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Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, which in turn govern
differentiation to Th1 and Th2 phenotypes. We have used this system to
confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1
interactions can suppress, differentiation of cells secreting the Th2
cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen
over a certain range of peptide doses. In addition, CD28 and Ox-40
interactions also promote Th1 differentiation. In general, agonist Abs
to accessory molecules shifted the response curves for IFN-, IL-5,
and IL-13 to lower doses, whereas antagonist reagents resulted in
similar curves shifted toward the higher doses. We conclude that
ligation of cell surface accessory receptors enables low doses of Ag to
promote responses normally induced only by higher doses. Individual
receptors do not intrinsically regulate one cytokine phenotype or
another, suggesting that differentiation is controlled by the level of
expression of multiple accessory molecule pairs integrated with the
number and affinity of peptide/MHC complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strength of signaling also appears to govern differentiation of T cells
into distinct cytokine-secreting effectors, and in many cases this may
work by modulating the balance of IL-2, IL-4, IL-12, and IFN- or
-
available to the T cell (reviewed in Ref. 9).
However, in contrast to early activation, the more is better paradigm
may not apply completely. We have recently shown, using altered
peptides at varying concentrations, that a low level stimulation (very
low Ag dose or low affinity peptide) of CD4 cells only favors
differentiation of uncommitted IL-2-producing cells. A moderate/high
level of stimulation favors differentiation of Th2-like cells which
produce IL-4 and IL-5, whereas a very high level of stimulation favors
Th1-like cells which produce IFN- (10). Data from other
laboratories have directly or indirectly supported such a model
(11, 12, 13, 14, 15, 16). We also hypothesized that accessory molecules
would integrate with the dose and affinity of Ag to regulate
differentiation by effectively increasing or decreasing the strength of
signal depending on their presence or absence on the T cell and APC.
This scenario would only apply if one accessory molecule did not have a
distinct action from another molecule. In contrast to this hypothesis,
a number of reports over the past few years have suggested that
individual molecules selectively regulate differentiation into Th1 or
Th2 phenotypes. For example, interaction of CD28 with B7-2 has been
suggested to promote Th2 differentiation but not Th1 differentiation
(17, 18, 19, 20), as has the interactions of CD4 with class II MHC
(21, 22), and Ox-40 with Ox-40 ligand (23, 24). In contrast, LFA-1 interacting with ICAM-1 or -2 (25, 26), and CD28 interacting with B7-1 (18, 27), have
been proposed to be inhibitory for Th2 differentiation and therefore
integral to Th1 differentiation. One caveat to these studies is that
they were largely performed in situations where the TCR signals were
not varied, i.e., either one concentration of mitogen or one
concentration of Ag was used.
In this report, we have revisited the question of whether Ag dose
integrates with accessory molecule signaling by using agonist Abs to
CD28 and Ox-40 or reagents that block CD4, LFA-1, and CD28 in a defined
model of differentiation that uses naive TCR transgenic T cells
responding to a peptide of moth cytochrome c
(MCC).3 We confirm
that CD28, Ox-40, and CD4 ligation can promote Th2 differentiation, and
that LFA-1 ligation can suppress Th2 cytokines. However, we demonstrate
that multiple effector cytokine phenotypes are generated depending on
the initial dose of peptide and the efficiency of stimulation or
inhibition provided by the accessory molecule Abs. In general, agonist
Abs shifted the response curves for IFN-, IL-5, and IL-13 to lower
doses, whereas antagonist reagents resulted in similar curves shifted
toward the higher doses. We conclude that accessory molecules largely
function to regulate how well a naive T cell responds to a given
peptide, and that the individual receptors studied here do not
intrinsically regulate one cytokine phenotype or another.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AND TCR transgenic mice bearing T cells reactive with peptides
of MCC or pigeon cytochrome c (PCC), and expressing the
Vß3/V11 TCR, were bred on a B10.BR background as previously
described (28). B10.BR mice were bred at the La Jolla
Institute for Allergy and Immunology (San Diego, CA) and used at 8–12
wk of age.
Peptides
T102S, a variant peptide of MCC, bearing a single amino acid substitution of S for T at position 102 (aa 88–103, ANERADLIAYKQASK), was synthesized in the peptide facility at the La Jolla Institute for Allergy and Immunology. This peptide has heteroclitic (super agonist) activity, being 5- to 10-fold more stimulatory for a naive CD4 cell than MCC, as described previously (10).
Antibodies
Abs (or Fc fusion protein) to the following Ags were added to cultures at 0.1–20 µg/ml as described in the figure legends. Control Ig (rat IgG1, rat IgG2a, rat IgG2b, and hamster IgG) and anti-LFA-1 (M1/74, rat IgG2a) were purchased from PharMingen (San Diego, CA). Anti-CD28 (37.51, hamster IgG) was purified from hybridoma supernatant and was a kind gift of Dr. J. Allison (University of California, Berkeley, CA). Anti-Ox-40 (CD134) (Ox-86, rat IgG1) was a gift from Dr. A. Weinberg (Earle Chiles Institute, Portland, OR). Anti-CD4 (GK1.5, rat IgG2b) was a gift from Dr. S. Schoenberger (La Jolla Institute for Allergy and Immunology). Fusion protein CTLA4-Ig, which blocks CD28 and CTLA4 interaction with CD80/CD86, was a gift from Dr. P. Linsley (Rosetta Inpharmatics, Kirkland, WA) and was purified from a hybridoma supernatant by passage over a Gammabind column (Pharmacia Biotech, Piscataway, NJ).
T cells
CD4+ T cells were purified from spleen and
lymph nodes of TCR-transgenic mice (28) by nylon wool
depletion, followed by Ab and complement treatment. T cells were
incubated with Abs to CD8 (3.155), heat-stable Ag (J11d), class II MHC
(M5/114 and CA-4.A12), B cells (RA3.6B2), macrophages (M1/70), NK cells
(PK136), and dendritic cells (33D1) and then cross-linked with mouse
anti-rat 
-chain (MAR18.5). Any residual APC and any in
vivo-activated T cells were removed by isolating high density cells
spun through a Percoll (Sigma, St. Louis, MO) gradient (45%, 53%,
62%, 80%). The resultant cells were resting (low forward scatter) and
>95% CD4+. In addition, >95% of these cells
possessed a phenotype associated with naive CD4 cells
(CD45RB+, CD62L+,
CD44low) along with expression of the
Vß3/V11 TCR (28, 29). T cells were further purified
by positive selection with anti-CD62L (Mel-14) using magnetic beads
(Miltenyi Biotech, Sunnyvale, CA) as in previous studies
(10) to ensure that responses were only generated from
naive T cells.
Ag-presenting cells
Spleen cells from B10.BR mice were depleted of T cells using complement fixation with Abs to Thy1.2 (F7D5 and HO.13.4), CD4 (RL172.4), and CD8 (3.155). Fibroblast cells (DCEK-ICAM) transfected with I-Ek and ICAM-1 (CD54), and constitutively expressing B7-1 (CD80), were used for restimulation of T cells. APC populations were treated with mitomycin C (50–100 µg/ml) for 30 min at 37°C before use.
Cell cultures
Cultures were set up in 1 ml of 10% FCS-RPMI in 48-well plates (Sarstedt, Newton, NC). Naive CD4 T cells were plated at 5 x 105/ml with 4 times as many T-depleted spleen APCs and various concentrations of T102S peptide. Abs to cell surface molecules were added on day 0 of culture. Anti-Ox-40 and CTLA4-Ig were added again on day 2. Stimulation was conducted for 4 or 12 days, after which viable cells were recovered and counted by trypan blue exclusion. T cells (3 x 105/ml) were replated in 0.25-ml volumes in 96-well plates in quadruplicate with half as many DCEK-ICAM fibroblast APC, prepulsed for 2–4 h with 20 µM PCC peptide as described previously (10). Supernatants were collected for cytokine analyses 24–48 h later. SDs between replicates were <15% of the means.
Cytokine secretion
Duplicate supernatants were recovered from quadruplicate
cultures at 24 h (IL-2) and 48 h (IFN-, IL-5, and IL-13)
after T cell stimulation to assess cytokine content. Cytokines were
measured by ELISA as in previous studies (10) using the
Abs JES6-1A12 (PharMingen) and biotin-JES6-5H4 (PharMingen) for IL-2,
R46-A2 and biotin-XMG1.2 (PharMingen) for IFN-, and TRFK5 and
biotin-TRFK4 for IL-5 detection. Standard curves were constructed with
purified IL-2, IL-5, and IFN- (supernatants from the respective
X63.Ag cell lines). Anti-IL-13 (clone 38213.11, MAB413),
biotin-anti-IL-13 (BAF413), and recombinant human IL-13 were
purchased from R&D Systems (Minneapolis, MN). The sensitivity of each
assay was similar, with levels of detection being 50–100 pg/ml for
IL-2, IL-5, and IFN- and 200 pg/ml for IL-13.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that differentiation of effectors
secreting Th1 (IFN-) or Th2 (IL-4, IL-5) cytokines can be controlled
by the dose or affinity of peptide initially presented to the naive CD4
cell (10). This is again illustrated in Fig. 1
, and we now show that IL-13 is also
regulated by Ag dose in a manner similar to IL-5. Naive CD4 cells from
AND TCR transgenic mice were stimulated with varying doses of the MCC
analogue, T102S, presented by syngeneic T-depleted splenic APCs.
Cultures were left for 4 or 12 days (Fig. 1, upper panel and
lower panel, respectively), and then an equivalent number of
live T cells were restimulated with a single dose of Ag and assayed for
the ability to secrete cytokines. Peak IFN- secretion was seen from
effectors generated with high doses of peptide (10–100 µM), whereas
peak IL-5 and IL-13 secretion were observed from effectors elicited at
low/intermediate doses (0.1–1 µM). At the early time point of 4
days, IL-13 was produced in much higher amounts than IL-5 and was the
best indicator of Th2-type cells. Very little IL-5 was detected from
cells generated after 4 days in culture (Fig. 1, upper
panel). In contrast, IL-5 levels were greatly increased with
differentiation over time (12 days; Fig. 1, lower panel),
showing the typical Th2 cytokine bell-shaped profile, whereas IL-13
levels were down-regulated, although still produced with a similar
dose-response curve. IL-5 production was also more variable than IL-13,
and in some experiments, very little IL-5 (or IL-4; data not shown) was
detected. IFN- was similar regardless of the length of
differentiation, although on average produced at slightly lower levels
over time.
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In a previous report we showed that expression of CD80 (B7-1) and
CD54 (ICAM-1) on APCs allowed naive CD4 cells to up-regulate activation
Ags (CD25 and CD69), secrete IL-2, and proliferate more efficiently to
low doses of peptide or peptides of low affinity (7). To
determine the effect of accessory molecules on T cell differentiation
and how they integrate with Ag dose, we utilized several agonist Abs
(anti-CD28, anti-Ox-40) and antagonist Abs (anti-LFA-1,
anti-CD4) or Ig fusion protein (CTLA4-Ig) to various molecules. To
confirm their stimulatory or inhibitory capacities, and thus agonist or
antagonist properties, we initially assessed naive T cell IL-2
production, shown in Fig. 2, and the
effects on T cell survival over time (Table I).
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, addition of anti-CD28 induced higher IL-2
secretion at all peptide doses, whereas anti-Ox-40 only slightly
increased IL-2 in this system. Anti-CD4 resulted in a 10- to 20-fold
shift in IL-2 secretion to the higher peptide concentrations, whereas
anti-LFA-1 and CTLA4-Ig resulted in more profound inhibition (100-
to 1000-fold shift). These results agree with many others who have used
these particular Abs. We have previously shown that Ox-40 stimulation
alone at the naive T cell stage has only slight effects on IL-2,
whereas more profound effects are seen at late time points and for
promoting clonal expansion (8). In addition, we have
confirmed that this Ab is stimulatory in vivo (unpublished data).
Agonist and antagonist reagents induced a corresponding increase or
decrease in T cell recovery after a 4-day culture (Table I). Anti-CD28
and anti-Ox-40 enhanced cell recovery, especially with low doses of
peptide, whereas anti-CD4, anti-LFA-1, and CTLA4-Ig reduced
cell recovery at these doses. We previously demonstrated that over
time, cell survival is optimal when naive T cells are stimulated with
intermediate doses (0.1–1 µM) of the high affinity peptide with
reduced recovery seen in cultures that initially received high doses of
peptide (10). This is illustrated in Table I for day 12
cultures. Anti-CD28 and anti-Ox-40-treated cells showed higher cell
recoveries at all Ag doses (except 0.001 µM with anti-Ox-40);
however, optimal recovery was still seen with 0.1–1 µM. In contrast,
anti-LFA-1-, anti-CD4-, and CTLA4-Ig-treated cells did not
survive well at low doses of peptide, and reduced recoveries were seen
at other doses. The exception was with anti-LFA-1 where cell
survival was greatly enhanced with high dose peptide (1–100 µM).
Therefore, blocking or stimulating accessory molecule interactions can
reduce or enhance cell survival depending on the Ag dose. There was
some correlation, although not strict, with initial IL-2 production
(Fig. 2), and overall, it could be argued that provision of
costimulation allowed naive T cells to respond and survive when less Ag
was presented, whereas inhibition of costimulation suppressed response
and survival when Ag dose was suboptimal.
Accessory molecule interactions can promote or suppress Th1/Th2 effector development but in a strictly Ag dose-dependent fashion
The ability of accessory molecules to modulate Th1 and Th2
development was initially assessed with three doses of Ag. Fig. 3 shows cytokine secretion in a single
representative experiment from equivalent numbers of cells restimulated
after 12 days in culture. In all cases, the cells recovered were CD4
positive and all expressed the transgenic TCR, Vß3/V11.
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(Fig. 3, left). None of the Abs significantly
affected IFN- at this dose. Anti-CD28 and anti-Ox-40 enhanced
IL-5, to a limited extent, and IL-13, to a great extent. In contrast,
Abs to CD4, LFA-1, and fusion protein CTLA4-Ig almost completely
inhibited development of both IL-5- and IL-13-secreting cells. With an
intermediate dose of Ag (1 µM; Fig. 3, center), some
similar and some distinct changes were seen. Anti-CD28 and
anti-Ox-40 again resulted in enhanced levels of IL-5 and IL-13, but
additionally promoted more IFN- secretion. Anti-CD4, CTLA4-Ig, and
anti-LFA-1 inhibited cells secreting IFN-, but had differing
effects on IL-5 and IL-13. CTLA4-Ig had little effect on either
cytokine, whereas increases were seen with anti-CD4, and
particularly with anti-LFA-1. The trend was similar with 100 µM
compared with 1 µM, although again differences were noted (Fig. 3, right). Both anti-CD28 and anti-Ox-40 resulted in
enhanced IFN-, although a much higher level was now seen with
anti-CD28. Again, anti-CD4, CTLA4-Ig, and anti-LFA-1
inhibited cells secreting IFN-. In contrast, anti-LFA-1 to a
great extent, anti-Ox-40 less, and anti-CD28 and anti-CD4
even less, augmented IL-5 and IL-13 production.
In summary, anti-CD28 and anti-Ox-40 could promote
differentiation of T cells secreting both Th1 and Th2 cytokines.
Anti-CD4 and anti-LFA-1 could suppress development of T cells
secreting IFN-; however, they had differential effects on Th2
cytokines (IL-5 and IL-13). CTLA4-Ig could suppress secretion of both
Th1 and Th2 cytokines. Thus, agonist Abs could enhance both Th1 and Th2
cytokines, whereas antagonist reagents showed differential effects on
Th1 and Th2 cytokines. The most important finding was that all
responses were dependent on the initial dose of peptide.
These data suggested that enhancing or suppressing the actions of
accessory molecules was primarily affecting the Ag dose-response
patterns. We therefore set up full dose-response titrations in the
presence or absence of the various Abs added at the initiation of
culture. Figs. 4 and
5 show the mean cytokine secretion from
effector T cells restimulated after 4 and 12 days from at least three
experiments.
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, addition of anti-CD28 and anti-Ox-40 enhanced
development of T cells secreting both Th1 (IFN-) and Th2 (IL-5,
IL-13) cytokines over a wide range of peptide concentrations. For both
Abs, at day 4 (upper panel), the dose-response curve for
IFN- and IL-13 was shifted 
10- to 100-fold to the lower Ag
concentrations. Anti-CD28 was more potent than anti-Ox-40 in
shifting the cytokine dose response. Minor differences were observed
for the Th2 cytokines in that anti-CD28 had greater effects at very
low doses (0.001 µM) and little/no effect at very high doses (10–100
µM). However, the quantity of cytokine and shape of the curve were
similar for both anti-CD28 and anti-Ox-40-treated T cells.
The enhancement of IFN- and IL-13 secretion, by anti-CD28 and
anti-Ox-40 was retained when T cells were restimulated at 12 days
(Fig. 4, lower panel) although the overall levels of each
cytokine was lower than at day 4 (upper panel). Both
anti-CD28 and anti-Ox-40 also enhanced IL-5 secretion,
especially in cultures that initially received low doses of peptide
(0.001–0.01 µM) with peak production occurring at 10-fold lower
doses vs control cultures (0.01 v. 0.1 µM). Therefore, both
anti-CD28 and anti-Ox-40 could enhance the ability to produce
Th1 and Th2 cytokines in addition to altering the dose-response curves,
with peak production of cytokine occurring in cultures that received
lower doses of priming Ag.
The effect of antagonist reagents (anti-CD4, anti-LFA-1, and
CTLA4-Ig) on Th1/Th2 development is shown in Fig. 5. Cytokine secretion
is shown from T cells restimulated after 4 (top
panel) and 12 days (middle and bottom
panels). There was little effect on IL-2 secretion; however,
CTLA4-Ig could inhibit IL-2 secretion from cells that initially
received low doses of Ag (0.001–0.1 µM), in effect, shifting the
dose response 100-fold. After short-term culture (top
panel), both anti-CD4 and anti-LFA-1 shifted the IL-13
response to higher Ag doses, whereas CTLA4-Ig greatly inhibited IL-13
secretion. Anti-LFA-1 and CTLA4-Ig also inhibited IFN- secretion,
whereas anti-CD4 had little effect, although in two of three
experiments anti-CD4 actually shifted the IFN- dose response to
higher Ag concentrations. Restimulation of cells after 12 days showed
that anti-CD4, anti-LFA-1, and CTLA4-Ig inhibited development
of IFN--secreting cells at all Ag concentrations, effectively
shifting the dose-response curves to the higher concentrations. These
Abs similarly inhibited IL-5 production when low doses of peptides were
used (0.01–0.1 µM) and either inhibited IL-13 or had no effect. At
high doses of priming Ag (1–100 µM), both anti-CD4 and
anti-LFA-1 treatments resulted in augmented IL-5 and IL-13 vs
control cultures. This increase was particularly striking with
anti-LFA-1. In contrast, CTLA4-Ig inhibited or had no effect on
subsequent IL-5 and IL-13 secretion. Although the absolute amounts of
cytokines varied, these data suggest that anti-CD4 and
anti-LFA-1 can alter Th2 dose response while inhibiting and/or
shifting the dose response for Th1 (IFN--secreting) cells. CTLA4-Ig
profoundly inhibited Th2 development and partially Th1 development
though lower doses of CTLA4-Ig appeared to shift the Th1/Th2 Ag
dose-response curves (data not shown).
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Multiple cytokine phenotypes result depending on the amount of Ab reagent used to modulate accessory molecules
In initial experiments to assess the effects of blocking various
accessory molecules, we had noted that differing cytokine profiles
result depending on the amount of Ab used, presumably related to the
extent of blocking achieved, and the expression and use of the
molecules in this particular system. This is illustrated in Fig. 6 for anti-LFA-1. With lower amounts
of anti-LFA-1 (0.1 µg/ml), which partially blocked naive IL-2
secretion (data not shown), a more subtle phenotype was seen than
previously shown in Figs. 3 and 5. IFN- was blocked at the lower
concentrations of peptide, but not significantly at very high
concentrations. Similarly, IL-13 and IL-5 were inhibited at very low
peptide doses (0.01–0.1 µM) but not at high doses, and IL-5 was
increased although only modestly. With increasing amounts of
anti-LFA-1, it was obvious that a shift in the peptide dose
response was occurring with all three cytokines, peaking at higher
peptide concentrations. This effectively resulted in lower levels of
all cytokines at the low Ag doses and higher levels of IL-5 and IL-13
at the high Ag doses. IFN- was blocked at 50 µM (and 100 µM,
Fig. 5) with 10 µg/ml anti-LFA-1; however, it could be argued
that increased levels may have been seen if we could have used
sufficient amount of peptide (100–200 µM is the maximum that is
feasible in these cultures).
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IL-5, and IL-13 were shifted 10-fold to lower Ag doses when 10 times
more anti-CD28 was used (1 µg/ml vs 0.1 µg/ml anti-CD28).
Overall, it can therefore be seen that multiple phenotypes result
depending on the amount of agonist/antagonist Ab used, and that the
overall conclusion as to the role of various accessory molecules can be
heavily biased depending on the extent of intervention. Again, most
importantly, modulation of the cytokine phenotype was strictly
correlated with the dose of Ag. The kinetics of development of IL-5- and IL-13-secreting cells can be altered by suppressing or promoting costimulation
Our previous studies showed that IL-5 was only produced at high
levels following a long differentiation period. More recently, we also
noted that the kinetics of IL-13 were opposite to IL-5 in that higher
levels of IL-13 were seen earlier (on day 4) rather than later (on day
12) (see Fig. 1). We therefore assessed whether modulating
costimulation would affect the kinetics of these responses. Fig. 7 shows IL-5 production at day 4 vs day
12 when costimulation from anti-CD28 was provided, and IL-13
production when anti-LFA-1 was used to inhibit costimulation.
Again, the conclusions were dependent on the dose of Ag used initially,
with multiple effects on cytokine phenotypes being seen depending on
the amount of peptide presented. However, significantly, provision of
CD28 costimulation resulted in enhanced or rapid differentiation to
IL-5 production. Cells treated with anti-CD28 and restimulated
after 4 days showed and IL-5 response pattern similar to that in
control cultures at 12 days (compare anti-CD28, day 4 to control,
day12). In addition to IL-5, the kinetics of IL-13 secretion which is
opposite to that of IL-5 (see Fig. 1), was also altered. Instead of
peak production on day 4, peak production of IL-13 was seen on day 12
from T cells initially cultured with anti-LFA-1. Therefore,
inhibiting LFA-1 interaction prevented the down-regulation of IL-13 at
late times (day 12) or shifted the kinetics of peak IL-13 secretion.
The overall conclusions reached, regarding the role of each molecule in
regulating individual cytokines, therefore depend on the time of
analysis, the concentration of reagents used, and most importantly, the
initial dose of Ag presented to the responding T cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we did see some results that do not exactly fit our model when
manipulating certain accessory molecules, including differences in
total cytokine levels, it is clear that in many cases the effect of
providing or taking away costimulation was to alter the dose-response
curve to peptide. Thus, in general, agonist Abs produced a similar
dose-response curve, but skewed toward the lower peptide
concentrations, and antagonist Abs resulted in a similar dose-response
pattern shifted toward the higher peptide concentrations. Variations
were seen between anti-CD4, CTLA4-Ig, and anti-LFA-1 for
example, but it could be argued that this may have been determined by
the expression levels and use of each molecule in the normal response,
or proficiency of blocking of the reagents, rather than any intrinsic
differences between the actions of the molecules. Therefore, rather
than an individual accessory receptor/ligand interaction positively
regulating one cytokine and negatively regulating another, our data
favor a model depicted in Fig. 8 where
the overall strength of stimulation governs T cell differentiation.
This is dictated by the balance between the extent of TCR signaling
(dose and/or affinity of peptide) and the extent of accessory molecule
involvement at the time of T cell/APC interaction. In such a model,
differentiation to a Th2 phenotype (IL-5, IL-13) will be induced by a
low/moderate dose of Ag presented in the context of many accessory
interactions, or alternatively by a high dose of Ag presented with
little accessory molecule help. Reducing both sets of signals
sufficiently will result in largely uncommitted effector T cells being
generated over time (IL-2 primarily), whereas maximizing both sets of
signals will favor predominantly IFN--secreting cells.
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On the other hand, are there really distinct intracellular events that
occur when the TCR or accessory receptors are engaged? Studies from
Lanzavecchia and colleagues (2, 5) showed that CD28
ligation reduced the number of TCRs that needed to be engaged, and the
length of the engagement period required for T cell triggering.
However, a similar level of activation could be achieved in the absence
of CD28 if the length of stimulation and number of engaged TCRs were
increased (2, 5). Similarly, the presence of CD4 has been
shown to be crucial for efficient T cell activation, but only when weak
peptide agonists are presented (35, 36). In the absence of
CD4, a strong agonist produces signaling patterns identical to those
seen with a weak agonist (37). We have seen similar
results with ICAM/LFA interactions and weak agonists (7).
Although much is unknown, studies of intracellular signals from
accessory receptors now suggest that pathways converge, in many cases
before the level of the nucleus. CD28 can affect inositol phospholipid
hydrolysis, phospholipase C1 (PLC1) phosphorylation, and
activation of p21Ras and the mitogen-activated
protein and stress-activated protein kinases (reviewed in Refs.
38 and 39). It is now clear that all of these
are also recruited to some extent by TCR/CD3 ligation, including
PI-3-kinase. CD4 and LFA-1, which in many cases have been considered as
adhesion molecules, are also obviously capable of enhancing the
intracellular signals induced in a T cell. CD4 through its association
with p56Lck can feed into the PLC1 pathway,
and LFA-1 binding also results in PLC1 phosphorylation, leading to
inositol phospholipid hydrolysis and increases in intracellular calcium
(40, 41). The TNFR family of costimulatory molecules
initially could have been viewed as the exception to these common
signaling pathways, each acting through one or more of the
TNFR-associated factor (TRAF) molecules, such as TRAF2 and -3 in the
case of Ox-40 (42). However, it is now clear that one of
the results of TRAF binding is activation of NF-B, via
NF-B-inducing kinase (NIK), and its translocation into the nucleus.
The recent demonstration that CD28 can also activate the NIK/NF-B
axis through a kinase termed Cot (43), again implies that
all signals (TCR and accessory receptor) converge before or at the
level of the nucleus. Whether truly separate pathways are initiated
from individual receptors may therefore be unlikely, although many more
studies are obviously needed.
In light of the above arguments, it then makes sense that a single
accessory ligand/receptor interaction will not control one function
(e.g., Th2 cytokines) as suggested by a number of reports (17, 18, 21, 22, 23, 24, 25, 26). Rather, as implied by our data, the integration of
signals from the TCR and multiple accessory receptors will determine
the overall response pattern of the T cell, with the dominant response
being determined by the dose and affinity of Ag and the expression
characteristics of the various accessory molecules on the T cell and
APC. How then is Th1/Th2 differentiation controlled by strength of
signaling? At present we do not know, but think that it will be
determined in the initial days after activation by the balance of
signals received by the T cell. IL-2R signaling plays some role in this
process as exogenous IL-2 can shift the Ag dose-response patterns for
effector cytokines to the lower concentrations (10), and
blocking IL-2 can mimic to a certain extent the effects seen here when
blocking with anti-CD4 and CTLA4-Ig (unpublished observations).
This is not surprising as a major role of TCR and accessory signals is
to regulate IL-2 production, and signaling through the IL-2R also
involves some of the same intracellular substrates such as Lck, Fyn,
Ras, mitogen-activating protein kinase (MAPK), and PI-3-kinase. We also
know that in our system the balance of IFN- and IL-4 initially
produced by the naive T cell is critical to subsequent differentiation
into Th1 and Th2 phenotypes (10, 44, 45), and this has
also been seen in several other systems (16, 46, 47).
Thus, IFN-R signaling controls the ability to subsequently make high
levels of IFN-, and IL-4R signaling controls the ability to make
high levels of IL-5 and IL-13, as demonstrated using blocking Abs to
these cytokines (Ref. 10 , and data not shown). IL-12,
which potentially could override the action of IL-4, was found not to
be a major factor in these studies (10).
In summary, we therefore believe that the signals that control the
balance of autocrine IL-4 and IFN- made by the responding naive T
cell will provide the answer to how the strength of signaling regulates
Th1/Th2 differentiation. Although great strides have been made in
understanding the signaling events initiated through the IL-4R and the
IFN-R, there is no information on what regulates the first burst of
IL-4 and IFN-. As both cytokines are produced relatively late after
naive CD4 activation, with mRNA first seen around 24 h compared
with several h for IL-2 (44, 45, 46, 48), the amount of IL-2
and hence also the level of signaling through the IL-2R may be critical
to the level of transcription of IL-4 vs IFN-. Data from Yamashita
et al. (49, 50), using dominant negative transgenic mice,
recently showed that the Lck/Ras/MAPK pathway may be critical to Th2
differentiation. Thus, an intriguing possibility, that could involve
both TCR, accessory receptor, and IL-2R signaling, is that the ability
to preferentially activate this pathway may favor IL-4 over IFN-,
and therefore Th2 differentiation over Th1 differentiation. Obviously,
studies of this nature are still in their infancy and many more will be
needed before a clear picture emerges.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Croft, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Abbreviations used in this paper: MCC, moth cytochrome c; PCC, pigeon cytochrome c; PI-3-kinase, phosphatidylinositol-3-kinase; PLC1, phospholipase C1; MAPK, mitogen-activating protein kinase.
Received for publication September 8, 1999. Accepted for publication January 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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