Peptide Dose, Affinity, and Time of
Differentiation Can Contribute to the
Th1/Th2 Cytokine Balance
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J Immunol 1999; 163:1205-1213; ;
http://www.jimmunol.org/content/163/3/1205
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References
Paul R. Rogers and Michael Croft
Peptide Dose, Affinity, and Time of Differentiation Can
Contribute to the Th1/Th2 Cytokine Balance1
Paul R. Rogers and Michael Croft2
D
ifferentiation to Th1 and Th2 phenotypes is largely controlled by the action of the cytokines IL-12, IFN-g, and
IL-4, each having direct effects on the responding T cells
(1–3). IL-12 is primarily produced by macrophages and dendritic
cells, whereas the sources of IFN-g and IL-4 that modulate CD4
differentiation have been debated. A potential source of IFN-g and
IL-4 was suggested by a number of reports showing that both
cytokines could be produced in reasonable quantities by the responding CD4 cells themselves (4 –9). This promoted the idea that
the type or level of stimulus received by a T cell may affect the
balance of autocrine IFN-g and IL-4 and direct subsequent differentiation into Th1 or Th2 phenotypes. This hypothesis has been
termed the strength of signaling model of differentiation and may
encompass such variables as the nature of the Ag (affinity for
MHC, affinity of Ag/MHC for TCR) and the availability of Ag
(number of Ag/MHC complexes presented at a given time, duration of TCR ligation). In addition, other factors such as the avidity
of interaction between T cell and APC (number of Ag/MHC complexes presented over time, amount of adhesion between accessory
molecules and coreceptors) and the extent of costimulation (number of coreceptors ligated, availability of the ligands over time,
type and number of different ligand-coreceptor interactions) may
play roles in the responses seen.
Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, San
Diego, CA 92121
Received for publication December 3, 1998. Accepted for publication May 17, 1999.
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.
1
This work was funded by National Institutes of Health Grant AI36259 (to M.C.).
P.R. was supported by a National Research Service Award postdoctoral fellowship
from the National Heart, Lung, and Blood Institute, National Institutes of Health. This
is manuscript 253 from the La Jolla Institute for Allergy and Immunology.
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: [email protected]
Copyright © 1999 by The American Association of Immunologists
Reports in vitro and in vivo have given validity to the strength
of signaling model. However, the studies have also brought much
confusion to the field with seemingly contradictory conclusions
being reached. IL-4-secreting cells can be induced by repetitive
stimulation with anti-CD3 and IL-2 (5), anti-CD28 (10), antiCD40 ligand (L)3 (11), or B7-expressing fibroblast cells (8, 12,
13), and with peptide presented to TCR transgenic T cells on
highly costimulatory APC (7, 14 –16). These studies support the
hypothesis that a greater level of signaling is required for inducing
Th2-type responses. Similarly, multiple immunizations or high
level infection with the parasites Leishmania major, Trichuris
muris, and Schistosoma mansoni favor induction of Th2 cytokines
whereas single immunizations or low level infection favor Th1
cytokines (17–19). Additionally, if the major costimulatory pathway through CD28 is disrupted (20 –25) or CD4 interaction is
blocked (26, 27), impaired Th2-type responses result while Th1type responses are spared.
In contrast, several studies with variant peptides, which exhibited altered affinity for MHC or for the TCR, have suggested that
high intensity stimulation promotes Th1-like responses and less
stimulation favors Th2 responses (28 –32). These reports also correlate with those that showed that low dose Ag preferentially supported IL-4 over IFN-g secretion (14, 33, 34). Similarly, Th2-like
cells can be elicited with soluble protein, a protocol that is thought
to induce only a weak T cell response, whereas protein in adjuvant,
which produces much higher levels of clonal expansion and also
involves CD28 ligation (35, 36), often primes for Th1-like
cells (37– 43).
Thus, there is no consensus regarding a universal model of differentiation based on strength of signaling, and the reasons for the
apparently conflicting experimental outcomes are not clear. In this
3
Abbreviations used in this paper: L, ligand; PCC, pigeon cytochrome c; MCC,moth
cytochrome c.
0022-1767/99/$02.00
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Opposing viewpoints exist regarding how Ag dose and affinity modulate Th1/Th2 differentiation, with data suggesting that both
high and low level stimulation favors Th2 responses. With transgenic T cells bearing a single TCR, we present novel data, using
peptides differing in affinity for the TCR, that show that the time period of differentiation can determine whether Th1 or Th2
responses predominate as the level of initial stimulation is altered. Over the short term, IFN-g-producing cells were induced by
lower levels of stimulation than IL-4-producing cells, although optimal induction of both was seen with the same high level of
stimulation. Over the long term, however, high doses of high affinity peptides led selectively to IFN-g-secreting cells, whereas IL-4and IL-5-secreting cells predominated with lower levels of initial signaling, brought about by moderate doses of high affinity
peptides. In contrast, too low a level of stimulation at the naive T cell stage, with low affinity peptides at any concentration,
promoted only IL-2-secreting effectors or was not sufficient for long term T cell survival. These results demonstrate that the level
of signaling achieved through the TCR is intimately associated with the induction of distinct cytokine-secreting T cells. We show
that dose, affinity, time over which differentiation occurs, and initial production of IL-4 and IFN-g all can contribute to which T
cell subset will predominate. Furthermore, these data reconcile the two opposing views on the effects of dose and affinity and
provide a unifying model of Th1/Th2 differentiation based on strength of signaling and length of response. The Journal of
Immunology, 1999, 163: 1205–1213.
1206
PEPTIDE REGULATION OF Th1/Th2 DIFFERENTIATION
Table I. Differential naive T cell responses with peptides of varying
affinitiesa
Peptide
Dose (mM) for
Half-Maximal
Proliferation
Relative
Difference in
Reactivity
Mean IL-2
Secretion (ng/
ml) at 0.1 mM
T102S
K103R
MCC
Q100N
Y97K
K99A
0.0003
0.0005
0.003
0.06
3.0
.10.0
110
16
1
220
21000
23000
15.0
8.9
3.9
0.5
0.3
,0.02
a
T cells (5 3 105/ml) were stimulated in triplicate with 2.5 3 105/ml DCEKICAM APCs and soluble doses of peptide for 72 h. Proliferation was measured by
incorporation of thymidine during the last 12 h. Column 2 shows the dose of peptide
required for half-maximal proliferation. Column 3 shows the activity of each peptide
relative to MCC (which is assigned a value of 1) based on proliferation dose responses. Column 4 shows mean IL-2 production at a single peptide dose (0.1 mM).
Similar results were seen with 1.5 3 106/ml T-depleted splenic APC. For full details,
see Ref. 45.
Ag-presenting cells
Spleen cells were depleted of T cells using complement fixation with Abs
to Thy-1.2 (F7D5 and HO.13.4), CD4 (RL172.4), and CD8 (3.155). Fibroblast cells (DCEK-ICAM) transfected with IEk and ICAM-1, and constitutively expressing B7-1, were used for restimulation of T cells. APC
populations were treated with mitomycin C (75 mg/ml, Sigma, St. Louis,
MO) 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 (Costar,
Cambridge, MA). Naive CD4 cells were plated at 5 3 105/ml with 4 times
as many T-depleted spleen APC and various concentrations of MCC or
peptide analogues. Stimulation was conducted for 4 or 12 days after which
viable cells were recovered and counted. T cells (6 3 104) were replated in
0.2-ml volumes in 96-well plates in duplicate with 3 3 104 DCEK-ICAM
fibroblast APC, prepulsed for 2– 4 h with 20 mM PCC peptide. Supernatants were collected for cytokine analyses 24 – 48 h later. SDs between
replicates were generally ,15% of the means. In some cases, 5–10 mg/ml
of Abs to IFN-g (XMG1.2), IL-4 (11B11), and IL-12 (R&D Systems,
Minneapolis, MN) were added at the initiation of primary cultures, and
IL-2 (supernatant from the X63.IL-2 cell line) was added at 10 ng/ml.
Materials and Methods
Mice
AND TCR-transgenic mice bearing T cells reactive for their native peptide,
moth or pigeon cytochrome c (MCC, PCC), and expressing the Vb3/Va11
TCR, were bred on a B10.BR background (H2k) as previously described (44).
Altered peptide ligands
MCC (aa 88 –103, ANERADLIAYLKQATK), PCC (aa 88 –104, KAERA
DLIAYLKQATAK), and variant peptides thereof, were synthesized in the
peptide facility at La Jolla Institute for Allergy and Immunology (San
Diego, CA), as described previously (45). Each peptide was selected based
on similar binding to purified IEk, with single amino acid substitutions (of
MCC) T to S at aa 102, K to R at aa 103, L to A at aa 98, Q to N at aa 100,
Y to K at aa 97, and (of PCC) K to A at aa 99 (Table I). Binding analyses
used a competition assay with radioiodinated PCC (46).
T cells
CD41 T cells were purified from spleen and lymph nodes of TCR-transgenic mice (44) by nylon wool depletion, followed by complement treatment with Abs to CD8 (3.155), heat-stable Ag (J11D), class II MHC (M5/
114, Y17, and CA-4.A12), B cells (RA3.6B2), macrophages (M1/70), NK
cells (PK136), and dendritic cells (33D1), cross-linked with mouse anti-rat
k chain (MAR 18.5). Any residual APC and any in vivo-activated T cells
were removed by isolating high density cells spun through a Percoll (Pharmacia, Piscataway, NJ) gradient (45, 53, 62, 80%). The resultant cells were
resting (low forward scatter, CD692, CD712, CD40L2, CD252) and
.95% CD41. In addition, .95% of these cells possessed a phenotype
associated with naive CD4 cells (CD45RB1, CD62L1, CD44low) along
with expression of the Vb3/Va11 TCR (44, 47). T cells were further purified by positive selection with anti-CD62L (Mel-14) using magnetic
beads (Miltenyi Biotech, Sunnyvale, CA) as in previous studies (7) to
ensure that responses were only generated from naive T cells.
Duplicate supernatants were recovered 20 –24 h (IL-2 and IL-4) and 44 – 48
h (IFN-g and IL-5) after T cell stimulation and pooled to assess cytokine
content. IL-2 production was determined as before (44, 48) by titrating
pooled replicate supernatants onto NK.3 cells, in duplicate, in the presence
of anti-IL-4 (purified from the 11B11 cell line, ATCC). IL-4, IL-5, and
IFN-g were measured by ELISA as in previous studies (7) using the Abs
11B11 and biotin-BVD6 (PharMingen, San Diego, CA), TRFK5 and biotin-TRFK4, and R46A-2 and biotin-XMG1.2 (PharMingen), respectively.
Standard curves were constructed with purified IL-2, IL-4, IL-5, and IFN-g
(supernatants from the respective X63.Ag. cell lines). The sensitivity of
each assay was similar, with levels of detection being 10 pg/ml for IL-2,
50 –100 pg/ml for IL-4 and IFN-g, and 100 pg/ml for IL-5.
Intracellular cytokine staining
Intracellular staining for cytokines was performed as described previously
(49) with modifications. Briefly, T cells were harvested after 4 or 12 days
of culture and restimulated (at 5 3 105/ml) with one-half as many PCCpulsed DCEK-ICAM APCs for 6 h in the presence of 10 mg/ml brefeldin
A (Sigma). Cells were harvested, stained with cychrome-anti-CD4
(PharMingen), fixed with 2% paraformaldehyde, and permeabilized with
PBS containing 0.5% saponin (Sigma) and 1% BSA. Cells were doublestained (at 4°C) with 2.5 mg/ml FITC-anti-IFN-g (Caltag Laboratories,
Burlingame, CA) and PE-anti-IL-4 (Caltag) or PE-anti-IL-2 (PharMingen),
and the percentage of positive cells (gated on live CD41 cells) was determined by FACScan flow cytometry (Becton Dickinson, Mountain View,
CA) with Cellquest software. Nonstimulated cells (negative control) were
,1% positive for any cytokines.
Results
Native peptide induces effector cells secreting IL-4 and IFN-g in
a dose-dependent manner
Naive CD4 cells were stimulated with varying doses of MCC presented on splenic APC and cultured for 4 days. An equivalent
number of surviving T cells was restimulated with a single dose of
Ag and assayed for their ability to secrete Th1 and Th2 cytokines
(Fig. 1, Table II, day 4). Low dose MCC (0.001 mM) elicited
effector T cells that primarily secreted IL-2 (8.6 ng/ml), a low level
of IFN-g (0.6 ng/ml), and no detectable IL-4 (,0.05 ng/ml). At
doses ,0.001 mM, only 20% or fewer naive cells are stimulated
(45), and those cells that persisted produced only IL-2 when restimulated (data not shown). At higher doses (0.01 mM and above),
effector T cells producing IFN-g and IL-2 were induced, with increasing peptide concentrations eliciting higher levels of IFN-g.
IL-4 was similar, although the dose required for induction of cells
secreting this cytokine was higher than for IFN-g-secreting cells
(Fig. 1 and data not shown, but typically 1–10 mM MCC for IL-4
vs 0.01– 0.1 mM for IFN-g). IFN-g was always produced at greater
levels than IL-4 (tens of ng/ml compared with up to 1 ng/ml),
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Cytokine secretion
study, we present novel data suggesting that both arguments are
correct and that the time period over which T cells differentiate can
be critical to whether Th1 or Th2 cells predominate. We show that
a high level of stimulation, incorporating both dose and affinity, is
required to generate T cells secreting IL-4 and IFN-g, but only
during the initial stages of the naive T cell response. Generally,
higher doses of peptide were needed to generate or detect IL-4secreting vs IFN-g-secreting cells. In contrast, differentiation to a
Th2-like phenotype (with a striking increase in IL-5 production)
over time is favored by a lower level of stimulation, with peptides
of high affinity, at high dose, promoting cells with a Th1-like phenotype. These results further demonstrate that the nature and dose
of the Ag can be major contributors to Th1 and Th2 differentiation
and show that the period of differentiation may also have a profound effect on the phenotype achieved.
The Journal of Immunology
1207
tion is required over the short term to generate cells producing IFN-g
and IL-4 and that the higher the level of stimulation, the more IFN-g
and IL-4 are produced. IL-5 was produced only minimally (,1 ng/
ml) by T cells at day 4 (data not shown).
Low affinity peptides do not promote cells producing either IL-4
or IFN-g, whereas high affinity peptides promote development of
both IL-4- and IFN-g-secreting cells
which is typical for CD4 cells from many sources. However, the specific activity of IL-4 is much greater than that of IFN-g, and previous
results have shown that these low levels of IL-4 are functionally significant. These data suggest that a moderate to high level of stimula-
Production of IL-2 by the naive T cell is critical for the
subsequent differentiation into Th1 or Th2 phenotypes and in
part accounts for the different cytokine profiles induced with
peptides of varying affinity
A major difference between weak and strong agonist peptides is
the ability to promote IL-2 secretion from naive cells, which in
Table II. T cell survival over time is differentially affected depending on peptide dose and affinitya
% of T Cells Recovered at Peptide Concentration (mM)
Peptide
Day 4
T102S
K103R
MCC
Q100N
Y97K
K99A
K99A 1
IL-2
Day 12
T102S
K103R
MCC
Q100N
Y97K
K99A
K99A 1
IL-2
0.001
0.01
0.1
1
10
100
50
96
64
NA
NA
NA
NA
540
348
66
36
NA
NA
NA
1088
608
284
56
24
28
68
808
1032
400
216
48
28
60
648
928
864
284
70
50
328
704
760
888
484
120
96
380
,10
NA
NA
NA
NA
NA
NA
248
90
,10
,10
NA
NA
NA
1296
388
28
10
,10
,10
,10
648
420
232
90
,10
,10
10
164
272
578
60
,10
,10
88
64
172
264
42
,10
,10
140
a
T cells were stimulated as in Figs. 1 and 3 and cell recoveries were measured by trypan blue exclusion on day 4 and day
12 of culture. Values represent the percent of T cells recovered based on the input. A similar pattern of recovery was seen in
three independent experiments. Recovery in the absence of Ag was 20 – 40% after 4 days and ,10% after 12 days. NA, not
assayed.
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FIGURE 1. a, High doses of agonist peptides are required to generate
IFN-g- and IL-4-secreting effector cells over 4 days. Naive CD4 cells from
TCR-transgenic mice were stimulated at 5 3 105/ml in 1-ml cultures with
4 times as many T-depleted spleen APC and varying doses of MCC (native) peptide, heteroclitic (T102S, K103R), and weak agonist (Q100N,
Y97K, K99A) peptides. After 4 days, equivalent numbers of viable T cells
were recultured at 3 3 105/ml in 0.2-ml cultures in duplicate with one-half
as many DCEK-ICAM fibroblast APC prepulsed with 20 mM PCC peptide.
Supernatants were recovered 24 – 48 h later, and duplicates were pooled
and assayed for cytokine content. Results are representative of at least two
experiments per peptide with SDs being within 15% of the means of the
replicates. The limit of detection for both IL-4 and IFN-g was the same, in
the range of 0.05– 0.1 ng/ml. n.a., not assayed.
To determine the effect of peptide dose and affinity on Th1/Th2
development, naive T cells were cultured with peptide analogues
that displayed similar binding to IEk but that varied in stimulatory
capacity (Table I) (45). Reactivities are most likely attributable to
varying affinities of interaction between the TCR and the peptide/
MHC complex as shown by Davis et al. (50, 51). The very weak
agonistsY97K and K99A induced little or no expansion of the T
cell population over 4 days (Table II). Similar to MCC at low dose,
Y97K and K99A at any dose elicited effector cells that largely
made IL-2 (Fig. 1). Small amounts of IFN-g were produced with
high doses, although much less than with high dose MCC, and
IL-4 was not detected. The third weak agonist peptide (Q100N)
produced intermediate results with IFN-g and IL-4 seen only at
high peptide doses (1–100 mM), which were greater than or equivalent to those required with the native peptide MCC. The heteroclitic (more stimulatory than native) peptides T102S and K103R
resulted in a shift in the dose response for effector cytokine generation by 10- to 100-fold. IL-4- and IFN-g-secreting cells were
now produced with peptide concentrations at much lower doses
compared with the native peptide. Similar results (not shown) were
also seen with a third heteroclitic peptide, L98A, which is also
;10-fold more stimulatory than MCC, showing that the effects
produced were not due to particular amino acid substitutions or
only for certain residues.
1208
FIGURE 2. Exogenous IL-2 can compensate for the inability of low
affinity peptides to induce IL-4-and IFN-g-secreting cells. Naive T cells
were cultured over 4 days with low affinity peptide, K99A, in the presence
of 10 ng/ml IL-2, or with MCC and then restimulated as in Fig. 1. Similar
results were seen in three other experiments and with the other low affinity
peptide, Y97K.
The length of the differentiation period affects the cytokine
profile induced by varying doses of native peptide
Differentiation in vivo can occur over an extended period of time
with primary T cell responses often lasting for 15 days or more. To
assess whether the parameters of dose and affinity established during the initial 4 days also applied over time, naive T cells were
stimulated with MCC for 12 days (Fig. 3). In the absence of Ag
and with MCC at 0.01 mM or less, T cells did not persist in culture,
indicating that a high level of stimulation is required for long term
cell survival (Table II, day 12). The pattern of response for IL-2 or
IFN-g was similar to that of day 4, with IL-2 levels being equivalent at all high doses (1–100 mM) and the amount of IFN-g rising
with increasing dose. IL-4 followed a pattern similar to that of
IFN-g; however, it was evident that the relative ratio of IFN-g to
IL-4 with medium dose of MCC (;1:1 at 1 mM in the example
shown) was significantly different from that at higher doses of
peptide (;4:1 and 6:1 at 10 and 100 mM, respectively), and in
complete contrast to that at day 4 (ratios of .25:1 for doses of 1,
10, and 100 mM; see Fig. 1). More striking was IL-5, which was
now produced at very high levels by T cells derived over 12 days
with 1–100 mM MCC. Again, the intermediate dose of 1 mM induced T cells that were highly skewed toward this Th2 cytokine
with an IFN-g-IL-5 ratio of 0.1:1 compared with 2.5:1 at 100 mM.
The absolute levels of IFN-g at day 12 varied between experiments
but were generally similar to or lower than at day 4, whereas
FIGURE 3. Strong agonist peptides when used at low/intermediate
doses preferentially elicit IL-5-secreting cells whereas high doses elicit
IFN-g-secreting cells after long term differentiation. Naive T cells were
stimulated as in Fig. 1 with varying doses of peptide on T-depleted splenic
APC. After 12 days, viable cells were recovered, and equivalent numbers
were restimulated for cytokine analyses. SDs from each assay were less
than 15% of the means. Results are representative of three separate experiments. n.r., too few/no cells recovered; n.a., not assayed.
IL-4/IL-5 levels were always elevated over time. Thus, whereas
higher priming doses of Ag favor increasing production of Th1 and
Th2 cytokines after 4 days, a lower priming dose induces greater
production of IL-4 and particularly IL-5 after 12 days relative to
IFN-g.
Over an extended differentiation period, lower doses of high
affinity peptides favor generation of T cells secreting Th2
cytokines
To determine the effect of time on differentiation, T cells were
cultured for up to 12 days using various doses of weak agonist and
heteroclitic peptides (Fig. 3). With the low affinity peptides Y97K
and K99A either no cells persisted over 12 days or too few to be
assayed (,10% of the input, Table II, day 12). This showed again
that long term T cell survival required a high level of stimulation
at the naive stage. The other weak agonist, Q100N, resulted in a
dose-response curve at day 12 that was shifted at least 10-fold to
the higher concentrations vs MCC. Moderate production of IFN-g,
IL-4, and IL-5 were seen only at the highest dose of priming Ag
(100 mM).
With higher doses of heteroclitic peptides, fewer T cells were
recovered than at lower doses (Table II, day 12). This suggests that
too high a level of initial stimulation may be detrimental to survival. The heteroclitic peptides, T102S and K103R, had a doseresponse pattern similar to that of MCC but shifted 10 –100 fold to
the lower concentrations. IL-2 production was similar to that seen
after 4 days with peak responses from T cells derived with doses
of 0.01–1 mM, whereas high dose peptide resulted in T cells producing less IL-2. The secretion of IFN-g was again dose dependent, with the greatest levels seen with T cells elicited with the
highest doses. In contrast, a bell-shaped profile was evident for
both IL-4 and IL-5 secretion with low to moderate doses (0.01–1
mM) of heteroclitic peptides promoting T cells capable of producing the highest levels of these Th2 cytokines and significantly
more than seen with larger doses (10 –100 mM).
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turn governs proliferation over time (45). To assess whether IL-2
played a role in differentiation, cultures were set up with varying
doses of the weak agonist K99A, and IL-2 (10 ng/ml) was added
at a level similar to that normally promoted by higher doses of
native or heteroclitic peptides. Addition of exogenous IL-2 to
K99A cultures resulted in a cytokine and dose-response profile
similar to that of MCC or K103R, with significant levels of IL-4
and IFN-g being produced at 1–100 mM (Fig. 2). In contrast,
K99A alone resulted in only IL-2-secreting cells (Fig. 1). Similar
results were seen with Y97K when supplemented with IL-2 (data
not shown). These results show that differentiation from T cells
secreting only IL-2 to those secreting Th1 and Th2 cytokines is
directly related to the dose and affinity of peptide seen by the naive
T cell. In addition, the ability to generate cells expressing IL-4
and/or IFN-g is dependent on the extent of stimulation of the naive
population and the capacity to promote IL-2 secretion.
PEPTIDE REGULATION OF Th1/Th2 DIFFERENTIATION
The Journal of Immunology
The ratio of Th1 to Th2 cytokines at day 12 was similar to that
of native peptide and was dramatically different depending on the
dose of heteroclitic peptides used. For example, in the experiment
shown in Fig. 3 and quantitated in Fig. 4a, the IFN-g-IL-4 ratio
with 0.01 and 0.1 mM T102S was 0.1:1 and 2:1, respectively;
whereas with 10 and 100 mM, it was 25:1 and 94:1, respectively.
Again, the most striking effect was seen with IL-5, which was
produced at very high levels from T cells derived after stimulation
with lower doses of high affinity peptides (Fig. 4a). Whereas 100
mM K103R and T102S induced T cells secreting predominantly
IFN-g (IFN-g-IL-5 ratios of 68:1 and 48:1 in the examples shown),
1 mM induced cells secreting near equivalent levels of both IFN-g
and IL-5 (ratios of ;1.5 for both peptides), and 0.01 mM resulted
in T cells highly skewed toward IL-5 (ratios of 0.3:1 and 0.02:1,
respectively). The relationship between dose and affinity was illustrated by comparing responses with the native peptide (Fig. 4b).
With 100 mM MCC T cells resembled those induced with 1 mM
K103R or T102S (IFN-g-IL-5 ratio, 2.5:1), and with 1 mM T cells
resembled those with 0.01 mM heteroclitic peptides (ratio, 0.1:1).
Results similar to those of K103R and T102S were seen with a
third heteroclitic peptide, L98A (data not shown), demonstrating
that the effects observed were a true reflection of peptide reactivity
and affinity. A similar relationship was seen using 100 mM con-
FIGURE 5. Increasing Ag dose and affinity induce greater numbers of IL-41 and
IFN-g1 T cells. T cells were cultured as in
Fig. 1 and restimulated with PCC-pulsed
APCs for 6 h. Live CD41 cells were gated
and the percentage of cytokine positive cells
(indicated in FACS plots) was determined by
flow cytometry. Th1 and Th2 cells were generated in vitro and used as positive controls.
Data from day 4 and day 12 restimulations
are summarized in Table III.
centrations of the weak agonist K99A in the presence of exogenous IL-2. Here, T cells were elicited that behaved similarly to
those derived with lower doses of wild-type peptide (1 mM MCC)
or heteroclitic peptides (0.01 mM K103R or T102S) (Fig. 4b).
In summary, with increasing dose and affinity (in Fig. 4b), there
is a change in T cell subset development after long term culture.
Higher affinity peptides are more effective than low affinity peptides at eliciting secretion of both Th1 and Th2 cytokines. Increasing the concentration of high affinity peptides results in an increased ability of T cells to make IFN-g, whereas a bell-shaped
response is seen with IL-4/IL-5. As a consequence, at lower doses
of moderate to high affinity peptide, IL-4/IL-5 predominate over
IFN-g; but at higher doses IFN-g predominates. In contrast, lower
doses of weak agonist peptides (1 mM K99A 1 IL-2) generate
only IL-2-secreting cells, but as the dose is increased there is a
change from a Th2-like phenotype (IL-2 1 IL-5) to a mixed Th1/
Th2 phenotype.
Intracellular cytokine staining reveals Ag dose/affinity changes
in Th0, Th1, and Th2 development over time
In an attempt to correlate cytokine secretion with actual Th0, Th1,
and Th2 phenotypes, we used intracellular cytokine staining on T
cells cultured for 4 or 12 days with various doses of peptides. Fig.
5 shows flow cytometry plots (IFN-g vs IL-4 on top, IFN-g v. IL-2
on bottom) of T cells stimulated with the indicated doses of peptide for 4 days and then restimulated with APC and native peptide
(PCC) as in Fig. 1. Th2-like and Th1-like populations generated in
vitro with IL-4 or IL-12, respectively, were included as positive
controls. Similar to the bulk cytokine responses in Fig. 1, high
doses of agonist peptides were required to generate IL-41 and
IFN-g1 cells. Results from day 4 and day 12 FACS analyses are
summarized in Table III. Over 4 days, maximal numbers of IL-41
and IFN-g1 positive cells were seen with high doses of heteroclitic
peptide T102S (and L98A, data not shown) and approached the
level of staining seen with Th1- and Th2-like cells made with
exogenous factors. In addition, this peptide also induced a number
of Th0 (IL-41IFN-g1) cells initially; however, this was a transient
phenotype given that few if any were detected in day 12 cultures
(data not shown). The dose-response pattern of IFN-g1 cells was
the same in long term (day 12) cultures; however, similar to data
measuring cytokine levels, maximal numbers of IL-4-producing
cells were seen with intermediate doses of T102S. Higher doses of
peptide resulted in reduced levels of Th2 (IL-4) development. The
loss of IL-4 was not due to outgrowth of nontransgenic cells because the cells remaining were 95–98% positive for Va11 and
Vb3 chains of the transgenic TCR regardless of peptide dose (data
not shown). As in previous studies (49), we were not able to efficiently visualize IL-5-secreting cells with the available reagents
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. Comparison of cytokine profile of effector T cells generated
over 12 days with high, intermediate, and low doses of peptide analogues.
Naive T cells were stimulated for 12 days as in Fig. 3 with MCC, K103R,
T102S, or K99A 1 IL-2, at doses of 0.01, 1, and 100 mM. a, Ratios of
IFN-g: IL-4 and IFN-g: IL-5 produced after restimulation of day 12 cultures; b, total cytokine profile after restimulation. Too few cells were recovered from cultures that received 0.01 mM K99A 1 IL-2 or 0.01 mM
MCC. Data are from Fig. 3.
1209
1210
PEPTIDE REGULATION OF Th1/Th2 DIFFERENTIATION
Table III. IL-4- and IFN-g-positive cells increase with Ag dose over the short term, but numbers of IL-4producing cells decrease in long term culture with heteroclitic peptide (T102S)a
% Positive
Day 4
Peptide Dose (mM)
MCC
0.1
1
10
100
T102S
0.01
0.1
1
10
100
Day 12
IL-2
IFN-g
IL-4
IL-2
IFN-g
IL-4
37
58
54
59
2.5
11
15
17
2.0
2.7
2.9
10
14
32
47
40
1.5
1.3
1.2
3.5
0.5
0.6
1.6
2.6
59
72
51
54
40
0.6
5.2
14
27
33
1.6
2.1
5.2
14
22
30
52
65
71
74
1.2
1.2
1.8
2.8
2.5
1.4
8.2
21
12
8.7
a
T cells were stimulated with MCC or T102S as in Figs. 1 and 3 for 4 or 12 days and then restimulated with PCC-pulsed
DCEK-ICAM APCs for 6 h. Live CD41 cells were gated, and the percent of cytokine-positive cells was determined by flow
cytometry. Control T cells that did not receive pulsed APCs were #1% positive for all cytokines.
Endogenous IL-4 and IFN-g partially dictate the Th2/Th1
phenotype of T cells induced with varying peptide doses, but
IFN-g or IL-12 does not account for the lack of Th2 cytokines
at high dose
Prior studies have established that autocrine production of IL-4
and IFN-g by responding naive T cells can be responsible for
inducing effector T cells secreting variable levels of these cytokines (7, 8, 52). However, very little if any IL-4 or IFN-g can be
detected in primary cultures of naive T cells by conventional protein assay (7, 44). To determine whether endogenous IL-4 and
IFN-g were responsible for the differential cytokine profiles elicited, Abs to these cytokines were added at the start of culture (Fig.
6). For these data, similar scales were used to compare day 4 and
day 12 to emphasize the changes in cytokine levels with time. In
Fig. 6, a (top vs bottom panels) and b, there was an increase in Th2
cytokines (IL-4 and especially IL-5) at day 12 vs day 4 with a
corresponding decrease in Th1 cytokines (IL-2 and IFN-g).
Anti-IFN-g significantly inhibited generation of T cells secreting IFN-g at both 4 and 12 days (.50% at most doses and .85%
at high dose); similarly, anti-IL-4 almost completely inhibited cells
producing IL-4 and IL-5 (.80% at all doses). These data suggested that the balance of endogenous IL-4 and IFN-g could dictate the Th1/Th2 profile elicited. Anti-IFN-g did not alter the levels
of Th2 cytokines with high dose Ag but did result in enhanced
production of both IL-4 and IL-5 with low dose (.01 mM) at day
12. Blocking IL-4 had little effect on IFN-g secretion except for an
increase with high Ag doses. Anti-IL-12 revealed a limited role for
this cytokine in influencing Th1/Th2 development in this system.
IFN-g was reduced ;30% at days 4 and 12, but no consistent
effects on IL-4 and IL-5 production were seen in this and in two
other experiments.
The effects of blocking Abs on IL-2 secretion were less dramatic; however, IL-2 secretion from day 4 effectors was increased
slightly with anti-IFN-g (in three experiments), whereas blocking
IL-4 or IL-12 slightly inhibited or had no effect on IL-2 (in three
repeat experiments). IL-2 secretion was lower at day 11 but largely
unaffected by blocking Abs (Fig. 6a, bottom).
Fig. 6b illustrates that IL-5 was greatly up-regulated between
days 4 and 12 and that peak production over time occurred at lower
doses of the initiating Ag (0.1 mM). Significantly, the low levels of
IL-5 produced with high doses of priming peptide were not increased by anti-IFN-g or anti-IL-12 in contrast to those at low Ag
doses where anti-IFN-g and anti-IL-12 resulted in greater IL-5
production (Fig. 6b, right). Thus, different mechanisms appear to
govern generation of IL-5-secreting cells with low vs high Ag
doses.
FIGURE 6. Endogenous IL-4 and IFN-g are required to generate effector cells secreting Th2 and Th1 cytokines, respectively. Naive T cells were
stimulated as in Fig. 1 with 0.1–100 mM T102S, on T-depleted splenic
APC, and cultured for 4 (a, upper graphs; b, left graph) or 12 days (a, lower
graphs; b, right graph) in the presence or absence of anti-IL-4 (10 mg/ml),
anti-IFN-g (10 mg/ml), anti-IL-12 (5 mg/ml), or control Ab (10 mg/ml rat
IgG1) added at time 0 and 48 h. Equivalent numbers of viable T cells were
recovered and restimulated for cytokine analyses (a, IL-2, IFN-g, IL-4; b,
IL-5). Cells were replated in duplicate, and supernatants were assayed by
ELISA. Values are the mean 1 SD from duplicate cultures for each dose.
Similar results were seen in two repeat experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
even though high levels of IL-5 were produced in culture. Although the dose-response patterns seen when measuring the number of cytokine-secreting cells was fairly similar to the results with
bulk cytokine levels, they were not identical. These data suggest
that the amount of cytokine produced per cell can vary among T
cell populations, as we have described previously (49), and that
this can be just as important for the ultimate response as the absolute frequency of cells secreting a particular cytokine.
The Journal of Immunology
Discussion
In this study, we show that the dose of Ag and its affinity for the
TCR can be major regulators of T cell differentiation and that the
length of the differentiation period also can determine the phenotype elicited. The results are summarized in the models presented
in Fig. 7, which are based on the overall strength of signaling
received by a naive T cell. In the case here, the variables were dose
and affinity, although it is reasonable to also include cosignaling in
this scheme as suggested by many of the published experiments
assessing CD28-B7 interactions. Below a certain undefined level,
the naive T cell either does not receive sufficient signals to be
triggered at all or alternately to survive over a period of days. A
low overall level of stimulation above this threshold induces short
term effector T cells making IL-2 with little commitment to either
Th1 (IFN-g) or Th2 (IL-4) cytokines. A moderate strength of signaling, above the threshold level for naive T cell activation and
short term effector generation, is necessary for T cell survival over
an extended time (see Fig. 7, arrow), a conclusion similar to those
from recent studies assessing the requirement for self peptide/
MHC (53, 54). A high level of overall signaling is also required at
the naive stage before efficient induction of cells secreting either
Th1 or Th2 cytokines, but promotion of IL-4 responses over the
short term relies on a greater initial strength of signaling than promotion of IFN-g responses. In contrast, over time, the differentiation of cells secreting Th2 cytokines is favored by a moderate/
high level of signaling at the naive stage, but if the strength of
signaling (dose and affinity) is increased sufficiently, differentiation of Th1 cytokine-secreting cells is favored with a concomitant
reduction in the ability to produce IL-5 and IL-4.
Is the data in this report in conflict with other published studies
of the effects of Ag dose, Ag affinity, and strength of signaling on
immune deviation, or does it go some way in resolving the two
sides of the argument regarding induction of Th1 or Th2 cells? We
believe the latter, although obviously our study cannot explain all
of the previous dichotomous results. With the studies in which
repetitive stimulation was required for eliciting IL-4 (7, 8, 10 –12,
16), in many cases this was not accompanied by a loss of IFN-g,
and these results essentially mimic those seen here over the short
term with medium/high dose, high affinity peptides. In the reports
contending that CD28-B7 interactions are essential for Th2-type
responses (20 –23, 25), it can be argued that the absence of CD28
signaling primarily resulted in lower level production of IL-2, as
this and long term cell survival are the main facets of CD28 action.
This would then have translated to an overall lower level of response of the T cell. In a physiological setting, this may result in
presentation of medium/high dose, high affinity peptides in a situation where they behave as if low dose, low affinity, a phenomenon that we recently demonstrated, occurs when there is a lack of
cosignaling (45). Because the initial induction of IL-4-secreting
cells is dependent on higher levels of signaling than induction of
IFN-g-secreting cells, in the latter examples it may not have been
surprising that the Th2 aspect of response was preferentially lost.
Our novel data on long term differentiation under conditions of
very high level stimulation (high dose, high affinity) which show
preferential IFN-g secretion are also similar to those reports of
systems in which the peptide/MHC density encountered by the T
cell was varied (28 –30). In these cases, a very high ligand density
favored IFN-g-producing cells, whereas lower densities largely resulted in mixed responses, a phenotype largely reproduced in our
studies with lower doses of high affinity peptides. Our data are also
directly in line with others that used peptides with the same binding capacity for MHC but that differed in affinity for the TCR (31,
32). In these studies, lower affinity peptides, in the presence of
either IL-2 or anti-CD28, promoted cells secreting IL-4 and IFN-g,
whereas if the wild-type peptide was used with IL-2 or anti-CD28,
the IL-4 response was lost and the IFN-g response was enhanced.
Whether our results can also be used to interpret differential
responses in the presence (Th1) or absence (Th2) of adjuvant is not
clear (37, 38, 41– 43). Deviation to a Th2 response is primarily
seen with whole protein, whereas soluble peptide often tolerizes
completely rather than merely the IL-2/IFN-g arm (40, 42). It is
possible that protein in adjuvant leads to an overall very high level
of stimulation which would push the T cell response beyond Th2
cytokines and be of such intensity that largely IFN-g-producing
cells are generated (Fig. 7). Protein without adjuvant may result in
a moderate level of stimulation, or stimulation that is not as prolonged, which would then favor Th2 cytokines, particularly IL-5.
In contrast, peptide without adjuvant may give only a near threshold stimulation as depicted in Fig. 7, because presentation is not
for a sufficient length of time or in the context of costimulation.
Thus, in the latter case, T cell activation may either not be promoted at all, or more likely only induced with minimal IL-2 production. These conditions may result in some form of tolerance
either through cell death or through anergy.
At present, the mechanism by which strength of signaling affects
the generation of cells secreting such varying patterns of cytokines
is not clear. Because experiments were performed with transgenic
T cells bearing a single invariant TCR, selection of populations of
cells with TCR of higher or lower affinity is not as likely as it
potentially is with in vivo experiments of this nature. Such a phenomenon was recently demonstrated by Kuchroo and colleagues
(55, 56) where an altered peptide of PLP was shown to select
Th2-like cells the TCR of which recognized different residues as
primary contacts than the Th1-like cells normally elicited with
wild-type peptide. In addition, it is unlikely that responses in our
study reflected the outgrowth of a minor contaminating population
of cytokine-precommitted cells, first because of the homogeneous
nature of the CD62L1CD44low T cells used, and secondly because
of the pattern of reactivity that was seen throughout the range of
peptide doses and affinities at day 4 vs day 12.
Our previous data showed that a major difference between
higher and lower affinity peptides was their ability to elicit IL-2
secretion from naive T cells, which in part governed the extent of
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FIGURE 7. Model of Th1/Th2 differentiation based on strength of signaling. Model is based on the overall levels of stimulation achieved at the
naive T cell stage and is derived from data in Figs. 1–3. Cytokine levels are
relative and not based on actual quantities elicited. Dashed arrow indicates
threshold level of signaling required for T cell survival over time. Early
differentiation is based on data at 4 days; late differentiation is based on
data at 12 days.
1211
1212
and cause either up- or down-regulation of the transcription factors
NIP45, GATA-3, and c-Maf, all of which are linked to Th2 cytokine production (62). The relative levels of these may then dictate
the extent of differentiation to Th1 or Th2 phenotypes in combination with both growth (IL-2) and directive (IL-4, IFN-g) cytokine influences. In line with this are preliminary data suggesting
that the long term dose-response phenotype seen in Fig. 3 can be
reproduced over the short term if cosignaling molecules are
blocked or triggered, a situation in which differential cell selection/
survival is unlikely to occur.
In conclusion, we have presented a model showing that the
strength of signaling to a naive CD4 cell may modulate its ability
to differentiate and produce effector cells with the potential for
both Th1 and Th2 cytokines, or predominantly one or the other.
These data attempt to reconcile two opposing viewpoints on the
effects of Ag dose and affinity on Th1 and Th2 responses and
provide a rationale for some of the data generated in the area of
immune deviation. Finally, because of the spectrum of cytokine
profiles that can be produced depending on concentration, affinity,
and length of response, these studies also caution against varying
a therapeutic immunization regime (based on dose, and Ag analogues), unless sufficient in-depth preliminary studies are available
to suggest in which direction the response may proceed.
Acknowledgments
We thank Howard Grey, for helpful discussions and advice concerning
peptide analogues; and Vipin Kumar and Linda Bradley, for critical reading of the manuscript and comments.
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