CD4-Dependent Signaling Is Required for a Late Checkpoint
during Th2 Development Associated with Resistance to
Activation-Induced Cell Death1
Zohreh Tatari-Calderone,* Jennifer L. Brogdon,†Kevin W. Tinsley,* Anahita Ramezani,*
and David Leitenberg2*‡
Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene
have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following
stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2
effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of
Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2
development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of
AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support
the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of
Th2 development, which is related to dysregulation of AICD.
Over the last several years it has become clear that Th subset
development is regulated by a complex interplay between TCR,
costimulatory and cytokine receptor signaling events, which in
turn regulate the expression and activity of a variety of transcrip-
tion factors that control the distinct patterns of gene expression
necessary for Th2 effector cell function (1–3). Despite extensive
study, many issues regarding the relative roles, timing, and poten-
tial cross-talk between TCR, costimulatory and cytokine receptor
signals involved in controlling Th development remain unclear.
CD4 plays an important role in regulating T cell activation, by
serving as a coreceptor for the T cell Ag receptor during initial Ag
recognition and promoting the activation and recruitment of the src
family kinase lck to the CD3/TCR complex. In addition, CD4 has
been shown to play a less well-understood role in regulating the
function and development of effector and memory T lymphocytes
(4–6). Several years ago, we and other groups independently
found that CD4-dependent signaling was required for the optimal
development of IL-4-secreting Th2 effector cells in vitro and in
vivo but not for the development of Th1 effector cells (7–9). The
specific failure in Th2 development was observed in class II MHC-
restricted, CD4-deficient T cells (or CD4-deficient T cells recon-
stituted with a tail-less CD4 transgene), as well as in situations
The Journal of Immunology, 2005, 175: 5629–5636.
he development of Th lymphocyte effector function is a
multistep process that occurs over several days following
initial recognition of peptide ligand presented by APCs.
where wild-type CD4?T cells were primed with peptide presented
by mutant class II MHC molecules unable to interact with CD4.
Surprisingly, the requirement for CD4 expression was not over-
come by the addition of large amounts of exogenous IL-4 and/or
following stimulation with anti-CD3 or strong agonist peptides
which do not require CD4 coreceptor function (8, 9). These data
suggest that the role of CD4 in promoting Th2 development is a
relatively late event in the differentiation pathway.
In support of this hypothesis, we have found that CD4 mutant
and wild-type T cells produce similar levels of IL-4 and GATA-3
messenger RNA early after priming with a strong agonist peptide
and exogenous IL-4, despite exhibiting a defect in Th2 cytokine
production upon restimulation. These data further indicate that ini-
tial Th2 priming events are intact in the absence of CD4 signaling
and that CD4-dependent signaling pathways define a previously
unrecognized late checkpoint in Th2 development, which is inde-
pendent of a coreceptor requirement for initial TCR activation.
Coincident with the defect in Th2 development there is also a
significant defect in Fas signaling and activation-induced cell
death (AICD)3in CD4 mutant T cells. Our data suggest that the
defect in AICD is linked to Th2 development because inhibition of
AICD after T cell priming in wild-type T cells inhibits Th2 de-
velopment and IL-4 production similar to that seen in CD4 mutant
T cells. We propose a model in which the differential susceptibility
to AICD during Th development is a critical selection step, which
enriches for committed Th2 effector cells from a diverse pool of
Th2 precursor cells.
Materials and Methods
AND TCR transgenic mice in which CD4?T cells express a TCR specific
for carboxyl terminus of moth cytochrome c (pMCC) peptide in the context
of I-Ekor I-Ebhave been previously described (10), and are maintained in
our breeding colony as heterozygotes on a B10.BR background. The
CD4?/??cyt mice were originally provided by D. Littman (New York
*Department of Immunology, The George Washington University, Washington, DC
20037;†Department of Immunobiology, Yale University, New Haven, CT 06520; and
‡Department of Pediatrics, The George Washington University and Childrens Re-
search Institute, Childrens National Medical Center, Washington, DC 20037
Received for publication November 22, 2004. Accepted for publication July 27, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from the Arthritis Foundation and the
National Institutes of Health (AI42963).
2Address correspondence and reprint requests to Dr. David Leitenberg, Department
of Immunology, The George Washington University, 2300 I Street, NW, Washington,
DC 20037. E-mail address: email@example.com
3Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas
ligand; DISC, death-inducing signaling complex.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
University, New York, NY) (11) and were backcrossed seven to nine times
onto a B10.BR background with the AND TCR transgenic mice as previ-
ously described (9).
Purification of APC and CD4?T cells
T cell-depleted APC were prepared by Ab-mediated complement lysis
from total splenocytes as described (9). The APC were treated with 50
?g/ml mitomycin C (Sigma-Aldrich) before use. CD4?CD8?T cells from
lymph nodes and spleens of transgenic mice were isolated by immuno-
magnetic negative selection, using Abs against CD8, CD32/CD16, B220,
and MHC class II, followed by incubation with anti-mouse and anti-rat
Ig-coated magnetic beads (Polysciences). Purity of the recovered V?11?/
CD4?T cells as determined by staining with anti-CD4 and anti-V?11 mAb
is usually 85–95%.
T cell stimulation and detection of IL-4 and IFN-? production
Induction of naive T cell differentiation was performed as previously de-
scribed with slight modifications. Briefly, purified T cells (0.5 ? 106/ml)
from AND TCR transgenic wild-type or ?cyt mice and mitomycin C-
treated, T cell-depleted splenocytes (1 ? 106cell/ml) were incubated with
5 ?g/ml pMCC (agonist peptide of moth cytochrome c (81–103), pMCC ?
VFAGLKKANERADLIAYLKQATK) under neutral conditions in the
presence of rmIL-2 (25 U/ml) or under Th2 skewing conditions (with IL-2,
rmIL-4 (10–20 ng/ml) (Endogen), and anti-IFN-? (XMG1.2) (2.5 ?g/ml)
(Harlan Bioproducts), or Th1 skewing conditions (with IL-2, rmIL-12 (10
ng/ml), anti-IL-4 (11B11)). After 4 days of priming, T cells were har-
vested, and dead cells were removed by using gradient centrifugation. Vi-
able cells were then incubated for a rest period of 2 days with fresh APCs
only. For the secondary culture, rested T cells (0.5 ? 106/ml) were restim-
ulated with pMCC (5 ?g/ml) and fresh APC (1 ? 106/ml) for an additional
period of 2 days. Cytokine concentrations in the supernatants were deter-
mined by ELISA kit (Pierce-Endogen). Supernatants were diluted serially
in duplicate, and the concentration of cytokine determined in relation to a
reference standard supplied by the manufacturer. In some experiments, we
added Jo-2 anti-mouse Fas mAb or anti-Fas ligand (FasL) mAb (BD
Pharmingen) at varying times after initial stimulation.
Intracellular cytokine analysis was performed following 12–16 h of
stimulation with agonist peptide and the addition of monensin (Golgistop;
BD Biosciences) for the final 4–6 h of culture. Cells were harvested and
surface labeled with anti-CD4 before fixation and permeabilization (Fix
and Perm; Caltag Laboratories), and labeling with Abs to IL-4 and IFN-?
RNase protection assay
Two or 3 days after stimulation of CD4?T cells with APC and peptide, the
cells were harvested and dead cells removed by gradient centrifugation. In
some experiments, CD4?T cells were further isolated by immunomagnetic
negative selection as described above. The purity of CD4?T cells was then
determined by FACS analysis and was ?98% by CD4 and V?11 labeling.
Total RNA was isolated using TRIzol (Invitrogen Life Technologies).
AND TCR transgene were primed with 5 ?g/ml agonist peptide (pMCC) and 25 U/ml IL-2 under Th2, Th1, or neutral skewing conditions for 4 days as
described in Materials and Methods. Cells were then washed and rested for an additional 2 days before challenging with agonist peptide and APC. A, After
48 h, culture supernatants were collected and analyzed for IL-4, IFN-?, IL-5, and IL-10 by ELISA. This experiment is representative of more than three
independent experiments. B, Flow cytometric intracellular cytokine analysis of cells primed under Th2 or Th1 conditions after overnight stimulation with
agonist peptide and APC. These data are representative of three independent experiments.
Specific defect in Th2 development in the absence of CD4 signaling. Purified CD4 T cells from wild-type (WT) or ?cyt mice expressing
dependent upon CD4 signaling. Purified WT or ?cyt CD4 T cells were
stimulated with pMCC and the indicated IL-4 concentrations. A, After 48 h,
the CD4 T cells were harvested, and total RNA was isolated. Cytokine
RNA levels were analyzed by RNase protection assay (RPA); 10 ?g of
total RNA were used in each reaction. Equal loading of RNA was assessed
using the housekeeping genes L32. B, Purified CD4 WT or ?cyt T cells
were stimulated as above, and total RNA was extracted at day 3 or day 4
and assessed for GATA-3 expression by Northern blot; 10 ?g of RNA
were loaded per lane, and equal sample loading was confirmed by UV light
shadowing of 28S and 18S rRNA. These data are representative of two
Induction of early IL-4 and GATA-3 transcription is not
5630CD4 REGULATION OF AICD AND Th2 DEVELOPMENT
RNase protection assays were conducted according to the manufacturer’s
protocol (BD Biosciences), using multiprobe template sets from the Ribo-
quant multiprobe kit (BD Biosciences).
For semiquantitative RT-PCR, 2 ?g of total RNA were used to prepare first
strand cDNA using SuperScript first strand RT-PCR kit (Invitrogen Life
Technologies) following manufacturer’s protocol. The specific primers for
FLIP were (from 5? to 3?) GTC ACA TGA CAT AAC CCA GAT TGT and
antisense (from 5? to 3?) GTA CAG ACT GCT CTC CCA AGC ACT. The
primers for FasL were (sense 5?to 3?) ATC CCT CTG GAA TGG GAA
GA and (antisense 5?to 3?) CCA TAT CTG TCC AGT AGT CG. Primers
for ?2-microglobulin were (sense 5? to 3?) TGA CCG GCT TGT ATG
CTA TC and (antisense 5? to 3?) CAG TGT GAG CCA GGA TAT AG.
Concentrations of input cDNAs were equalized by comparing different
dilutions with the band intensities of ?2-microglobulin amplification prod-
ucts. The integrity of the PCR was controlled by parallel amplification of
plasmid pMus3 (gift of Dr. D. Shire, Sanofi Recherche, Labe `ge, France,
and N. Noben-Trauth, George Washington University, Washington, D.C.).
Northern blot analysis
After 2 days in the primary culture, cells were harvested and dead cells
were moved by gradient centrifugation. Total cellular RNA was isolated
using TRIzol reagent (Invitrogen Life Technologies), and 10 ?g of total
RNA from each sample were fractionated on 1.2% agarose/formaldehyde
gels, transferred to ?-Probe GT membranes (Bio-Rad), and hybridized with
the indicated cDNA probes in QuickHyb buffer (Stratagene). The GATA-3
construct was kindly provided by R. Flavell (Yale University, New Haven,
CT). UV shadowing of the membrane to visualize 28S and 18S rRNA was
performed to ensure equivalent sample loading.
Immunoprecipitation and Western blotting
CD4?T cells (106/ml) have been primed with agonist peptide and APC
(2 ? 106) for 3 days. Cells were harvested and starved for 2 h in a medium
culture without cytokines. They are then stimulated with different concen-
trations of IL-4 for 10 min. Cells were then 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 mM H20, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 10
?g/ml aprotinin). Cell lysates are pretreated with anti-STAT-6 and JAK-3
Abs (Santa Cruz Biotechnology and Upstate Biotechnology, respectively).
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) followed by goat anti-mouse IgG HRP conjugate (Bio-
Rad) and detected by ECL as described by the manufacturer (Amersham
T cell from TCR transgenic mice, wild-type, or ?cyt were stimulated with
mitomycin C-treated T cell-depleted splenocytes (106/ml) and 5 ?g/ml
pMCC under neutral or Th2 skewing conditions as described above for 3
days. T cells were harvested and dead cells were removed by using gradient
centrifugation. Viable cells were then incubated for an additional 24 h with
10 U/ml rmIL-2. For the secondary culture, T cells (5 ? 105/ml) were
restimulated with fresh APC (106/ml) and pMCC (5 ?g/ml) for 16 h. Cells
are then harvested and stained with annexin-FITC and propidium io-
dide-PE using annexin V-FITC apoptosis detection kit II (BD Biosciences)
and CD4-allophycocyanin (BD Biosciences) according to the manufactur-
er’s instructions. In some experiments, 150 ng/ml rhsFasL and 1 ?g/ml
cross-linker (Alexis Biochemicals Axxora) were added at the time of re-
stimulation. Analysis was based on a CD4?cell gate. Data were collected
on a FACSCalibur (BD Biosciences) and analyzed with CellQuest
Caspase 8 activity analysis
To evaluate caspase 8 activation after restimulation with agonist peptide,
CD4 wild-type and ?cyt blasts were generated and restimulated with ag-
onist peptide as described above in the AICD assay. After 16 h of restimu-
lation, the cells were washed and stained for caspase 8 activity using car-
(Immunochemistry Technologies). The apoptotic cells containing cleaved
caspase substrate were detected in the FL1 channel. Cells are analyzed by
gating on CD4?T cells.
CD4 regulation of Th subset differentiation
To investigate the role of CD4 in regulating peripheral T cell dif-
ferentiation, we have used T cells from CD4-deficient mice, re-
constituted with a mutant CD4 transgene, which contains a dele-
tion of the cytoplasmic tail of CD4 (CD4 ?cyt) and inter-crossed
with AND TCR transgenic mice. T cells from AND TCR trans-
genic mice recognize an agonist peptide from moth cytochrome c
(pMCC) in the context of I-EkMHC molecules and are activated
in the absence of CD4 coreceptor function (9, 12). Importantly,
thymic development of functional “CD4 lineage” AND TCR trans-
genic T cells also occurs relatively efficiently in the absence of
CD4 signaling when selected on a homozygous I-Ekbackground
To evaluate the developmental potential of these cells, purified
CD4?CD8?T cells from the spleen and lymph nodes were primed
in vitro with agonist peptide (pMCC) under neutral or Th2 skew-
ing conditions. After 4 days of stimulation, and a 2 day “rest”
period, the cells were then harvested and normalized for cell num-
ber and restimulated with pMCC for an additional 48 h. In cell
cultures stimulated under Th2 skewing conditions, there is a sig-
nificant decrease in IL-4, IL-5, and IL-10 production in CD4 ?cyt
T cells compared with the wild-type control cells, whereas IFN-?
production is suppressed similarly in both groups (Fig. 1). In con-
trast, when the cells were primed under neutral or Th1 skewing
IL-4 signaling is independent of CD4 expression. Purified CD4 ?cyt or WT
controls were stimulated with agonist peptide in the presence or absence of
IL-4. Three days after stimulation, the viable T cells were isolated and
rested for 3 h and then 5 ? 106cells group were restimulated with the
indicated dose of IL-4 for 10 min and immunoprecipitated with antisera to
STAT-6 or JAK-3. Immunoprecipitated proteins were separated by 8%
SDS-PAGE and assessed for tyrosine phosphorylation by Western blot.
The same membranes were reprobed for total STAT-6 or JAK-3. These
data are representative of three independent experiments.
Tyrosine phosphorylation of STAT-6 and JAK-3 following
CD4?or CD4 ?cyt T cells were labeled with 0.5 ?M CFSE and stimulated
with agonist peptide (pMCC) plus IL-2 under neutral or Th2 skewing con-
ditions as described in Fig. 1. Dilution of CFSE labeling is dependent on
cell division and was assessed by flow cytometry at 3 days after initial stim-
ulation. The solid line denotes wild-type cells and the dotted line CD4 ?cyt T
cells. These data are representative of three independent experiments.
Increased cell division in CD4 mutant T cells. Purified
5631The Journal of Immunology
conditions, both wild-type CD4 and CD4 ?cyt cells produce a sim-
ilar amount of IFN-? (Fig. 1). Th2 cytokines are not significantly
induced in both wild-type and CD4 mutant T cells following stim-
ulation with agonist peptide under neutral conditions.
Consistent with previous results, this data suggests that CD4-
dependent signaling is not required for the development of Th1
effector cells, yet is critical for the optimal development of Th2
effector cells (9). Although the production of Th2 cytokines is
decreased in the CD4 mutant T cells, it is important to note that
Th2 development is not completely abrogated. As seen in the
ELISA data and following intracellular single cell analysis of cy-
tokine production when the CD4 mutant T cells are primed under
Th2 conditions, IFN-? production is suppressed and there is con-
tinued production of Th2-associated cytokines, although at a much
lower level than in wild-type CD4 T cells (Fig. 1, A and B). Thus,
it appears that in the absence of CD4-dependent signaling, cells are
not converted into Th1 or Th0 cells but simply fail to develop as
efficiently into a population of Th2 effector cells capable of high
rate cytokine synthesis.
Defect in Th2 development is independent of initial TCR signal
The defect in Th2 differentiation after priming with a strong ago-
nist peptide does not appear related to a defect in initial TCR-
mediated signals, because the agonist peptide used in these studies
is able to stimulate early biochemical signaling events in the ab-
sence of CD4 expression (9). Consistent with this finding, we also
found no defect in cytokine gene transcription early after priming.
As shown by RNase protection assays, wild-type and CD4 ?cyt
cells produced equivalent levels of IL-4 mRNA 48 h after initial
priming with agonist peptide plus exogenous IL-4 (Fig. 2A).
As GATA-3 is both a critical regulator as well as a hallmark of
Th2 development, we also examined GATA-3 expression in the
CD4 mutant T cells. Similar to the RNase protection assay data,
induction of GATA-3 transcription following agonist peptide and
IL-4 stimulation is also unchanged in wild-type and CD4 ?cyt cells
(Fig. 2B). Thus, these results suggest that the initial signaling
events induced by TCR recognition of peptide by CD4 ?cyt T cells
in conjunction with exogenous IL-4 are sufficient for the develop-
ment of a Th2 precursor population. It also suggests that the defect
in Th2 differentiation, seen in the absence of CD4 signaling, occurs
during the maturation and/or clonal expansion phase of Th2 de-
velopment. In total, this data lead to a model in which there are at
least two checkpoints that regulate Th2 development. One is dur-
ing the initial development of a Th2 precursor population, which is
CD4-signaling independent if T cells are activated using a high
potency stimulus. The other is at a later checkpoint that promotes
the selection of a mature Th2 effector cell population and is CD4
for AICD. Purified CD4 ?cyt and wild-type
(WT) T cells were primed with agonist
peptide (pMCC) for 3 days under neutral
Th2 skewing conditions as described in
Fig. 1, then harvested and recultured with
10 U/ml IL-2 in the absence of peptide
stimulation of 24 h. AICD was then in-
duced by restimulation of CD4 T cells with
5 ?g/ml agonist peptide (pMCC) (A and B)
or following exposure to soluble FasL plus
cross-linking (C). After 16 h, cells were
harvested and stained with FITC-annexin
V, PE-propidium iodide, and allophyco-
cyanin-CD4. CD4?cells were gated for
analysis. Apoptotic cells were defined by
annexin V and propidium iodide labeling.
CD4 signaling is required
and regulatory proteins in wild-type and CD4 mutant T cells. A, Purified
wild-type and mutant CD4 T cells were stimulated with agonist peptide for
3 days. Total RNA was extracted and subjected to an RNase protection
assay using commercially available reagents (mAPO-3 kit; BD Pharmin-
gen). B, RNA from the same cell populations was assessed for FasL and
FLIP mRNA using a semiquantitative RT-PCR technique using the pMus3
plasmid-containing ?2-microglobulin as an internal competitor.
Similar expression levels of AICD signaling components
5632 CD4 REGULATION OF AICD AND Th2 DEVELOPMENT
To examine factors important in Th2 development that may be
influenced by CD4 signaling, we initially focused on IL-4R sig-
naling. As indicated in Figs. 1 and 2, several aspects of IL-4R
signaling appear grossly intact as shown by efficient suppression of
IFN-? production and induction of GATA-3 transcription. A more
direct analysis of early IL-4R signaling events also reveals that
STAT-6 and JAK-3 phosphorylation are induced similarly in both
CD4 wild-type and CD4 ?cyt T cells (Fig. 3). This data indicates
that proximal IL-4R signaling events are not affected in the ab-
sence of CD4 signaling.
CD4 signaling regulates AICD
Consistent with our previous observations that CD4 mutant T cells
are efficiently activated by the agonist peptide used in these stud-
ies, CFSE labeling before stimulation with peptide in the presence
of exogenous IL-2 reveals that CD4 ?cyt T cells exhibit a signif-
icantly greater rate of cell division in cells primed under both neu-
tral and Th2 skewing conditions (Fig. 4). A possible explanation
for this finding is that the CD4 mutant T cells were more resistant
to AICD than wild-type cells resulting in prolonged survival and
enhanced cell division. Indeed, when wild-type or CD4 ?cyt T cell
blasts were restimulated with agonist peptide, the wild-type cells
underwent apoptotic cell death to a much greater extent than the
CD4 mutant T cells (Fig. 5A). Importantly, although the overall
level of cell death is lower in cells primed for 3 days under Th2
conditions there remains a significant decrease in death in the ab-
sence of CD4 signaling compared with wild-type CD4 T cells (Fig.
5B). This defect in AICD is also seen when the CD4 mutant blasts
are stimulated with recombinant soluble FasL followed by cross-
linking (Fig. 5C). These data indicate that there is a defect in AICD
in the CD4 mutant T cells, which is at least due in part to a defect
in the Fas signaling pathway.
One possible explanation for the decreased AICD in the CD4
mutant T cells is failure to express components of the death re-
ceptor signaling pathways (e.g., FasL), and/or overexpress nega-
tive regulators of AICD (e.g., FLIP). In semiquantitative RT-PCR
assays, and/or in RNase protection assays we have not found any
evidence for decreased expression of Fas, FasL, Fas-associated
death domain protein, caspase 8, TRAIL, TNFR, or increased ex-
pression of FLIP (Fig. 6). Consistent with this data, we have found
that FasL expression as well as expression of other death receptor
signaling components is more sustained over time in CD4 mutant
cells compared with wild-type T cells. Typically, expression of
FasL is transient on T cells because membrane expression allows
cis-interactions with Fas resulting in cell death of the FasL ex-
pressing cells. When FasL membrane expression was examined in
wild-type and CD4 mutant T cells, we found that expression
peaked at day 1 after restimulation of wild-type T cells but was not
detectable at day 2. In contrast, the CD4 mutant T cells exhibited
sustained detectable levels of FasL expression through day 2 (Fig.
7). Flow cytometric analysis of Fas expression was also equivalent
in CD4 wild-type and CD4 mutant T cells (data not shown). In
total, this data suggests that in the absence of CD4-dependent sig-
naling, there is a defect in the signaling events mediating AICD
independent of Fas and FasL expression.
The defect in Fas signaling in the CD4 mutant T cells appears to
occur before activation of caspase 8 in the cell death pathway,
because caspase 8 activation was also significantly decreased in
the CD4 mutant cells (Fig. 8). Activation of caspase 8 occurs early
on in the death receptor signal transduction cascade upon assembly
of the death-inducing signaling complex (DISC). This process re-
quires appropriate oligomerization of death effector domains
within Fas-associated death domain protein following association
with Fas. Thus failure to activate caspase 8, in conjunction with
appropriate Fas and FasL expression, suggests that the defect in
AICD in the CD4 mutant T cells may be related to dysregulation
of the assembly or composition of the DISC.
CD4 mutant T cell defect in AICD is linked to the defect in Th2
In experiments designed to link the defect in AICD described
above to the defect in Th2 development described earlier, we have
inhibited AICD using anti-Fas or anti-FasL Abs in wild-type CD4
cells. Purified wild-type and dcyt CD4 T cells were stimulated as in Fig. 5
and then restimulated with agonist peptide. Flow cytometric analysis of
FasL expression in wild-type (dark line) and ?cyt (pale line) CD4 T cells
was done 1 and 2 days after restimulation. Data shown are FasL expression
using a “gate” on CD4?T cells. The shadowed histogram represents la-
beling with an isotype control Ab.
Sustained increase in FasL expression in CD4 mutant T
neutral conditions as described in Fig. 5 and then left unstimulated (?) or
restimulated with agonist peptide (?) for 14 h. Caspase 8 activation was
detected using a fluorescent reagent specific for active caspase 8 (FAM-
LETD-FMK) obtained from Immunochemistry Technologies. This reagent
is cell permeable and binds to active caspase 8. In the absence of caspase
8 activity unbound reagent is washed away. The percentage of cells con-
taining fluorochrome-bound active caspase 8 was determined by flow cy-
tometric analysis after gating on CD4?T cells.
Purified wild-type or dcyt CD4 T cells were primed under
5633The Journal of Immunology
cells and assessed the subsequent impact on Th2 development.
Initial experiments were done to confirm that anti-Fas and anti-
FasL Abs would block AICD in wild-type CD4 T cells to the low
levels seen in the ?cyt T cells following Ag restimulation (Fig. 9,
A and C). Subsequent experiments went on to assess the effect of
blocking AICD with Fas or FasL Ab during Th2 priming. In these
experiments, wild-type CD4?T cells were first stimulated under
Th2 cytokine skewing conditions as described in Fig. 1. One or 2
days after the initial stimulation, anti-Fas (Jo-2) or anti FasL
(MFL3) was added. When cells were analyzed for IL-4 production
after being treated with Ab during priming only and then restim-
ulated with peptide, there was a defect in IL-4 production similar
to that seen in the CD4 ?cyt T cells after challenge (Fig. 9, B and
D). There was little or no effect on Th1 development as shown by
IFN-? production in cells primed under neutral or Th1 skewing
conditions following treatment with anti-Fas or anti-FasL (Fig. 9,
B and D). No significant IFN-? production was detected in cells
primed under Th2 conditions upon treatment with anti-Fas or anti-
FasL as shown by ELISA or intracellular cytokine analysis (data
In total, these data suggest that inhibition of AICD after initial
priming of naive CD4 T cells specifically inhibits the development
of Th2 effector cells. Furthermore, this data suggests that the de-
fects we have described in the regulation of AICD in the absence
of CD4 signaling are related to the defect in Th2 development.
The role of CD4 as a coreceptor that promotes initial T cell Ag
receptor-mediated activation events is well established. However,
the role of CD4 in regulating previously activated T cells and
ongoing immune responses is less well understood. In the present
study, we have found that CD4 signaling is required for a distinct
checkpoint during Th2 effector cell development, which is inde-
pendent of initial T cell stimulation. Although production of Th2-
associated cytokines is significantly diminished in T cells primed
under Th2 conditions in the absence of CD4 signaling, initial Th2
development appeared intact as defined by the appropriate induc-
tion of IL-4 and GATA-3 transcription. These findings are quite
distinct from other studies in itk, SAP, and fyn-deficient mice in
which Th2 development was compromised in cells primed under
5 in the presence or absence of anti-Fas (2 ?g/ml Jo-2) or anti-FasL (5 ?g/ml) added during restimulation with agonist peptide. B, Purified CD4 T cells
from AND TCR transgenic mice were stimulated under neutral or Th2 skewing conditions as in Fig. 1. Two days after initial stimulation, 2 ?g/ml anti-Fas
or isotype control Ab were added to the cultures. The cells were maintained in culture for an additional 2 days, then harvested, washed, and rested for 2
days before restimulation with agonist peptide and APC. After 48 h, culture supernatants were collected and analyzed for the detection of IL-4 or IFN-?
production by ELISA. IFN-? production was below detectable limits in cells primed under Th2 skewing conditions under all conditions. D, Cells were
stimulated under Th2 or Th1 skewing conditions as described in Fig. 1, and 1 day after priming 5 ?g/ml anti-FasL or isotype control Ab were added to
the cultures. Following 4 days of stimulation, the cells were harvested, washed, and restimulated for 2 additional days with agonist peptide and APC only.
Supernatants were collected and analyzed for IL-4 and IFN-? by ELISA. These data are representative of at least two independent experiments.
Inhibition of AICD causes decreased Th2 development. A and C, WT and ?cyt CD4 T cells were assayed for AICD as described in Fig.
5634 CD4 REGULATION OF AICD AND Th2 DEVELOPMENT
neutral conditions, but was recovered upon the addition of exog-
enous IL-4 in conjunction with a high potency TCR stimulus (14–
16). In these cases, the defect in Th2 development appears related
to initial T cell signal transduction events that are required for IL-4
production. In contrast, CD4 mutant T cells appear to be initially
primed to undergo Th2 differentiation after stimulation with a
strong agonist peptide and exogenous IL-4, but do not become a
fully developed Th2 effector population when compared with wild-
type CD4 T cells.
In addition to the defect in Th2 development, T cells unable to
signal through CD4 are relatively insensitive to AICD and Fas-
mediated death receptor signaling. The defect in Th2 development
and AICD appear linked as shown in experiments in which inhi-
bition of Fas-mediated cell death in wild-type T cells also results
in a defect in Th2 development. These data suggest that the ap-
propriate regulation of AICD during Th development is an impor-
tant factor in controlling the selection of different effector cell sub-
set populations. It has been recognized for some time that mature
Th2 effector populations are relatively resistant to AICD (17, 18).
We propose that the development of this resistant phenotype is an
important factor in enriching for committed Th2 effector cells in a
mixed population. In this model, cells that are more committed to
the Th2 developmental pathway will be correspondingly more re-
sistant to AICD and will have a survival advantage over less com-
mitted Th cells. In Th precursor cells that do not signal efficiently
through CD4, there is an inappropriate resistance to AICD remov-
ing the survival advantage of the more committed Th2 cells. This
creates a more mixed population of effector cells after several days
of cell expansion (and cell death), with a relative decrease in the
proportion of high-rate IL-4 producing Th2 effector cells.
This model is supported by another report describing the paradox-
ical effect of IFN-? in promoting Th2 development (19). Bocek et al.
found that cells in an IFN-?-deficient environment were less efficient
in developing Th2 effector cells. Similar to our model, the authors
proposed that IFN-? promotes susceptibility to AICD in uncommitted
Th precursors, which allows more committed Th2 cells that are rel-
atively resistant to AICD to have a selective advantage. The regula-
tory role of Fas and AICD in selecting for Th effector cell subsets is
also consistent with data in young MRL/lpr (Fas deficient) mice
which exhibit a propensity toward Th1 development and IFN-? pro-
duction compared with MRL control mice (20).
The defect in AICD seen in the absence of CD4 signaling is
similar to that observed in some previous reports; however, the
molecular basis for this defect has not been clear (21, 22). In con-
trast to the results of Hamad et al. (22), we do not find any evi-
dence of a decrease in FasL expression in the CD4 mutant T cells.
Instead, our data suggests that CD4 signals are important to render
Fas competent to initiate death receptor signaling and initiate
caspase activation. It has been known for some time that expres-
sion of Fas and FasL is not sufficient for the induction of cell death
in T lymphocytes, but that additional signals from the CD3/TCR
complex provide a critical competency signal (23, 24). The precise
nature of this signal is not yet known; however, it closely corre-
lates with the oligomerization of Fas molecules and Fas residence
in lipid raft membrane microdomains (25). Our current data sug-
gest that CD4 signaling provides a necessary part of this compe-
Although CD4 signaling may affect other components of death
receptor signaling, our hypothesis that CD4 promotes oligomer-
ization of the Fas signaling macromolecular complex, is consistent
with our previous data indicating that CD4 signaling is required for
the association of the TCR complex with lipid rafts after T cell
activation (26). CD4 may play a similar role in promoting Fas
association with lipid rafts and/or in promoting Fas oligomeriza-
tion. The mechanism(s) by which CD4 (and presumably CD4-
associated lck) may promote oligomerization and effective Fas sig-
naling are also not yet clear. One possibility is that CD4 signaling
provides a positive signal required for DISC oligomerization. For
example in the absence of CD4 there may be a defect in protein
kinase C activation, which has been suggested to be an important
signaling event in developing competence for Fas-mediated cell
death following CD4 and/or CD3/TCR cross-linking (24, 27). Al-
ternatively, CD4 signaling may be required to inhibit signaling
pathways that constitutively down-regulate Fas signaling path-
ways. One possibility in this scenario would be inappropriate
activation of the ras/raf/ERK pathway due to a requirement of
CD4-associated lck for the activation of dok family members and
ras-GAP activation, which normally would inhibit ERK activation.
Inappropriate activation of ERK has been associated with resis-
tance to Fas-mediated cell death (28); thus CD4-dependent signal-
ing may be normally required to down-regulate ERK activation,
which would in turn enhance sensitivity to Fas-mediated cell
Regardless of the mechanism by which CD4 promotes AICD,
our data suggest that this is a critical step in the development and
selection of Th effector populations. These data may have impor-
tant implications for controlling Th development in vivo. Follow-
ing initial T cell priming, developing T cells will typically be in a
complex microenvironment with a variety of competing signals
and cytokines capable of influencing Th1 and Th2 effector cell
development. Our finding that dysregulation of AICD in CD4 mu-
tant T cells affects Th2 development, suggests that the differential
development of susceptibility and resistance to AICD seen in Th1
and Th2 cells can be a factor in selecting for biased Th1 and Th2
effector populations. Therapeutic strategies that alter susceptibility
to AICD during the Th development may be an effective means of
biasing outcomes of T cell activation.
We thank Drs. Mark Williams, Achsah Keegan, Stan Vukmanovic, and
Stephanie Constant for critical review of the manuscript and for helpful
The authors have no financial conflict of interest.
1. Leitenberg, D., and K. Bottomly. 1999. Regulation of naive T cell differentiation
by varying the potency of TCR signal transduction. Semin. Immunol. 11:
2. Murphy, K. M., and S. L. Reiner. 2002. The lineage decisions of helper T cells.
Nat. Rev. Immunol. 2: 933–944.
3. Ho, I.-C., and L. H. Glimcher. 2002. Transcription: tantalizing times for T cells.
Cell 109: S109–S120.
4. Farber, D., M. Luqman, O. Acuto, and K. Bottomly. 1995. Control of memory
CD4 T cell activation: MHC class II molecules on antigen presenting cells and
CD4 ligation inhibit memory but not naive CD4 T cells. Immunity 2: 249–259.
5. Stumbles, P., and D. Mason. 1995. Activation of CD4?T cells in the presence of
a non-depleting monoclonal to CD4 induces a Th2-type response in vitro. J. Exp.
Med. 182: 5–13.
6. Chirmule, N., A. Avots, S. M. Lakshmitamma, S. Pahwa, and E. Serfling. 1999.
CD4-mediated signals induce T cell dysfunction in vivo. J. Immunol. 163:
7. Fowell, D. J., J. Magram, C. W. Turck, N. Killeen, and R. M. Locksley. 1997.
Impaired Th2 subset development in the absence of CD4. Immunity 6: 559–569.
8. Brown, D. R., N. H. Moskowitz, N. Killeen, and S. L. Reiner. 1997. A role for
CD4 in peripheral T cell differentiation. J. Exp. Med. 186: 101–107.
9. Leitenberg, D., Y. Boutin, S. Constant, and K. Bottomly. 1998. CD4 regulation
of T cell receptor signaling and T cell differentiaion following stimulation with
peptides of different affinites for the T cell receptor. J. Immunol. 161: 1194–1203.
10. Kaye, J., M.-L. Hsa, M.-E. Sauron, S. C. Jameson, N. Gasciogne, and
S. M. Hendrik. 1989. Selective development of CD4?T cells in transgenic mice
express a class II MHC restricted antigen receptor. Nature 341: 746–749.
11. Killeen, N., and D. R. Littman. 1993. Helper T-cell development in the absence
of CD4–p56lck association. Nature 364: 729–732.
5635 The Journal of Immunology
12. Yelon, D., K. L. Schaefer, and L. J. Berg. 1999. Alterations in CD4-binding
regions of the MHC class II molecule I-Ek do not impede CD4?T cell devel-
opment. J. Immunol. 162: 1348–1358.
13. Matechak, E. O., N. Killeen, S. M. Hedrick, and B. J. Fowlkes. 1996. MHC class
II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity
14. Miller, A. T., H. M. Wilcox, Z. Lai, and L. J. Berg. 2004. Signaling through Itk
promotes T helper 2 differentiation via negative regulation of T-bet. Immunity 21:
15. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols,
A. Antonellis, G. A. Koretzky, K. Gardner, and P. L. Schwartzberg. 2004. SAP
regulates T(H)2 differentiation and PKC-? mediated activation of NF-?B1. Im-
munity 21: 693–706.
16. Davidson, D., X. Shi, S. Zhang, H. Wang, M. Nemer, N. Ono, S. Ohno,
Y. Yanagi, and A. Veillette. 2004. Genetic evidence linking SAP, the X-linked
lymphoproliferative gene product to src-related kinase FynT in T(H)2 cytokine
regulation. Immunity 21: 707–717.
17. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato,
J. C. Reed, D. Green, and S. L. Swain. 1997. Unequal death in T helper cell (Th)
1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated
apoptosis. J. Exp. Med. 185: 1837–1849.
18. Varadhachary, A. S., S. N. Perdow, C. Hu, M. Ramanarayanan, and P. Salgame.
1997. Differential ability of T cell subsets to undergo activation-induced cell
death. Proc. Natl. Acad. Sci. USA 94: 5778–5783.
19. Bocek, P., G. Foucras, and W. E. Paul. 2004. Interferon ? enhances both in vitro
and in vivo priming of CD4?T cells for IL-4 production. J. Exp. Med. 199:
20. Zhang, X. R., L. Y. Zhang, S. Devadas, L. Li, A. D. Keegan, and Y. F. Shi. 2003.
Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of ap-
optosis in T helper subset differentiation. Cell Death Differ. 10: 203–210.
21. Maroto, R., X. Shen, and R. Konig. 1999. Requirement for efficient interactions
between CD4 and MHC class II molecules for survival of resting CD4?T lym-
phocytes in vivo and for activation-induced cell death. J. Immunol. 162:
22. Hamad, A. R., A. Srikishnan, P. Mirmonsef, C. P. Broeren, C. H. June,
D. Pardoll, and J. P. Schneck. 2001. Lack of coreceptor allows survival of chron-
ically stimulated double-negative ?/? T cells: implications for autoimmunity.
J. Exp. Med. 193: 1113–1121.
23. Hornung, F., L. Zheng, and M. J. Lenardo. 1997. Maintenance of clonotype
specificity in CD95/Apo-1/Fas-mediated apoptosis of mature T lymphocytes.
J. Immunol. 159: 3816–3822.
24. Wong, B., J. Arron, and Y. Choi. 1997. T cell receptor signals enhance suscep-
tibility to Fas-mediated apoptosis. J. Exp. Med. 186: 1939–1944.
25. Muppidi, J. R., and R. M. Siegel. 2004. Ligand-independent redistribution of Fas
(CD95) into lipid rafts mediates clonotypic T cell death. Nat. Immunol. 5:
26. Balamuth, F., D. Leitenberg, J. Unternaehrer, I. Mellman, and K. Bottomly. 2001.
Distinct patterns of membrane microdomain partitioning in Th1 and Th2 cells.
Immunity 15: 729–738.
27. Algeciras, A., D. H. Dockrell, D. H. Lynch, and C. V. Paya. 1998. CD4 regulates
susceptibility to Fas ligand- and tumor necrosis factor-mediated apoptosis. J. Exp.
Med. 187: 711–720.
28. Holmstrom, T. H., I. Schmitz, T. S. Soderstrom, M. Poukkula, V. L. Johnson,
S. C. Chow, P. H. Krammer, and J. E. Eriksson. 2000. MAPK/ERK signaling in
activatied T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC
assembly. EMBO J. 19: 5418–5428.
5636CD4 REGULATION OF AICD AND Th2 DEVELOPMENT