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*
Howard Hughes Medical Institute, Section of Immunobiology, and
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510
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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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components associated with the TCR, which is
thought to be mediated by the src family tyrosine kinases lck and/or
fyn. Following tyrosine phosphorylation of the CD3/
components, a second protein tyrosine kinase associated with the TCR
complex, ZAP70, is activated, which in conjunction with a series of
adapter proteins can activate the phosphatidyl inositol second
messenger pathway resulting in protein kinase C activation and the
mobilization of intracellular calcium stores. The regulation of these
early biochemical signaling events generated following TCR/CD3
stimulation has been characterized in various models of T cell
activation using long term T cell cloned lines or T cell leukemic lines
primarily in relation to IL-2 gene transcription and proliferation.
However, relatively little is known about the regulation of early
signaling events generated by peptide stimulation of naive T cells, and
the relation of these events to T cell differentiation into Th1 and Th2
effector cell subsets.
Several groups of investigators have shown in T cell cloned lines that
the initial TCR-mediated signaling pathways can be modified by
stimulating T cells using peptides with low affinity (having high
dissociation rates) for a particular TCR (3). These peptides, termed
altered peptide ligands
(APL),3 can be generated by
substituting specific amino acid residues of an agonist peptide at the
TCR contact area, and can induce a distinct signaling pattern
compared with stimulation with agonist peptides: there is an altered
pattern of phosphorylation, absent ZAP70 phosphorylation, and low
amplitude, transient calcium mobilization (4, 5, 6). These signaling
events generally do not result in T cell proliferation and IL-2
production, but correlate with other possible outcomes of T cell
activation including antagonist function and IL-4 secretion in T cell
cloned lines (7, 8) and, in naive T cells, with differentiation into
Th2 effector cells (9). Because these variant peptides induce different
outcomes of T cell activation and because the altered pattern of
signals cannot be mimicked by low doses of agonist peptide (3, 4, 5, 9),
it has been proposed that the signaling events associated with variant
peptides with a low affinity for the TCR are qualitatively different
compared with the events induced by agonist peptides. However,
the molecular basis for the different signaling patterns induced by
these variant peptides is not well understood. Furthermore, it remains
unclear how these different signals are interpreted by the T cell to
result in different outcomes of activation. It has been demonstrated in
several model systems, in vivo and in vitro, that providing a weak
TCR-mediated signal can preferentially generate IL-4-producing cells
and Th2 differentiation (10, 11, 12, 13, 14, 15, 16, 17, 18). These data suggest that the pattern
of signaling events induced by low affinity peptides is sufficient to
induce Th2 differentiation; however, it remains uncertain whether these
events are necessary or whether the full signaling pattern seen
following agonist peptide stimulation simply inhibits Th2
differentiation.
One hypothesis that has been proposed to explain the different
signaling patterns seen following APL stimulation is that CD4 and the
associated src family tyrosine kinase, lck, are not effectively
recruited to the TCR complex, either because of a failure in the
formation of TCR/CD4 oligomeric complexes or due to a change in TCR
conformation that prevents or disrupts effective TCR/CD4
association (3, 19, 20). Failure to recruit and/or activate lck
to the TCR complex could then result in a truncated pattern of TCR
-chain phosphorylation and a failure to phosphorylate ZAP70. In
support of this hypothesis are recent experiments by Madrenas and
coworkers using T cell cloned lines. These experiments indicate that
prevention of CD4 interaction with MHC class II by using anti-CD4 mAbs
or MHC class II mutants which cannot bind CD4 results in conversion of
an agonist signaling phenotype and a proliferative response into an APL
signaling phenotype, failure to proliferate, and anergy (21).
These data indicate that in fully differentiated T cell cloned lines,
the failure to recruit CD4/lck to the TCR complex can modulate TCR
signaling pathways.
However, since CD4 is expressed normally in these experiments, the role of CD4 signaling apart from the TCR complex remains uncertain. Furthermore, although the above-referenced data suggest that in the absence of CD4 recruitment to the TCR, the response to a high affinity peptide can resemble the response to an APL, the role of CD4 during low affinity, APL-induced T cell stimulation was not determined. Indeed, data from several different systems suggest that peptides with a poor affinity for the TCR may be more dependent on the presence of a coreceptor to stabilize TCR/MHC class II interactions and to enhance the efficiency of TCR signaling (22, 23). If this is the case, then APL signaling and subsequent Th2 differentiation may be abrogated in the absence of CD4. Indeed, recent studies using CD4-deficient mice have suggested that CD4 expression on peripheral T cells is required for the generation of a Th2 effector response in vivo and in vitro (24, 25). However, changes in TCR signaling pathways were not examined in these studies. Thus, the mechanism for the CD4 requirement to induce a Th2 effector response remains unclear.
Since we had previously shown that stimulation with APLs is associated
with Th2 differentiation (17), we have now examined the requirement for
CD4 signal transduction following activation with peptides of different
affinity and analyzed the role of CD4 in early signaling events, in T
cell proliferation, and in priming of naive T cells to differentiate
into Th1 and Th2 subsets. To do this, we bred mice with a transgenic
TCR specific for moth cytochrome c onto CD4-deficient mice
or CD4-deficient mice reconstituted with a CD4 transgene lacking the
cytoplasmic tail of CD4 (CD4
cyt). We then
stimulated CD8- TCR transgenic T cells isolated from these
mice with peptides of varying affinity and evaluated the effect of CD4
and CD4-associated lck on early signaling events in naive T cells and
on subsequent differentiation into Th1 and Th2 effector cell
populations. These experiments showed that, although signaling with
agonist peptide in the absence of functional CD4 leads to a moderate
reduction in responsiveness as measured by proliferation, it does not
lead to any qualitatively distinct changes in the biochemical signaling
profile ( phosphorylation, ZAP70 phosphorylation, and calcium
mobilization) similar to that induced by APLs. In contrast, T cell
responses to APL stimulation were severely compromised in the absence
of CD4 coreceptor function as measured by loss of calcium mobilization
and a marked decrease in proliferation. In addition, we found that in
the absence of CD4 coreceptor function, differentiation of naive T
cells into Th2 cells was severely impaired, while Th1 differentiation
remained unchanged. These experiments show that a component of CD4
signaling is required for the differentiation of CD4 T cells into Th2
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.A (5R) mice were obtained from The Jackson Laboratory (Bar
Harbor, ME). The TCR transgenic mice in which CD4+ T cells
express a TCR specific for the carboxyl terminus of pigeon cytochrome
c have been previously described (26) and were bred in our
facilities and maintained as heterozygotes on a B10.A (5R), B10.BR, or
C57BL/6 background. The CD4-/- and CD4-/-
cyt mice were generously provided by D. Littman (New York
University, New York, NY) and were originally on a B6 background
(27). These mice were backcrossed four to six times on to a B10.BR
background, and then heterozygote CD4-/+ or
CD4-/+ cyt mice were crossed to obtain
homozygous CD4-/- mice. All mice used in these studies
were 6 to 10 wk old.
Peptides
Moth cytochrome c (MCC; peptide 81–103), pMCC = VFAGLKKANERADLIAYLKQATK, K99R = VFAGLKKANERADLIAYLRQATK, and pBSA (peptide 141–154) = GKYLYEIARRHPYF were synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory (New Haven, CT). All peptides were purified by HPLC before use.
Preparation of APC and CD4+ T cells
T cell-depleted APC were prepared by Ab-mediated complement
lysis of 5R splenocytes as previously described (28). The APC were
treated with 50 µg/ml of mitomycin C (Boehringer Mannheim
Biochemicals, Indianapolis, IN) before use.
CD4+CD8- T cells from lymph nodes and spleens
of transgenic mice were isolated by immunomagnetic negative selection
(28) using Abs against CD8, CD32/CD16, B220, and MHC class II, followed
by incubation with anti-mouse and anti-rat Ig-coated
magnetic beads (PerSeptive Biosystems, Framingham, MA). Purity
of the recovered V
11+, CD4+ T cells (or
V11+, CD4-CD8- T cells in
CD4-/- mice) was usually 85 to 95% as determined by
staining with ant-CD4 and anti-V11 mAb.
Flow cytometric analysis
Blood leukocytes were labeled with mAbs to V11 (PharMingen,
San Diego, CA), CD8 (Life Technologies, Gaithersburg, MD), and CD4
(Sigma, St. Louis, MO), conjugated with FITC, phycoerythrin, and Red
670, respectively, after water lysis. The cells were labeled with
saturating amounts of the indicated Abs diluted in PBS supplemented
with 5% FCS/0.02% sodium azide and 1 mg/ml mouse Ig and
incubated on ice for 30 min. Fluorescence analysis was done using a
FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and the
data analyzed using the LYSYS 2 software package (Becton Dickinson).
Proliferation assays
Proliferation of Ag-stimulated T cells was determined as
previously described (29) Briefly, 105 V11+,
CD8- T cells were cultured in triplicate in 96-well
flat-bottom microtiter plates and stimulated with different doses of
peptide in the presence of 105 mitomycin C-treated APC.
After 3 days, the cells were pulsed with 1 µCi/well of
[3H]thymidine, harvested 18 to 24 h later, and
the level of incorporation determined by scintillation counting.
In vitro differentiation of naive T cells
Induction of naive T cell differentiation was performed as
previously described (17) with slight modifications. Briefly, mitomycin
C-treated, T cell-depleted splenocytes (2 x 106
cell/ml) were incubated with 20 µM of agonist peptide or APL for
2 h at 37°C. T cells (5 x 105/ml) from
TCR-transgenic mice were then cocultured with peptide-primed APC
(1 x 106/ml) and rIL-2 (25 U/ml). After 4 days of
priming, T cells were harvested and dead cells were removed by gradient
centrifugation. Viable T cells were then incubated for a rest period of
2 days with fresh APC only. For the secondary culture, rested T cells
(5 x 105/ml) were restimulated with pMCC (2 µM) and
fresh APC (5 x 105/ml) for an additional period of 2
days. The presence of IL-4 and IFN-
in the supernatants of primary
and secondary cultures was determined using ELISA kits from Endogen
(Cambridge, MA). The supernatants were diluted serially in duplicate
and the concentration of cytokine determined in relation to a reference
standard supplied by the manufacturer.
Protein biochemistry and immunoprecipitation
CD4+ T cells (5 x 106) were added
to peptide-primed APC (5 x 106) and incubated at
37°C for indicated times. At the indicated time points, cells were
harvested and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.2, 1%
Nonidet P-40, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA)
containing protease and phosphatase inhibitors (10 mM
Na4P2O7·10
H2O, 1 mM Na3VO4, 50 mM NaF, 1 mM
PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin), and nuclear
material was removed as previously described (30). Cell lysates were
incubated for 1.5 h with protein A-Sepharose CL-4B beads
(Pharmacia Biotech, Piscataway, NJ) that had been pretreated with
anti--chain or ZAP70 rabbit antisera prepared in our laboratory.
Immunoprecipitates were washed four times and analyzed for tyrosine
phosphorylation by Western blot. Phosphotyrosine-containing proteins
were detected by blotting with anti-phosphotyrosine mAb (4G10;
Upstate Biotechnology, Lake Placid, NY) followed by goat anti-mouse
IgG horseradish peroxidase conjugate (Bio-Rad, Hercules, CA) and
detected by ECL as described by the manufacturer (Amersham,
Buckinghamshire, England). Densitometric analysis was determined using
a digital imaging system, model IS-1000 ( Innotech, San Leandro,
CA).
Calcium mobilization
Calcium signaling following Ag-specific stimulation was monitored as described previously (31). Briefly, CD4+ T cells loaded with 5 µM fluo-3/AM ester (Molecular Probes, Eugene, OR) were plated by centrifugation in 96-well plates at a concentration of 5 x 105 cells/50 µl. The cells were then scanned using the ACAS 570 video laser cytometer (Meridian Instruments, Okemos, IL). After initiation of scanning, 4 x 106 T-depleted splenocytes pulsed with 20 µM of peptide were added to the CD4+ T cells. The initial average fluorescence of each cell was digitized and normalized to 1, and the results are expressed as changes in normalized fluorescence intensity of individual cells over time. The percentage of responding cells was determined by dividing the number of cells demonstrating an increase in intracellular calcium of >50% by the total number of scanned cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown in several systems that varying the
signal strength of an antigenic peptide during the initial priming of
naive CD4 T cells can regulate the development of Th1 and Th2 effector
cell populations (11, 12, 13, 17, 32). To investigate the molecular basis
for this result, we have recently developed an in vitro model of T cell
differentiation in which naive CD4 T cells expressing a transgenic TCR
specific for MCC are induced to differentiate into Th1 and/or Th2 cells
depending upon the nature of the T cell ligand used during priming. In
this system, naive MCC-specific CD4 T cells are primed with peptides
that have differing affinities for the TCR while maintaining similar
association for MHC class II, and then all groups are restimulated with
a high affinity (agonist) peptide and assessed for differences in T
cell effector populations induced by the peptide used during priming.
Using this system, we can analyze the early signaling events that occur
during priming and correlate them with the subsequent differentiation
of the T cells into different effector subsets (results summarized in
Table I
). The low affinity peptide (or
APL) used in most of these experiments has a substitution of arginine
for lysine at position 99 (K99R) and is a characteristic APL in that it
has a diminished affinity for the cytochrome c-specific TCR
with a high dissociation rate (33) and, in naive T cells, induces an
altered pattern of T cell signaling events similar to that seen
following APL signaling in T cell cloned lines (3, 4, 5). Most
interestingly, we have shown that this peptide can induce the
differentiation of naive CD4 T cells into Th2 effector cells as
compared with stimulation with the agonist peptide, which can only
induce Th1 differentiation (17). In the present experiments, we tested
whether CD4 coreceptor signaling was required for these differential
effects.
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To investigate the role of CD4 in naive T cell signaling and
differentiation, we bred mice transgenic for the MCC-specific TCR with
CD4-deficient mice and subsequently with mice expressing a transgene
for CD4 with a deletion of the cytoplasmic tail (CD4cyt),
originally described by Killeen and Littman (27). It has previously
been shown that the CD4cyt transgene encoding the full
ectodomain of CD4, but lacking the cytoplasmic domain, can
significantly restore selection of functional
CD4+CD8- MHC class II-restricted T cells in
CD4 knock-out mice (27), and it appears to be a useful model for
examining the role of CD4-associated signaling molecules during
peripheral T cell activation. The MCC-specific TCR transgene used in
these experiments is positively selected into the CD4 lineage in mice
that express either I-Ab and I-Ek (26, 34).
Although initial experiments were performed using C57/BL6
(I-Ab) mice, most of the following data are derived from
I-Ek-expressing mice, which select the MCC-specific TCR
more efficiently in the absence of CD4 signaling (data not shown) (34).
The results were similar regardless of the mouse strain, although the
yield of purified MCC-specific TCR+ CD8- cells
from spleen and lymph nodes is better when B10.BR mice are used. As
shown in Figure 1, A and
B, selection of "CD4- lineage" MCC-specific
cells (V11+ CD8-) is relatively efficient
in the CD4-deficient and CD4cyt mice and is 
30 to 50%
of the level seen in CD4 wild-type mice. Furthermore, as shown in
Figure 1A, the expression level of the CD4cyt
transgene is similar to the expression of wild-type CD4.
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CD4 regulation of T cell proliferation following stimulation with peptides with different affinities for the TCR
Initial experiments were performed to assess the proliferative
capacity of purified CD8- V11+ cells
isolated from CD4cyt, CD4-deficient, and wild-type
cytochrome c-specific-TCR mice following high (agonist) and
low affinity (APL) peptide stimulation. In this way, we could determine
the relative dependence of the MCC-specific TCR transgenic T cells on
CD4 for a late event of T cell activation and compare two peptides with
differing affinities for the TCR, which have known differences in their
ability to induce proliferation (17). As expected, in T cells from the
two CD4 mutant mice, proliferation to the agonist peptide (pMCC) was
diminished compared with T cells from CD4+ wild-type mice
(Fig. 2A), although it
remained significantly above the level of proliferation induced by the
APL (K99R) in T cells from CD4+ wild-type mice (Fig. 2B, note different scale). As shown in Figure 2B,
when the APL (K99R) was used to stimulate wild-type and CD4 mutant TCR
transgenic CD4 T cells, Ag-specific T cell proliferation was completely
lost in the absence of CD4 and in the absence of the cytoplasmic
portion of CD4 (CD4cyt mutant).
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C). However, in contrast
to agonist peptide stimulation, proliferation in response to the APL
(K99R) remained impaired in the CD4 mutant mice despite the addition of
exogenous IL-2 (Fig. 2D). Although proliferation was
reproducibly observed in the T cells from the CD4cyt mice
in the presence of IL-2, it remained significantly below the level of
proliferation observed in T cells from CD4 wild-type mice. Furthermore,
no proliferation in response to K99R was observed in T cells from
CD4-deficient mice. In total, these data suggest that the requirement
for CD4 is more stringent when a peptide with lower affinity for the
TCR is used. This is supported by the observation that the specific
induction of CD69 expression was markedly reduced 24 h after
stimulation with K99R in the CD4 mutant T cells compared with the CD4
wild-type cells (data not shown). CD4 regulation of early signaling events following stimulation with peptides of different affinities for the TCR
We next examined the role of CD4 coreceptor function on three
peptide-induced signaling events (ZAP70 phosphorylation, calcium
mobilization, and CD3/ phosphorylation) that are known to differ
depending on the affinity of the peptide for the TCR and have been
shown to correlate with different outcomes of T cell activation. In
these experiments, we asked whether the early biochemical signaling
events initiated upon CD4 T cell stimulation with high affinity
peptides required CD4 signaling function and whether, in the absence of
CD4 signaling, these events would resemble stimulation with an APL.
Furthermore, these experiments examined changes in the relative
dependence of T cell signaling pathways on CD4, based on varying the
affinity of the peptide for the TCR.
Since ZAP70 phosphorylation is induced in wild-type CD4 T cells
following stimulation with the high affinity peptide (pMCC), but not
after stimulation with an APL (K99R), we asked whether CD4-associated
signaling function was required for ZAP70 phosphorylation following
stimulation with the high affinity peptide. As shown in Figure 3B, agonist peptide-induced
ZAP70 phosphorylation is detected in T cells isolated from wild-type
mice and mice with the CD4 cytoplasmic deletion mutant. Similar results
are seen in T cells from CD4-deficient mice (data not shown; Fig. 5).
Although the level of ZAP70 phosphorylation is somewhat reduced in this
experiment compared with that seen upon stimulation of T cells from
wild-type CD4 mice, it is readily detectable compared with the absence
of ZAP70 phosphorylation seen following stimulation with the APL (Fig. 3A). These data suggest that CD4 signaling ability is not
required for ZAP70 phosphorylation and that the absence of CD4
recruitment does not necessarily result in the signaling phenotype seen
following APL stimulation.
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, A
and B). In addition, there was no significant change in the
pattern of calcium mobilization: most of the responding cells exhibited
a strong, sustained calcium flux characteristic of stimulation with
agonist peptide (Fig. 4B). This is in marked contrast to the
pattern of calcium flux seen following stimulation with an APL, which
induces a small amplitude, transient calcium flux in the majority of
responding cells (9) (Fig. 4, C and D). When T
cells from the CD4 mutant mice were stimulated with APC pulsed with the
APL, calcium mobilization was reduced to near background levels
compared with T cells from normal control mice (Fig. 4, C
and D).
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However, some signaling events in response to stimulation with APLs did
occur in the absence of CD4, as seen by -chain phosphorylation. When
T cells from wild-type and CD4 mutant mice were stimulated with either
agonist or APL, the pattern of p18 and p21 phosphorylation was
essentially unchanged in the T cells from CD4 mutant mice compared with
the wild-type mice (Fig. 5, A
and B). In cells from all three mouse groups, agonist
peptide stimulation induced strong phosphorylation of the p21
phospho- band, with concomitant phosphorylation of ZAP70, while
stimulation with the APL characteristically induced less p21
phosphorylation relative to the p18 isoform and failed to induce
association with phosphorylated ZAP70. Densitometric analysis revealed
a similar ratio of p18:p21 after stimulation with K99R in both
wild-type mice and CD4 mutant mice that was approximately half that
induced with the agonist peptide (Fig. 5B). These data
indicate that in the absence of CD4 signaling, APLs can stimulate early
signaling events such as -chain phosphorylation and that the pattern
of this phosphorylation is not changed. However, the altered pattern of
phosphorylation induced by the APL is not sufficient to initiate
the transient calcium pattern characteristic of APL signaling.
In summary, the signaling data indicate that agonist peptide signaling in the MCC-specific TCR transgenic mice is qualitatively unchanged in the presence or absence of CD4 signaling function. Although early proliferation was somewhat diminished in the CD4 mutant mice, early signaling events remained intact and did not resemble the signaling pattern seen following stimulation with APLs. In contrast, signaling events following APL stimulation were qualitatively changed in cells lacking CD4 signaling capacity, as exhibited by the failure to induce significant calcium flux, which correlates with a marked reduction in the ability to proliferate to the APL even in the presence of exogenous IL-2.
Th1 and Th2 differentiation following agonist and APL stimulation
Although the signaling phenotype of the CD4 mutant T cells after
agonist stimulation was not qualitatively different from wild-type CD4
T cells and did not resemble APL-induced signaling, the decrease in
proliferation in both the CD4-deficient and CD4cyt mice
suggested that the signal received by the TCR in these mice was
quantitatively diminished due to the lack of CD4-associated signaling
capacity. To determine whether this would affect differentiation into
Th1 and Th2 effector subsets, naive CD4 T cells were primed with either
agonist or altered peptide in the presence of exogenous IL-2, allowed
to differentiate for 4 to 6 days, then restimulated with agonist
peptide and assayed for the ability to produce Th1 or Th2 effector
cytokines. As we have previously shown, stimulation with agonist
peptide primes the cells to secrete IFN-, but not IL-4, while
stimulation with the APL, K99R (with decreased affinity for the TCR),
primes the cells to produce IL-4 in addition to IFN- upon secondary
stimulation (Fig. 6). However, in
contrast to CD4 T cells from wild-type mice, when T cells from the
CD4cyt mice were primed with the APL, Th2 differentiation
was not induced, although, interestingly, IFN- production was not
affected and was similar to that seen in T cells from CD4 wild-type
mice. No defect in the ability of the agonist or APL to prime for
IFN- production was observed in the absence of CD4 signaling (Fig. 6A). Furthermore, despite the decrease in T cell
proliferation, there was no significant production of IL-4 following
priming with the high affinity peptide (Fig. 6B). Since the
CD4-deficient cells did not significantly proliferate upon stimulation
with K99R, we could not reproducibly examine these mice in the double
culture system because insufficient cells were available after
primary stimulation.
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Effect of exogenous IL-4 on Th2 differentiation in CD4 mutant mice
We next determined whether the addition of exogenous IL-4 could
overcome the failure in Th2 differentiation seen in cells defective in
CD4 signaling. In this way, we could determine whether the inability of
CD4 mutant T cells to differentiate into Th2 cells is due to their
failure to produce sufficient IL-4 during the primary culture or if it
is due to a general failure of these cells to respond to IL-4 and
undergo Th2 differentiation. As shown in Figure 7, when exogenous IL-4 is added to T
cells from CD4cyt mice primed with the APL (K99R), Th2
differentiation is significantly restored. This suggests that the
signaling failure seen in CD4 mutant T cells after stimulation with an
APL results in part from a failure to produce sufficient endogenous
IL-4 required for Th2 differentiation normally seen following APL
priming (17).
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A). These data suggest that
signaling through the IL-4R may be modulated by the CD4 coreceptor. It
appears that some signaling through the IL-4R is intact, however, since
IL-4 is able to suppress Th1 differentiation, as shown by the decrease
in IFN- production in T cells from the CD4 mutant mice (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we have examined the role of CD4 signaling in naive T cell activation and differentiation following stimulation with specific Ag. Using peptides with different affinities for a TCR specific for a peptide of MCC, we have shown that naive T cell activation with a strong agonist peptide is qualitatively unchanged in the absence of CD4. Although there is a quantitative difference in the proliferative response, no qualitative changes in early signaling events were detected, and there was no alteration in the subsequent differentiation of CD4 T cells. In contrast, the activation characteristics following stimulation with an APL are markedly affected both quantitatively and qualitatively by the absence of CD4. The T cells fail to significantly mobilize calcium, and they have a profound defect in proliferation that cannot be fully recovered by the addition of IL-2. Most interestingly, in the absence of CD4 signaling ability, T cells fail to differentiate into Th2 effector cells, while Th1 differentiation is unaffected. This provides support for the hypothesis that the TCR signaling pathways that lead to Th1 and Th2 differentiation are distinct and can be regulated at the level of early T cell signaling events in the absence of exogenously provided cytokines.
The experiments presented here demonstrate that a failure in CD4
recruitment or signaling during stimulation with an agonist peptide
does not necessarily result in an APL signaling phenotype, but rather
CD4 signaling appears to be required to generate at least some of the
signals characteristic of APLs. The relative CD4 independence of
agonist signaling seen in our experiments argues against the model of
APL signaling proposed by Madrenas et al. in which the APL-signaling
phenotype arises from a failure to induce CD4 recruitment to the TCR
complex (21). They showed that exposing T cell clones to anti-CD4
Abs during agonist stimulation, or preventing CD4-MHC class II
interaction using MHC class II mutants unable to interact with CD4,
resulted in an APL signaling phenotype as demonstrated by the failure
to phosphorylate ZAP70, an altered -chain phosphorylation, and
anergy induction. One possible explanation for the different results is
that the agonist peptide used in our studies may be of higher affinity
than the agonist stimuli used in the studies of Madrenas et al., which
may have been more dependent on the CD4 coreceptor. In cases of
relatively weak agonist signaling, CD4 coreceptor function may be more
important for the generation of a full signaling pattern, and in the
absence of coreceptor, inefficient signaling may occur that
resembles APL signaling. This conclusion is consistent
with several independent experiments in which sensitivity to
anti-CD4 inhibition was dependent on the strength of the ligand to the
TCR: strong agonists are generally resistant to anti-CD4 treatment
compared with weaker agonists, which are more sensitive (23, 35, 36).
Thus, one mechanism for inducing an altered peptide signaling phenotype
may be absence of coreceptor recruitment to the TCR following
stimulation with a weak agonist; however, our data indicate that the
absence of CD4 does not necessarily result in APL signaling if there is
a sufficiently strong signal to the TCR. Alternatively, the differences
observed between studies may be a reflection of differences in the
regulation of signaling in long term T cell clones vs primary CD4 T
cells.
The CD4 dependence of APL signaling observed in our experiments is consistent with other correlative data showing that low affinity TCR signals appear to be more dependent on the expression of coreceptors (22, 23, 37) and the increased susceptibility of T cell stimulation to anti-CD4 inhibition with partial agonists compared with agonist peptide (23, 35). The increased requirement of CD4 for APL signaling may be due, in part, to the increased dissociation rate of APLs for the TCR, making TCR/peptide interactions less stable and potentially making CD4 adhesion to MHC class II more important for stabilizing the interaction with specific peptide. In addition to adhesion, CD4 signaling function appears to be important, since CD4 T cells with an intact ectodomain but deleted cytoplasmic domain were also impaired in APL signaling. Although the CD4 cytoplasmic domain may interact with a variety of signaling molecules, its interaction with lck is particularly well defined and disruption of CD4-lck association is a potential mechanism for the effects observed in these experiments. Thus, one can infer that APL signaling may be especially dependent on CD4-associated lck, while other tyrosine kinases or non-CD4-associated lck may be sufficient for agonist peptide signaling. Again, this may simply be due to the APL requirement for increased signaling efficiency, which is generated by CD4-associated lck being more effectively targeted to the TCR complex. However, this does not fully explain the qualitatively distinct signals generated by APLs and their subsequent effects on T cell effector function. An alternative hypothesis is that CD4-associated lck may have access to distinct substrates compared with non-CD4-associated lck, and preferential use of CD4-associated lck during APL signaling may result in the qualitatively distinct pattern of signals generated compared with agonist signaling.
It should be noted, however, that while APL-induced calcium signaling
and Th2 differentiation are impaired in CD4 mutant T cells, the altered
pattern of TCR -chain phosphorylation is essentially unchanged in
the absence of CD4 signaling. This result is consistent with the data
of Rabinowitz et al., who proposed a hierarchy of signaling events
based on the strength of TCR signals with phosphorylation being the
least sensitive to changes in TCR signaling efficiency (35). However,
the mechanism behind the qualitatively distinct pattern of
phosphorylation seen during APL signaling relative to agonist signaling
remains unclear. Our data showing that the APL-induced altered pattern
of phosphorylation is largely unchanged in the absence of CD4
suggests that CD4-associated lck is not necessary for the differential
regulation of -chain phosphorylation seen in T cells stimulated with
APL. It remains possible that non-CD4-associated lck, or another
tyrosine kinase such as fyn, is still involved. Alternatively,
differential tyrosine phosphorylation patterns may also be regulated by
the presence of a tyrosine phosphatase such as CD45. It is possible
that tyrosine phosphatase association with the TCR complex may be
differentially regulated during agonist and APL stimulation, which may
affect -chain phosphorylation directly, or indirectly by regulating
src family tyrosine kinase activity.
Signaling events required for Th2 differentiation
Perhaps the most striking aspect of our data is the failure to induce Th2 differentiation in the absence of CD4 signaling ability. This is especially interesting in light of the experiments of Stumbles and Mason in which in vitro administration of whole anti-CD4 Ab induced Th2 differentiation of naive T cells in a mixed lymphocyte reaction (38). It was unclear from these studies whether the mechanism of CD4 regulation of T cell differentiation was by blocking MHC class II/CD4 interactions or whether the CD4 Ab was directly signaling the T cell. In support of the latter possibility are other studies by Konig and colleagues in which the in vivo administration of peptides that inhibit CD4/MHC class II interaction promote Th1 development (39). In conjunction with our own results, these data imply that CD4 signaling may be required for the initiation of Th2 differentiation.
The experiments presented in the present report clarify the nature of the signals required to induce Th2 differentiation. We have previously proposed a signal strength model of T cell differentiation in which low avidity signals to the TCR promote Th2 differentiation, while high avidity signals promote Th1 differentiation. This model was based on in vivo and in vitro experiments in which CD4 T cell differentiation was regulated by varying the ligand density on the APC (11, 12, 13, 14) or by varying the affinity of the TCR/peptide interactions (16, 17). In both instances, Th2 differentiation was correlated with low avidity signaling to the TCR, while Th1 differentiation was seen during high avidity signaling. In the studies of APL-induced Th2 differentiation, we have shown that this is dependent on the production of IL-4 by primary T cells 2 to 3 days after initial priming. These data suggest that low avidity TCR signaling can induce IL-4-driven differentiation of Th2 cells, which is similar to data derived from studies of T cell clones in which APL signaling was sufficient for the selective production of IL-4, but insufficient to induce proliferation (8). Our current data indicate that, at least in terms of APL with a decreased affinity for the TCR, the specific nature of the low avidity signal is important for the induction of Th2 differentiation and is not simply due to an overall weaker level of signaling.
The specific failure of APL to induce calcium flux in CD4 mutant T cells and their subsequent failure to undergo Th2 differentiation suggest that the altered pattern of calcium mobilization normally seen in wild-type CD4 T cells may be a necessary signal for Th2 differentiation. This is consistent with our observation that blockade of the calcium/calcineurin/NF-AT pathway with cyclosporin A during primary stimulation with APL inhibits Th2 development (9). In addition, we have previously shown that APL-induced Th2 differentiation is inhibited by inducing a sustained increase in intracellular calcium using a calcium ionophore. This indicates that the pattern of Ca2+ mobilization may mediate distinct signaling pathways and affect the differentiation of CD4 T cells.
In addition to changes in calcium signaling, there may be additional
alterations in signal transduction that occur in the absence of CD4
which influence T cell differentiation. As shown in Figure 8, and as
recently reported by others during the preparation of this report (24, 25), CD4 signaling seems to be important for efficient Th2 generation
driven by the addition of exogenous IL-4 even in the presence of
agonist peptide signaling. The role that CD4 plays in promoting Th2
differentiation in this situation remains uncertain. Optimal doses of
agonist peptide prime MCC-specific transgenic T cells to undergo
differentiation into Th1 effector cells only, without any detectable
Th2 activity (13, 17). Th2 differentiation can be induced, however, by
the addition of large amounts of exogenous IL-4 during primary culture
(Fig. 8) (40), and it is this step that appears to be diminished in the
absence of CD4, suggesting that CD4 signaling may be required for the
cells to be optimally receptive to signaling by IL-4. This may be
because IL-4R expression is not induced to the same level on the
surface of the T cell in the absence of CD4 signaling; or because IL-4R
signaling pathways are qualitatively affected by CD4 expression. Since
inhibition of Th1 differentiation by the addition of IL-4 is unchanged
in the absence of CD4, we currently favor the possibility that IL-4R
signaling occurs, but that the downstream effect of inducing Th2
differentiation may be differentially regulated in the absence of CD4.
In conclusion, it appears from our present data that CD4 expression can
regulate Th2 differentiation in two possible ways: by modulating TCR
signaling pathways and by modulating IL-4R signaling pathways. In our
experiments with APL-induced Th2 differentiation (and in the
experiments of Fowell et al.) (24), peptide normally induces
differentiation of both Th1 and Th2 effector populations, while Th2
differentiation is selectively lost in the absence of CD4 signaling
(Fig. 6), but can be substantially recovered upon the addition of
exogenous IL-4 (Fig. 7). These data suggest that CD4 primarily affects
TCR signaling pathways induced by APL, which are necessary for Th2
differentiation. This is quite different from the experiments using the
agonist peptide of MCC, which primes for Th1 differentiation only,
regardless of CD4 expression, while the addition of exogenous IL-4 will
allow Th2 differentiation to occur (Fig. 8). The absence of CD4
signaling in this case causes a significant decrease in Th2
differentiation, but it is not completely lost as is true with APL
stimulation. These experiments suggest that the CD4 coreceptor can
regulate Th2 differentiation by affecting the response to IL-4 in
addition to affecting TCR signaling pathways. In total, these
experiments demonstrate an important role for the CD4 coreceptor
in modulating TCR and cytokine receptor signaling pathways that are
important for Th2 differentiation.
|
Acknowledgments |
|---|
cyt mice, Patty Ranney for excellent
technical assistance with animal breeding, and Dr. Charles A. Janeway
for helpful comments on the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Kim Bottomly, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510.
3 Abbreviations used in this paper: APL, altered peptide ligand; MCC, moth cytochrome c; p, peptide (e.g., pMCC); CD4cyt, CD4 transgene lacking the cytoplasmic tail of CD4.
Received for publication October 15, 1997. Accepted for publication April 1, 1998.
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