCR/CD3-derived
signals.
The results presented support a paradigm for CD28
signaling/costimulation in which non-CD4-associated Lck plays a
critical and proximal role. It posits that the recruitment and
activation of Lck enable the involvement of phosphatidylinositol
3-kinase, Grb-2, and Itk (15, 48, 49, 50, 51, 52, 53). Furthermore, the
reported recruitment of p62, cbl, ras-GAP, and
rho (54) would be predicated by these initial
Lck-dependent events. Two nonexclusive sources of non-CD4-associated
Lck could accommodate CD28 signaling. Primary resting T cells contain a
significant pool of non-CD4-associated Lck (26, 55).
Alternatively, Ag-mediated coaggregation of CD4/Lck and TCR/CD3 may
result in both the activation of associated Lck, as well as its
dissociation from CD4, as has been described for mAb-mediated CD4
aggregation (55, 56, 57). This dissociated Lck may then become
available for CD28 signaling. However, this latter mechanism would not
explain the Lck dependence of TCR-independent CD28 signaling.
Thus, we propose that constitutive levels of non-CD4-associated Lck in
resting T cells support CD28-mediated costimulation.
Recent reports have suggested that CD28 costimulation induces a
reorganization or clustering of sphingolipid-cholesterol-rich rafts, as
well as an increased consumption of Lck (58, 59). This
reorganization correlates with efficient T cell activation by
increasing the local concentration of kinases, as well as excluding
regulatory phosphatases (60, 61, 62). Although the present
study supports a central role of Lck in CD28-mediated costimulation,
the direct involvement of lipid rafts in TCR-independent CD28
stimulation remains to be characterized.
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Acknowledgments |
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Footnotes |
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2 Current address: Fred Hutchinson Cancer Research Center, Molecular Medicine Division, Seattle, WA 98109.
3 Current address: Hematopoiesis Department, Holland Laboratory, American Red Cross, Rockville, MD 20855.
4 Address correspondence and reprint requests to Dr. Michael Julius, c/o 610 University Avenue, 620 University Avenue, Room 700B, Toronto, Ontario, Canada M5G 2 M9. E-mail address:
5 Abbreviations used in this paper: PTK, protein tyrosine kinase; DC, double cysteine; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; MSCV, murine stem cell virus; LN, lymph node; MIEV, MSCV-based IRES-EGFP virus; WT, wild-type.
Received for publication February 5, 1999. Accepted for publication May 25, 1999.
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) and wild-type mice (
) were either
unstimulated or stimulated with 100 µg/ml of indirectly immobilized
anti-CD28. Cultures were pulsed with 1 µCi of
[3H]TdR at 30 h and harvested 6 h later, and
levels of thymidine uptake were assessed by liquid scintillation
spectroscopy. The values represent the mean of duplicate wells with 1
SD indicated.
), WT CD4 (
