By
From the * Department of Medicine, Committee on Immunology and Gwen Knapp Center for Lupus
and Immunology Research, University of Chicago, Chicago, Illinois 60637; and Department of
Microbiology and Immunology, University of California, San Francisco, San Francisco, California
94143
Naive CD4+ T helper cells (Th) differentiate into one of two well-defined cell types during immune responses. Mature Th1 and Th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. CD4 serves a prominent role in potentiating antigen recognition by helper T cells. We have examined the role of CD4 in peripheral T cell differentiation by studying helper T cells from mice with a congenital defect in CD4 expression. After protein immunization or infection with Leishmania major, CD4-deficient mice were incapable of mounting antigen-specific Th2 responses, but retained their Th1 potency. CD4-deficient, T cell receptor transgenic T cells were also incapable of Th2 differentiation after in vitro activation. Expression of a wild-type CD4 transgene corrected the Th2 defect of CD4-deficient mice in all immune responses tested. To investigate the role of the cytoplasmic domain, mice reconstituted with a truncated CD4 molecule were also studied. Expression of the tailless CD4 transgene could not rescue the Th2 defect of CD4-deficient mice immunized with protein or CD4-deficient transgenic T cells activated in vitro, raising the possibility that the cytoplasmic domain of CD4 may influence Th2 generation. Expression of the tailless transgene was, however, capable of restoring Th2 development in CD4-deficient mice infected with L. major or CD4-deficient transgenic T cells activated in the presence of recombinant IL-4, demonstrating that the cytoplasmic domain is not absolutely required for Th2 development. Together, these results demonstrate a previously undescribed role of the CD4 molecule. The requirement for CD4 in Th2 maturation reflects the importance of molecules other than cytokines in the control of helper T cell differentiation.
Akey functional characteristic of CD4+ helper T cells is
their capacity to secrete cytokines when stimulated
with peptides presented by MHC class II molecules. During an immune response, a differentiation process gives rise
to one of two types of mature helper T cells (see reviews in
references 1 and 2). Th1 cells produce IFN- Although cytokines have a dominant role in helper T cell
differentiation, there is mounting evidence that TCR signaling and costimulation can also influence helper T cell fate
(2). Of the many cell surface molecules involved in T cell
activation, CD4 has a unique capacity to enhance TCR
signaling through direct interactions with MHC class II
molecules on antigen presenting cells (5) and through its
intracellular association with the src-related tyrosine kinase
p56lck (6). The possible involvement of CD4 in regulating the differentiation of helper T cells has been suggested
by several studies (11), but remains unclear.
We have studied the functional properties of a unique
population of helper T cells that develops in CD4-deficient
(CD4°)1 mice. Earlier studies have demonstrated the capacity of these cells to become Th1 cells (14). To examine the
role of CD4 in the full spectrum of peripheral differentiation, we have generated CD4° mice on the Th2-prone
BALB/c background and examined their response in several well-characterized models of helper T cell maturation.
Under all circumstances tested, we found that helper T cells
lacking CD4 are defective in their capacity to adopt the Th2 fate. CD4° mice were bred with mice expressing a
transgene encoding either wild-type CD4 or a mutant form
of the molecule lacking its cytoplasmic portion (15). While
the full-length molecule was capable of reconstituting Th2
development, the tailless molecule was only partially capable of rescuing Th2 maturation. Thus, our results define a
novel function for CD4 in the determination of peripheral
T cell fate through its selective potentiation of Th2 maturation.
CD4-deficient Mice.
CD4° mice used in these experiments
were from the fifth backcross to C57BL (C57BL/6 or B10.D2)
or fifth backcross to BALB/c. Heterozygote and wild-type littermates were used as controls. Mice expressing transgenes encoding a
wild-type or truncated CD4 molecule have been previously described (15). CD4° mice and CD4 transgenic mice were also bred
to mice expressing a transgenic TCR specific for a peptide from
chicken OVA presented by I-Ad (16; provided by Dennis Loh,
Washington University, St. Louis, MO). All TCR transgenic experiments were performed using cells from mice of the BALB/c
background. Mice were housed in a specific pathogen-free environment. Genotypes were determined by flow cytometry using
stains for commercially conjugated fluorescent mAbs against CD4
(CALTAG Labs., South San Francisco, CA). All work was performed in accordance with University of Chicago (Chicago, IL)
guidelines for animal use and care.
KLH Immunization.
BALB/c or C57BL mice plus normal
littermate controls were immunized in the hind footpads with
150 µg KLH (Calbiochem Corp., San Diego, CA) emulsified in
complete Freund's adjuvant (Sigma Chemical Co., St. Louis,
MO). 7-10 d after immunization, draining popliteal lymph nodes
were harvested and single cell suspensions were prepared in complete Iscove's media (supplemented with 10% fetal calf serum, 2 mM
1-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and
50 µM 2-mercaptoethanol). For the restimulation assay, 106 cells
were plated in each well of a flat-bottom 96-well microtiter plate
(Costar Corp., Cambridge, MA) with either media or KLH at 30 µg/ml. Supernatants were harvested at 48 h and analyzed for IL-4 and IFN- Infection with Leishmania major.
Infections were performed by
injection of 5 × 105 metacyclic promastigotes (WHOM/IR/-/
173) into each hind footpad. Lesion size was measured weekly
with a metric caliper. Leishmania-infected mice were killed at 6-8 wk
after infection for cytokine analysis. Single cell suspensions of
popliteal lymph node cells from individual animals were examined for antigen-specific cytokine production by restimulation in
vitro. In brief, 0.5-1 × 106 lymph node cells/well were cultured
in a 96-well round-bottom microtiter plate in complete media
with and without soluble Leishmania antigen (100 µg/ml). MHC
class II was blocked using mAb M5/114 (rat IgG2b anti-Ab,d and
-Ed,k), whereas mAb 10-2-16 served as an isotype control (mouse
IgG2b anti-Ak). Supernatants from the restimulation cultures
were removed at 48 h and analyzed for IL-4 and IFN- In Vitro Priming of Transgenic T Cells.
CD8 Anti-CD3 Stimulation of T Cells.
CD8 Competitive PCR Analysis.
106 purified CD8 To induce T helper subset differentiation in vivo, CD4°
mice were immunized with the protein antigen, KLH.
Draining lymph node cells were restimulated 7 d later with
antigen in vitro, and the cytokine profile was assessed. Cultures from CD4+ animals produced abundant IL-4, as well
as moderate amounts of IFN-
To examine T helper cell differentiation in response to
chronic infection with a parasite, we challenged mice with
L. major. Infected BALB/c CD4+ mice developed progressively, enlarging footpad lesions. In striking contrast, BALB/c
CD4° mice developed minimal footpad swelling, which resolved over time and was indistinguishable from genetically resistant controls (Fig. 2 A). Parasite cultures from the feet and spleens of infected mice confirmed that BALB/c CD4°
mice had resolved infection (data not shown). When lymph
node cells from infected animals were restimulated in vitro
with parasite antigens, the cells from CD4+ mice produced
high levels of IL-4 and low levels of IFN-
The absence of CD4 decreases positive selection in CD4°
mice resulting in a reduced number of helper T cells (14,
17). To correct for this deficiency in cell number, the capacity of CD4-deficient helper T cells to adopt the Th2
fate was examined using in vitro differentiation assays. CD4°
mice were bred to mice expressing a transgenic TCR specific for a peptide from chicken OVA presented by I-Ad
(16). The clonotypic TCR formed by pairing of the transgenic
As a further test of the intrinsic differentiation capacity of
CD4-deficient helper T cells, we established an antigen-
MHC-independent system of T cell activation thereby eliminating differences in T cell-APC adhesion of CD4+ and
CD4° T cells. Highly purified CD8
Despite the inability to generate a Th2 response, there
was no deficiency in the proliferative response of the CD4°
T cells (Fig. 4 C). Thus, an inability to proliferate normally
does not appear to account for the Th2 defect. Helper T cells
initially transcribe multiple lymphokine genes in response
to an activation stimulus (18). The addition of rIL-4 to cultures enhances IL-4 messenger RNA (mRNA) and suppresses IFN-
The association of CD4 with p56lck has been implicated in signaling
and in the recruitment of CD4 to the TCR complex (20,
21). To study the role of this association in the potentiation
of the Th2 fate, we used transgenic mice expressing wild-type CD4 (CD4WT) or a mutant lacking the cytoplasmic
tail (CD4
The results presented in this paper suggest that the function of CD4 influences the outcome of helper T cell differentiation. CD4° helper T cells exhibited a consistent bias
toward the Th1 fate in several types of immune responses,
even under optimal in vitro conditions of cell number and
in the presence of exogenous IL-4. Furthermore, the defect
in Th2 differentiation was observed for CD4° mice on
both the Th2-prone BALB/c background and for mice on
the C57BL/6 background. We cannot fully exclude the
possibility that CD4° T cells exit the thymus with an incomplete helper potential. However, CD4° T cells were
capable of proliferating and generating IL-4 mRNA early
in the response to TCR ligation, showing that the absence
of CD4 does not impair these common aspects of T helper cell activation. Rather, the results of in vitro assays suggest that the lack of CD4 results in an intrinsic defect in the capacity of helper T cells to generate signals for proper Th2
differentiation. Thus, the results support a novel role for
CD4 in the potentiation of the Th2 fate during immune
responses.
In an effort to explore the mechanism by which CD4 influences the fate of T helper cells, we used two previously
described lines of transgenic mice that express either CD4WT,
or CD4 The requirement for CD4 in T helper cell differentiation
may be consistent with models proposing that stronger interactions and activation signals favor Th2 differentiation
(2). In this regard, it is noteworthy that the Th2 defect
was evident even when CD4° T cells were activated in an
MHC class II-independent fashion using anti-CD3 mAbs.
This observation is perhaps paradoxical given the expectation that anti-CD3 stimulation should bypass the need for
CD4 in T cell activation. However, previous work has
shown that the activation of T cells with anti-CD3 antibodies can promote the recruitment of CD4 to the TCR
complex (23, 24). Thus, CD4+ T cells treated with anti-CD3 may receive a qualitatively or quantitatively distinct
activation signal from that received by CD4° T cells, due to
differential recruitment of CD4 and its associated p56lck.
Nonetheless, it is also possible that the absence of CD4 impairs or changes TCR/CD3 signaling in a distinct and crucial fashion, as has been suggested by studies on T cell hybridomas (25). Regardless of how CD4 exerts its effect on
the differentiation process, the data presented here would
still be consistent with a model that involves commitment
to the Th2 fate only when the character of antigen-specific
signaling is sufficiently complex or efficient.
As has been previously reported, the lack of CD4 expression on T helper cells did not impair the differentiation
of Th1 cells. Interestingly, CD4° T cells showed normal
levels of IL-4 mRNA, but enhanced levels of IFN- and provide
help for cell-mediated immunity, whereas Th2 cells produce IL-4 and support the generation of humoral immunity. These two types of helper cells derive from a common naive precursor that has a fate determined in large part
by exposure to cytokines. In particular, IL-12 is a potent
inducer of the Th1 fate while IL-4 drives cells toward the
Th2 fate (1, 2).
by ELISA using commercial mAb pairs (PharMingen, San Diego, CA) according to the manufacturer's instructions.
production by ELISA.
T cells were isolated from spleens and lymph nodes of fifth BALB/c CD4+ or
CD4° T cell receptor transgenic mice (16) by depletion of B220+
and CD8+ cells using mAbs and magnetic beads (PerSeptive Biosystems, Cambridge, MA). Where indicated, CD4° mice also expressed a transgene encoding either a full-length or truncated
CD4 molecule. An aliquot was stained with mAb KJ1-26 to calculate the percentage of cells expressing clonotypic antigen receptors. 2.5 × 105 clonotypic cells were cultured in 1 ml with 2.5 × 106 irradiated (2,500 rads) T-deficient splenocytes and 0.3 µM
OVA peptide in the presence or absence of rIL-4 (20,000 U/ml).
After 5 d, cells were washed extensively and 2 × 105 cells were
restimulated with 8 × 105 irradiated splenocytes and 7.5 µM
OVA peptide in 200 µl. Supernatants were harvested at 48 h for
quantitation of IL-4 and IFN-
by ELISA.
T cells (>95% pure)
were isolated from lymph nodes of B10.D2 CD4+ or CD4° mice
by depletion of B220+, CD8+, NK1.1+, and MHC class II+ cells
with mAbs and magnetic beads. 5 × 105 cells were cultured in
wells precoated with anti-CD3 (2C11; 10 µg/ml; a gift of Dr. Jeff
Bluestone, University of Chicago, Chicago, IL) plus rIL-4 (10,000 U/ml) in 1 ml. After 5 d, cells were washed extensively and 105
cells were restimulated in anti-CD3-coated 96-well flat-bottom plates in triplicate for an additional 48 h. Cell number and anti-CD3 concentrations vary where indicated. IL-4 and IFN-
were
quantitated from supernatants by ELISA. For determination of
thymidine incorporation, 1 µCi of methyl-[3H]thymidine (Amersham Life Science, Arlington Heights, IL) was added to wells in 25 µl after 48 h. Cultures were harvested 12-14 h later onto filters,
and radioactivity was measured (Betaplate 1205 counter; Wallac,
Turku, Finland).
lymph node
cells were cultured in the presence of rIL-4 (10,000 U/ml) on
plates precoated with anti-CD3 (10 µg/ml). After 48 h, the cells
were pelleted and RNA was extracted with TRIzol Reagent
(GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. RNA was reverse transcribed using random
hexamer primers (Pharmacia, Piscataway, NJ). Semiquantitative PCR was performed as described (14). In brief, cDNA was amplified in the presence of a polycompetitor construct that contains
addition mutations of the authentic cDNA. Resolved on agarose
gels, the larger molecular weight bands provide an internal standard for the relative amounts of the lower molecular weights experimental cDNAs. Concentrations of cDNAs are adjusted using
the housekeeping gene hypoxanthine phosphoribosyl transferase before assay of lymphokine gene transcription. Results were verified
by repetition of both individual samples and whole experiments.
Impaired In Vivo Th2 Responses in the Absence of CD4.
, whereas the cells from
CD4° mice made the opposite response, producing very
little IL-4 and accentuated levels of IFN-
(Fig. 1 A). The
addition of an anti-MHC class II mAb to the cultures blocked all antigen-driven cytokine production, confirming that the responses were indeed mediated by MHC class
II-restricted helper T cells (data not shown). Moreover, the
CD4-deficient helper T cells manifested the same Th2 defect in the absence of MHC class I expression in doubly deficient CD4°
2-microglobulin (
2m)° mice (Fig. 1 B). Thus,
in vivo Th2 differentiation in response to KLH immunization
is dependent on CD4 expression.
Fig. 1.
The absence of CD4 impairs Th2 differentiation during immunization. (A) BALB/c CD4+ or CD4° mice were immunized with
KLH, and draining lymph node cells were harvested 7-10 d later for a restimulation assay as described in Materials and Methods. Supernatants
were harvested at 48 h for quantitation of IL-4 (filled bars) and IFN-
(open bars) by ELISA. The results shown are the mean of triplicate determinations from two CD4+ and 3 CD4° mice, and are representative of 16 separate experiments involving 6 BALB/c CD4+, 9 BALB/c CD4°, 25 C57BL CD4+, and 24 C57BL CD4° mice. Cells cultured without the addition of KLH produced <25 U/ml of IL-4 and 0.5 U/ml of IFN-
. Standard deviations are expressed as y-axis error bars throughout the figures. (B)
2m-deficient mice (
2m°CD4+) and double mutant (
2m°CD4°) were immunized with KLH and restimulated as described above. Results are the mean cytokine measurements of triplicate determinations using
three animals in each group.
[View Larger Versions of these Images (15 + 15K GIF file)]
, whereas the
cells from CD4° mice produced high levels of IFN-
and
minimal IL-4 (Fig. 2 B). The addition of an anti-MHC
class II antibody to the cultures blocked all cytokine production, again confirming that class II-restricted helper T
cells mediated these effects (Fig. 2 B). Thus, like the response to KLH, L. major infection in BALB/c CD4° mice
is characterized by defective Th2 differentiation.
Fig. 2.
BALB/c CD4° mice
are resistant to L. major. (A)
BALB/c CD4+ (+/+, +/),
CD4° (
/
) littermates, and genetically resistant C57BL/6 mice were injected with 5 × 105
metacyclic L. major promastigotes in each hind footpad. Disease progression was assessed by
measuring footpad thickness
with a metric caliper. The results
shown are the mean footpad
measurements of individual
BALB/c mice and are representative of 10 separate experiments using 24 BALB/c CD4+, and 20 BALB/c CD4° mice. (B). Infected BALB/c CD4+/+, CD4+/
, or CD4° mice
were killed after 7 wk, and popliteal lymph node cells were cultured with either no addition (M) or with the addition of 100 µg/ml of soluble parasite
antigens in triplicate (Antigen). Wells receiving antigen were cultured without antibody [(
)] or with either anti-MHC class II (
II) or a control antibody
(iso). After 48 h, supernatants were harvested for quantitation of IL-4 (filled bars) and IFN-
(open bars) by ELISA. The results shown are the mean of triplicate wells from individual mice and are representative of eight experiments using 19 CD4+ and 14 CD4° mice.
[View Larger Versions of these Images (21 + 16K GIF file)]
and
chains is recognized by the mAb KJ1-26
(16). Equal numbers of CD4 lineage (CD8-negative) KJ1-26+ helper T cells were stimulated in vitro with OVA peptide and APCs. After 5 d, cells were washed extensively
and restimulated to assess their differentiation state. CD4+
T cells produced high levels of IL-4 and low levels of IFN-
, whereas CD4° T cells produced greatly reduced levels of
IL-4 and accentuated levels of IFN-
(Fig. 3). The addition
of rIL-4 to primary cultures strongly biases toward Th2 differentiation (1). CD4° T cells activated in the presence of
rIL-4 still yielded only minimal IL-4 production upon restimulation (Fig. 3). Thus, even when responding helper
T cell numbers were equalized, the CD4° cells were still
incapable of antigen-driven Th2 differentiation.
Fig. 3.
The absence of CD4 impairs antigen-specific Th2 responses
in vitro. CD8 T cells were isolated from spleens and lymph nodes of
CD4+ or CD4° BALB/c D011.10 TCR transgenic mice. An equivalent
number of clonotypic cells were cultured in 1 ml with irradiated T-deficient splenocytes and OVA peptide without (left) or with (right) the addition of rIL-4. After 5 d, cells were washed and restimulated with irradiated splenocytes and OVA peptide. Supernatants were harvested at 48 h
for quantitation of IL-4 (filled bars) and IFN-
(open bars) by ELISA. The
results shown are the mean of triplicate determinations and are representative of two separate experiments.
[View Larger Version of this Image (14K GIF file)]
helper T cells were
stimulated with plate-bound anti-CD3 mAb plus rIL-4 for
5 d. Cells were then washed and restimulated with plate-bound anti-CD3 to assess their maturational state. CD4+ T
cells were capable of significant IL-4 and modest IFN-
production, whereas the T cells from CD4° mice produced
high amounts of IFN-
with negligible IL-4 (Fig. 4 A).
We also attempted to augment the efficiency of Th2 maturation or IL-4 secretion by increasing the density of T cells
in the primary or secondary cultures, respectively. For
CD4+ T cells, increasing cell number in the primary culture was sufficient to promote Th2 maturation, even in the
absence of rIL-4 (Fig. 4 B). For CD4° T cells, even the
highest cell number plus the addition of rIL-4 failed to
elicit IL-4 production (Fig. 4 B). Moreover, neither the addition of rIL-2 nor co-culture of CD4+ with CD4° T cells
could rescue Th2 differentiation in the CD4° population
(data not shown).
Fig. 4.
The absence of CD4 impairs Th2 differentiation when T
cells are primed without APCs. (A) Purified CD8 T cells were isolated
from lymph nodes of CD4+ or CD4° mice, and 5 × 105 cells were plated
in wells precoated with anti-CD3 (10 µg/ml) plus rIL-4. After 5 d, cells
were washed extensively and 105 cells were restimulated on anti-CD3-
coated plates in triplicate for an additional 48 h. IL-4 (filled bars) and IFN-
(open bars) were quantitated from supernatants by ELISA. The results represent the mean of triplicate determinations and are representative of six
separate experiments. (B) Purified helper lineage T cells were primed as
described in A, using varying numbers of cells in the primary stimulation
(x-axis) and 2 × 105 cells/ml in the secondary stimulation (B, top), or 2.5 × 106 cells/ml in the primary stimulation and varying numbers of cells in
the secondary stimulation (x-axis; B, bottom). rIL-4 was withheld (B, left)
or added (B, right) to the primary cultures where indicated. (C) CD8
T
cells isolated from lymph nodes of CD4+ or CD4° mice were cultured on
plates precoated with the indicated concentration of anti-CD3. After 48 h,
[3H]thymidine was added, and plates were harvested 12-14 h later. The
mean of triplicate wells was used to determine the percent maximum thymidine incorporation for each group.
[View Larger Versions of these Images (15 + 29 + 14K GIF file)]
mRNA within 48 h (18, 19). Competitive
reverse transcriptase PCR was performed on highly purified helper T cells that had been stimulated for 48 h with
anti-CD3 plus rIL-4. Although the levels of IFN-
mRNA were higher in the CD4° T cells, both populations made
equivalent levels of IL-4 mRNA at this early time point
(Fig. 5). Furthermore, the viability of unstimulated CD4°
helper T cells was substantially augmented by the addition
of rIL-4 as a survival factor (data not shown). Taken together, these results suggest that the absence of CD4 impairs the intrinsic ability of T cells to commit to the Th2
fate, but does not impede their ability to proliferate, receive signals through the IL-4 receptor, or transcribe the IL-4
gene.
Fig. 5.
Early cytokine
mRNA from CD4° T cells. CD8
T cells from CD4+ (+/+) and
CD4° (
/
) mice were stimulated with plate-bound anti-CD3 plus rIL-4. After 48 h, RNA was
isolated for competitive reverse transcriptase PCR analysis. PCR
reactions were resolved on ethidium-stained 2% agarose gels.
Within each panel, the upper band
corresponds to amplification of
the internal standard competitor
molecule, whereas the lower
band corresponds to the amplification of experimental cDNAs.
The results are representative of
three separate experiments.
[View Larger Version of this Image (58K GIF file)]
cyt). Both of these transgenes are capable of rescuing helper T cell development in CD4° mice, although
overexpression of the tailless version is required for this effect (15). CD4WT and CD4
cyt transgenic mice were backcrossed onto the BALB/c background and then intercrossed
with CD4° mice expressing the OVA-specific transgenic TCR. The CD4WT transgene corrected the Th2 defect
when expressed in CD4° OVA-specific T cells, but the
CD4
cyt transgene was ineffective in promoting the Th2
fate (Fig. 6). When exogenous rIL-4 was added to the primary culture, however, either transgene was capable of reconstituting Th2 induction (Fig. 6). Similar results were
observed in vivo. The CD4WT transgene rescued the Th2
response to KLH immunization, whereas the CD4
cyt
transgene did not (Fig. 7). Both types of transgenes, however, restored susceptibility (Fig. 8 A) and Th2 responses
(Fig. 8 B) to leishmaniasis in BALB/c CD4° mice. Thus,
the Th2 defect caused by a null mutation in the CD4 gene
can be fully corrected by ectopic expression of wild-type
CD4, and partially corrected by a mutant form that lacks
the cytoplasmic domain.
Fig. 6.
Rescue of Th2 differentiation by CD4 transgenes. CD8 T
cells were isolated from spleens and lymph nodes of CD4° BALB/c
D011.10 TCR transgenic mice that had been additionally reconstituted
with transgenes encoding either full-length CD4 (WT) or a mutant CD4 lacking the cytoplasmic tail (
cyt). An equivalent number of clonotype cells were cultured in 1 ml with irradiated T-deficient splenocytes and
OVA peptide without (left) or with (right) the addition of rIL-4. After 5 d,
cells were washed and restimulated with irradiated splenocytes and OVA
peptide. Supernatants were harvested at 48 h for quantitation of IL-4
(filled bars) and IFN-
(open bars) by ELISA. The results shown are the
mean of triplicate determinations and are representative of two separate
experiments.
[View Larger Version of this Image (14K GIF file)]
Fig. 7.
The cytoplasmic
domain of CD4 is required for
Th2 responses after KLH immunization. CD4 transgene-reconstituted BALB/c CD4° mice
were immunized with KLH, and
draining lymph node cells were restimulated as described in Materials and Methods. The results shown are the mean of triplicate
determinations from two CD4°CD4WT (WT) and three CD4°CD4cyt
(
cyt) mice, and are representative of nine separate experiments involving
11 BALB/c CD4°CD4WT, 12 BALB/c CD4°CD4
cyt, 9 C57BL
CD4°CD4WT, and 11 C57BL CD4°CD4
cyt mice.
[View Larger Version of this Image (17K GIF file)]
Fig. 8.
The cytoplasmic domain of CD4 is not essential for susceptibility or Th2 responses after L. major infection. (A) BALB/c CD4° and
CD4° transgene-reconstituted mice (CD4°CD4WT, CD4°CD4cyt) were
infected with L. major as described in Materials and Methods. The results
shown are the mean footpad measurements of groups of two animals each
and are representative of five separate experiments using 9 CD4°CD4WT
and 11 CD4°CD4
cyt mice. (B) Popliteal lymph node cells from infected
BALB/c CD4° (
) and CD4° transgene-reconstituted mice (WT,
cyt)
were analyzed for parasite-specific cytokine secretion after restimulation
in vitro. The results shown are the mean of triplicate determinations from
individual mice, and are representative of four separate experiments using
eight CD4WT and nine CD4
cyt mice.
[View Larger Versions of these Images (20 + 13K GIF file)]
cyt (15). Th2 differentiation was fully restored by
expression of the former molecule, but only partially by the
latter. For example, Th2 responses to KLH required the
presence of the cytoplasmic domain of CD4, but Th2-dependent susceptibility to Leishmania did not. The partial rescue
of Th2 differentiation mediated by the tailless molecule indicates that the cytoplasmic domain of CD4 can be dispensable for Th2 differentiation under certain circumstances. This result implies that Th2 cells can be formed in
the absence of any unique signaling pathway that initiates
exclusively at the CD4 cytoplasmic domain. Thus, it is
possible that the primary Th2-promoting effect of the tailless molecule is to stabilize the TCR engagement of peptide-MHC complexes. In this way, the tailless molecule may
facilitate sustained or enhanced TCR signaling, and this
may be the determining factor for Th2 differentiation. However, since some Th2 responses were not restored in
the CD4
cyt mice, it is also possible that the cytoplasmic
domain of CD4 may have an intrinsic (but not exclusive)
capacity to promote the Th2 fate. This Th2-promoting activity could be mediated by direct signal transduction involving p56lck, or other proteins that may associate with
CD4, such as CD81 or CD82 (22). Although the results
obtained with the CD4
cyt transgenic mice preclude a precise determination of how CD4 influences the differentiation process, they still provide a striking example of how
the Th2 differentiative capacity of T helper cells can be
profoundly affected by a strategy that impairs the function of CD4.
mRNA
at 48 h after stimulation. In other published experiments,
the blockade of the CD4-MHC class II interaction with an
MHC class II peptide also impaired Th2 differentiation and
favored the Th1 fate (11). Although these and our experiments could be consistent with passive commitment to
Th1 differentiation in the absence of CD4, it is also possible that the involvement of CD4 in T cell activation normally serves an active Th1-suppressive function. This supposition may be consistent with the finding that the ligation
of CD4 with mAbs can suppress the production of IFN-
and the induction Th1 cells (12). Similarly, it is possible
that chronic ligation of CD4 by the HIV envelope in HIV-infected humans delivers signals that potentiate Th2 differentiation. This may be relevant to the controversial finding
that the progression to AIDS is accompanied by increased
Th2-mediated allergic complications and impaired Th1
immunity (26). If CD4 does have an active role in suppressing Th1 differentiation, then it may be possible to induce
Th2 differentiation in naive T helper cells by strategies that
would bypass CD4 and directly engage the putative Th1
suppressive pathway. A better understanding of the mechanism by which CD4 regulates T helper cell fate may ultimately suggest alternative therapies for many diseases. In
summary, the data support a novel role for CD4 in the determination of helper T cell lineage after thymic development. Despite the critical role of cytokines in controlling
helper T cell fate, the results of this work help to refocus
attention on TCR/CD4 signaling as being essential for proper Th2 development.
Address correspondence to S. Reiner, University of Chicago, 924 East 57th St., R420, Chicago, IL 60637-5420.
Received for publication 14 March 1997 and in revised form 25 April 1997.
D.R. Brown was supported by the University of Chicago Medical Scientist Training Program and Immunology Training grant AI-07090. This work was supported by National Institutes of Health grants AI-01309 to S.L. Reiner and AI-39506 to N. Killeen. S.L. Reiner is supported by the Burroughs Wellcome Fund.We are grateful to Jeff Bluestone, Robert Seder, Craig Thompson, and Chyung-Ru Wang for review of the manuscript.
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