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Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek.
We identified two altered peptide ligands of Hb(64-76) that retain
strong agonist activity but, at a given dose, induce cytokine patterns
distinct from the Hb(64-76) peptide. The ability of these peptides to
produce distinct cytokine patterns at identical doses is not due to an
intrinsic qualitative property. Each peptide can induce Th2 cytokines
at low concentrations and Th1 cytokines at high concentrations and has
a unique range of concentrations at which these distinct effects occur.
The pattern of cytokines produced from limiting dilution of naive T
cells demonstrated that the potential to develop an individual Th1 or
Th2 cell is stochastic, independent of Ag dose. We propose that the
basis for the observed effects on the Th1/Th2 balance shown by the
altered peptide ligands and the amount of Ag dose involves the
modification of soluble factors in bulk cultures that are the driving
force that polarize the population to either a Th1 or Th2
phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß or 
) with an antigenic ligand,
8–15 amino acids in length, bound to a self MHC molecule 1, 2, 3 .
Interaction of the TCR with its ligand in the presence of costimulation
provided by the APC leads to T cell activation, clonal expansion, and a
variety of effector functions 4 . Partial T cell activation is a
phenomenon in which a T cell is able to interact productively with a
suboptimal ligand. It was initially described in a 1991 report that a
2.102 CD4+ Th2 clone responded to hemoglobin
(Hb)4(64-76) peptide plus MHC
by producing IL-4 and proliferating but that the clone responded to an
altered peptide ligand (APL) containing a single-amino acid
substitution of Hb(64-76) by producing IL-4 in the absence of
proliferation 5 . This result directly demonstrated that subtle
changes in the peptide-MHC do not simply turn a given T cell on or off.
Instead, a T cell can respond to variants of the same antigenic peptide
differently by selectively activating some pathways but not others. To
extend our results from the clone and understand the biology of naive T
cells to different ligands, we made a TCR transgenic (Tg) mouse
expressing the 2.102 TCR. In this study, we wanted to examine the roles
of Hb(64-76) wild-type peptide ligand and variants of it in Th cell
differentiation and development.
Th cell subsets were first described by Mosmann and colleagues
based on the cytokine profiles and functional phenotypes of these cells
6 . Several studies have focused on Ag dose as a determining factor in
Th cell development 7, 8, 9, 10 . Stimulation of T cells specific for
OVA(323-339) from DO11.10 TCR ß Tg mice with a low or high dose of
Ag facilitated the differentiation of Th2 type cells, producing large
quantities of IL-4 and low levels of IFN-. In contrast, stimulation
with moderate levels of Ag directed the Th phenotype toward
IFN--producing Th1 cells. Furthermore, addition of neutralizing Abs
against IL-4 during the primary stimulation with either high or low
doses of Ag inhibited the differentiation toward the Th2 phenotype 7 .
This observation suggests that the levels of IL-4 produced during the
primary stimulation as a result of antigenic dose lead to Th cell
differentiation. Stimulation of T cells specific for pigeon
cytochrome c from AND Tg mice showed that low
doses of Ag lead to a Th2 phenotype, whereas high-dose stimulation
induced Th1-like cells 8 . The precise role of the MHC/peptide-TCR
interaction in Th cell development is still unclear. We sought to
pursue the role of Ag dose and APLs in Th cell development in a TCR Tg
model with a TCR specific for Hb(64-76). Our system has the advantage
of the availability of a wide panel of APLs to facilitate understanding
the TCR interaction with, and its affinity for, a variety of peptides.
Specifically, we wanted to address whether the differences in cytokine
profiles that we observe with various ligands in bulk culture
stimulation are reflected at the single-cell level. In
limiting-dilution experiments in this report, we observed that at the
single-cell level there is an equal chance to become an IL-4 or IFN-
producer independent of Ag dose. However, the simple rules governing
the phenotype of the single cells no longer apply to the complex
dynamics of cell-cell interaction at the population level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 2.102 Th2 cell clone was derived from a CE/J mouse recognizing
amino acids 64-76 of the d allele of the murine hemoglobin
ß-chain bound to an I-Ek molecule 5, 11 . The TCR -
and ß-chains were cloned from the cDNA, and sequencing revealed a
V4 and Vß1 variable-region usage. The sequences of these variable
regions of the - and ß-chains of the 2.102 T cell clone have been
previously reported (GenBank accession Nos. U46581 and U46842) 12 .
The TCR - and ß-chains were coinjected into C57BL/6 embryos, using
cDNA vectors that direct specific expression in Tg animals 13 . The Tg
founders were screened using both Southern blot analysis and PCR
amplification from tail digests 14, 15 , and two founders expressing
the cointegrated TCR and ß were identified. These mice were then
further backcrossed to B6.AKR containing the
H-2k allele to introduce the appropriate
restriction element. They were then crossed to RAG1-/-
mice to abrogate expression of endogenously rearranged receptors 16 .
The average thymus size from 2.102 TCR Tg RAG-/- mice is
1 x 108 cells, and the average number of splenocytes
is 3 x 107 cells.
Antigens
The variants of Hb(64-76) agonist peptide are referred to by a one-letter code representing the substituted amino acid followed by its position. For example, K69 refers to the Hb(64-76) peptide that has lysine substituted for threonine at position 69. The names and sequences of the peptides used in this study are as follows: Hb(64-76), GKKVITAFNEGLK; D73, GKKVITAFNDGLK; K69, GKKVIKAFNEGLK; and S70, GKKVITSFNEGLK. All peptides were synthesized on an Applied Biosystems (Foster City, CA; model 432A) or a Rainin (Woburn, MA) Symphony Multiplex peptide synthesizer. The peptides were purified by reverse-phase HPLC, and their amino acid composition was confirmed by mass spectrometry (Washington University Mass Spectrometry Facility, St. Louis, MO) and amino acid analysis (Beckman (Fullerton, CA) model 6300) 17, 18 .
Cell lines
The generation and characterization of the Th2 clone, 2.102, and the hybridoma cell line, G2, have previously been described 5, 11, 19 . T cell lines from the 2.102 TCR Tg RAG-/- mice were propagated as described 20 using 2.5 x 105 Tg splenocytes and 5 x 106 irradiated (2000 rad) non-Tg B6.AKR splenocytes as a source of APC in a 24-well culture plate. During the primary culture, splenocytes from the 2.102 TCR Tg RAG-/- mice were stimulated with various doses of Hb(64-76) or APLs. On days 3–4, cells were expanded threefold into fresh medium in 24-well plates. On day 7, the T cells were harvested, washed, counted, and restimulated at 2.5 x 105/well by 5 x 106 B6.AKR splenocytes presenting 1–10 µM Hb(64-76) peptide. Supernatants were collected at 48 h, and the cytokine profile was assayed by ELISA as described below. Tissue culture medium was RPMI 1640 containing 10% FCS (HyClone, Logan, UT), 2 mM Glutamax, 2 x 10-5 M 2-ME, and 50 µg/ml gentamicin.
T cell proliferation assay
T cell proliferation assays were performed in 96-well flat-bottom plates. T cells, at 2 x 104 per well, were cultured with 5 x 105/well irradiated (2000 rad) non-Tg or B6.AKR splenocytes and 0–100 µM Hb(64-76) or the APLs. The final volume per well was 200 µl, and the cultures were incubated at 37°C for various times with the addition of [3H]thymidine at 0.4 µCi/well during the last 20 h.
In vitro cytokine assay
The concentrations of IL-4 and IFN- in the supernatants
collected from the T cells that had been restimulated for 48 h
after full primary stimulation were measured by quantitative capture
ELISA 21, 22 . Purified mAbs to mouse cytokine IL-4 (11B11) and
IFN- (H22) were the kind gift of Dr. R. Schreiber (Washington
University School of Medicine, St. Louis, MO), and used to coat
Immulon 2 ELISA plates (Dynatech Laboratories, Chantilly, VA).
Recombinant mouse cytokines (Dr. R. Schreiber) were used as controls to
generate standard curves, and polyclonal rabbit anti-IL-4 (1:4,000)
and polyclonal goat anti-IFN- (1:4,000) were used as the
secondary Abs (Dr. R. Schreiber). For the IL-4 ELISA, biotinylated goat
anti-rabbit (1:20,000) and avidin D-peroxidase
conjugate (1:5,000) (Vector Laboratories, Burlingame, CA) were
developed with 3,3',5,5'-tetramethylbenzidine (Medic Biotech,
Foster City, CA) and read at 450 nm on a Microplate Reader (Bio-Tek,
Winooski, VT) after the addition of 0.5 M H2SO4
to stop the reaction. For the IFN- ELISA, goat anti-rabbit
peroxidase-conjugated Ab was used, and the ELISA was read at 414 nm
after the addition of 2,2'-azino-di-[3-athyl-benzthiazolin-sulfonate
(b)] (ABTS) plus hydrogen peroxide.
Intracellular cytokine staining
Naive 2.102 TCR Tg T cells were stimulated with either different
doses of Hb(64-76) or APLs in the presence (200 U/ml IL-4 or 5 U/ml
muIL-12) or in the absence of exogenous cytokines for 7 days. T cells
were then washed, and equivalent numbers of cells were restimulated for
18 h with 1 µM Hb(64-76) in the presence of APCs lacking
CD4+ and CD8+ T cells. During the last 4 h
of stimulation, 10 µg of brefeldin A was added to the culture. Cells
were then fixed with formalin and permeabilized with 0.5% saponin.
Subsequently, T cells were stained with phycoerythrin-conjugated
anti-IL-4 or fluorescein-conjugated anti-IFN- (PharMingen,
San Diego, CA) for FACS analysis.
Limiting dilution of 2.102 TCR Tg RAG-/- cells
Splenocytes from the 2.102 TCR Tg RAG-/- mice were diluted between 10–100 cells per well in a U-bottom 96-well plate. Based on FACS analysis, this resulted in 1 to 10 T cells per well. Irradiated exogenous splenocytes from B6.AKR were added (5 x 105) to each well as APCs. Cells were stimulated with different doses of Hb(64-76) for 10 days. After 10 days, cells were restimulated with 1 µM of Hb(64-76) and 2.5 x 105 APCs for 3 days. On day 3, cells were expanded in the presence of 50 U/ml of rIL-2 23 . On day 7, cells were pooled, washed with HBSS, and restimulated with 1 µM of Hb(64-76) for 48 h in the presence of 5 x 106 irradiated APCs in a 96-well U-bottomed plate. Supernatants were collected and analyzed further for cytokine production.
Flow cytometric analysis
Cells (1 x 106) were incubated at 4°C for 1 h with 100 ng of either phycoerythrin-conjugated anti-mouse CD4 or fluorescein-conjugated anti-mouse CD8 (PharMingen) in a total volume of 200 µl of staining buffer (PBS containing 2% BSA). Cells were then washed, resuspended in 400 µl of staining buffer and 1% paraformaldehyde, and analyzed by flow cytometry on a Becton Dickinson FACScan (Becton Dickinson, Braintree, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 2.102 TCR Tg mouse was generated as described in
Materials and Methods. Fig. 1
compares the CD4/CD8 profiles of thymus and spleen cells between
different haplotypes of 2.102 TCR Tg RAG-/- mice. T cells
expressing the 2.102 TCR were efficiently positively selected on the
H-2k haplotype. We had thus generated a TCR Tg mouse,
through the use of which we could examine Th cell differentiation in
the Hb(64-76) system.
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We wanted to confirm the cytokine profile produced by D73 and
Hb(64-76) in a more physiologic setting. Previously, it was shown that
stimulation of the 2.102 T cell clone with D73 peptide induced IL-4
production in the absence of proliferation 5 . Therefore, we extended
our observations to the Tg mice expressing the same TCR. Naive 2.102
TCR Tg RAG-/- cells were stimulated with 1, 0.1, or 0.01
µM of Hb(64-76); 100 µM of D73 peptide; or 1 µM ionomycin plus 50
ng/ml PMA. After 1 wk, an equivalent number of all the T cells were
restimulated with 1 µM of Hb(64-76). As shown in Fig. 2, D73 ligand induced a strong Th2
response in the 2.102 TCR Tg RAG-/- splenocytes, even
though the proliferation seen by this peptide during primary
stimulation was diminished more than 10,000-fold in comparison with
Hb(64-76). Our result with Hb(64-76) stimulation is consistent with the
data reported in other systems 7, 8 , in that, with a low dose of Ag,
the phenotype of the T cells is polarized toward a Th2 phenotype, and
with higher Ag doses, toward a Th1 response.
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To examine a broader spectrum of proliferative responses of the
2.102 TCR Tg RAG-/- splenocytes, we used a panel of APLs
that have substitutions at critical residues interacting with the TCR
11, 24 . To assess the phenotypic development of these Tg T cells
after the primary stimulation, we concentrated on ligands that had
either agonist or weak agonist properties. The previously described D73
ligand is a weak agonist ligand and requires a very high Ag dose (100
µM) to stimulate the T cells. We needed to find other altered ligands
for which we could vary the concentration during the primary culture.
Since we were unable to vary the concentration of D73, we used K69 and
S70 for subsequent experiments. As shown in Fig. 3A, stimulation of the Tg T
cells with Hb(64-76) and K69 resulted in very similar proliferative
responses, with K69 slightly more stimulatory at lower concentrations
of peptide. Stimulation with the S70 ligand resulted in a proliferative
response that was shifted about fivefold from the response to
Hb(64-76). All these ligands have their maximal proliferative response
at 1 µM of Ag. To examine the responses of multiple 2.102 TCR Tg
RAG-/- mice to these ligands, we compiled the data from
nine separate experiments and plotted these values based on the maximal
response of each peptide (Fig. 3B). We saw that much less
K69 peptide was needed to induce proliferation of 2.102 T cells than
Hb(64-76) and conversely, more of the S70 peptide was required to give
a similar proliferation pattern. Again, the maximum response with all
of these ligands was achieved at 1 µM of each peptide.
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Variants of Hb(64-76) induce different cytokine patterns
Previous reports have indicated that an APL can induce a cytokine
pattern that differs from that induced by the wild-type agonist ligand
9, 10, 26 . The presence or absence of IL-2 absolutely did not result
in any difference in phenotype development of our bulk cultures (data
not shown). Shown in this report, primary stimulation was done in the
absence of any exogenous cytokines, and production of IL-4 or IFN-
was assessed after secondary stimulation with Hb(64-76). Fig. 4 depicts values for IL-4 or IFN-
produced when naive 2.102 TCR Tg RAG-/- splenocytes were
stimulated with 1 µM wild-type or APL for 1 wk and subsequently
restimulated with Hb(64-76). As Fig. 3
shows, proliferative responses
for each of these three ligands was maximal at 1 µM concentration of
peptide, yet IL-4 production at this concentration was very different
(Fig. 4).
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Next, we wanted to examine whether the differential cytokine
production we observed with APLs was due to an intrinsic ability of the
peptide to transduce a qualitatively different signal to the T cell. We
altered the concentrations of the peptides used during the primary
stimulation as shown in Fig. 5 and
assessed IL-4 production. The results indicated that although there was
a lack of IL-4 produced by K69 at a 1 µM concentration of peptide
(Figs. 4 and 5), decreasing the concentration of K69 during the primary
culture increased IL-4 production (Fig. 5). While it is at 1 µM of
each peptide that the proliferative responses seen for each of the
three ligands is maximal, it is clear that the concentration for
optimal cytokine production for each peptide differs. Lower
concentrations are optimal for K69-stimulated IL-4 production than for
S70 or Hb(64-76). This argues against the idea that each ligand has an
absolute intrinsic ability to drive Th1 or Th2 differentiation, since
for each given peptide, a dose can be found at which it will stimulate
specific cytokine production. This study is consistent with those
reported by others, in which it has been shown that low doses of
peptide Ags will induce a Th2 response, while higher doses will direct
the differentiation toward a Th1 phenotype 7, 8, 9, 10 . However, these
results also show that for each given ligand, there is a range of
concentrations at which cells will be driven toward Th1 or Th2, but the
concentrations at which these occur are not necessarily identical for
each given ligand.
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-producing T cells
We then wanted to investigate whether the differences in cytokine
production seen in the bulk populations result from stimulating
different percentages of cells, each of which produces a constant
amount of cytokine, or from stimulating a constant percentage of cells
that produces a fluctuating amount of cytokine. To address this, we
used the intracellular staining of T cells for IL-4 or IFN- after
restimulation. The controls consisted of either no stimulation or
stimulation in the presence of exogenous IL-4 or IL-12 for Th2 and Th1
development, respectively. As shown in Fig. 6A, if cells were not
stimulated, no IL-4 or IFN--producing T cells were detected.
However, upon primary Ag stimulation in the presence of exogenous IL-4,
we detected about 13% IL-4-producing T cells and essentially no
IFN--producing cells. In the presence of exogenous IL-12, about 23%
of cells produced IFN-, and no cells made IL-4. The amounts of
cytokines produced under these conditions based on the ELISA assay
correlate very well with the percentages of different IL-4- or
IFN--producing T cells. In a Th2 environment, a large amount of IL-4
is made, and no IFN- is made. In a Th1 response, we detected a large
amount of IFN- and no IL-4 (Fig. 6A). In the case of
different doses of Hb(64-76) stimulation in the absence of any
exogenous cytokines, we observed that at low peptide stimulation there
was a higher percentage of cells producing IL-4 (Fig. 6B).
At extremely high concentrations, we detected more IFN--producing T
cells. Interestingly, also consistent with the proliferation data shown
in Fig. 3A, significantly increased numbers of cells were
invariably recovered from primary cultures stimulated with higher Ag
doses (data not shown). However, there is a dose of Hb(64-76) ligand at
which both IL-4- and IFN--producing T cells can be detected at
similar percentages. With D73 at 100 µM and S70 at 1 µM
stimulations, there was a skewing toward the IL-4-producing cells.
However, K69 stimulation at 1 µM favored increased percentages of
IFN- cells. Therefore, rather than having a constant number of cells
increasing or decreasing cytokine production, different stimulation
conditions resulted in changing percentages of cells producing each
cytokine.
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produces exclusively Th2 cells, while addition of
IFN augments differentiation of Th1 cells
To further examine the role of IFN- during Th cell
differentiation, we added either 50 µg/ml of anti-IFN- (H22)
or 1000 U/ml of exogenous IFN- to the primary stimulation with
different doses of Hb(64-76) (Fig. 7).
Although after restimulation there was not a remarkable difference
observed in IL-4 production across the Ag doses in the presence of H22,
the level of IFN- production was dramatically decreased, even at a
high Ag dose. In the presence of H22, T cells exclusively produced Th2
cytokines. In the presence of exogenously added IFN-, we no longer
attained a Th2 phenotype, even at the low doses of peptide stimulation,
as observed previously. There was a dramatic decrease in the production
of IL-4, with an increase in IFN- production by the T cells across
all of the Ag doses. These data support the idea that increasing the
amount of IFN- in the primary culture leads to Th1 phenotype
development. In combination with the results shown in Fig. 6B, this suggests that Th1 development is dependent upon a
critical number of cells producing IFN- activated in the primary
culture.
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at the
single-cell level is stochastic and independent of Ag dose
We wanted to understand whether the differential cytokine pattern
seen due to the effect of Ag dose on bulk cultures was reflected at the
level of single T cells. To address this issue, we performed
limiting-dilution assays and stimulated the individual T cells with
different concentrations of Hb(64-76) and a fixed number of APCs. As
shown in Fig. 8A, in contrast
to the bulk culture stimulation, independent of Ag dose, we identified
both IL-4- and IFN--producing T cells. Even under low- or high-dose
conditions, under which we only observe IL-4 or IFN- produced at a
population level, we saw that at the single-T cell level, this event
was stochastic. Further, the potential of a given T cell to develop
into a Th1- or Th2-type cell was independent of the stimulatory dose.
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B, stimulation of
single T cells in the presence of exogenous cytokines such as IL-4 or
IL-12 directed the phenotype development toward Th2 or Th1,
respectively. Taken together, our results show that Th1 and Th2
development is determined at the population level, at which the
establishment of cross-talk between T cells and APCs exists. There is a threshold for generation of a Th1 population
In the bulk culture stimulation, there is polarization of cytokine
production depending on the Ag dose or the APL used. One possible
explanation is that APLs do not deliver an intrinsically distinct
signal through the TCR, but rather act by having different thresholds
for cellular activation in bulk culture. We therefore propose a simple
model in which there is a threshold, correlating with the percentage of
IFN--producing cells, that determines the overall Th1/Th2 phenotype
of the population. Below this threshold of stimulation with any ligand,
a Th2 response predominates, whereas above this threshold a Th1
response is favored. There exists a zone in which both Th2 and Th1
cells coexist. To distinguish the possibilities, we measured the
percentage of the T cells producing IFN- as a function of Ag dose
(Fig. 9). At low concentrations of
Hb(64-76), the percentage of the IFN- cells was very low, whereas at
higher peptide concentrations the number of IFN--producing T cells
increased rapidly. The results of the studies using APLs correlate well
with this scenario. For example, at 100 µM stimulation with D73
peptide, the majority of the cells activated were IL-4 producing,
leading to the generation of a Th2 phenotype at the population level.
The same is true for S70 stimulation at 1 µM of Ag. However, as with
the Hb(64-76) peptide, we observed that increasing the dose of the S70
ligand would activate enough IFN--producing cells to shift the bulk
culture toward a Th1 response. At 1 µM of K69, the threshold of
IFN--producing cells had already been reached. Therefore, by
lowering its concentration, we could then induce a Th2 phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells dominate and a Th1 response is generated. APLs
may require lower or higher Ag doses to give the same phenotype as the
wild-type ligand but show the same basic dose dependence. For the very
weak agonist ligands, such as D73 in this study, the Ag dose necessary
to reach the threshold for induction of a Th1 phenotype may not be
attainable. We propose that it is not an absolute intrinsic ability of
individual ligands that causes Th1 or Th2 development, but rather that
the activation of a critical number of cells at high doses results in
the production of enough IFN- in the milieu to effect polarization
to Th1.
We believe that our results unify much of the data that has been put
forth by other colleagues. Several studies have shown that, in the case
of soluble Ags, a Th2 response results at low doses of priming Ag, and
a Th1 response occurs with higher Ag concentrations 27 . In the OVA
system, Hosken and colleagues showed that low Ag doses (<0.05 µM)
generated Th2 effector cells, intermediate doses induced Th1 cells, and
high doses (100 µM) produced Th2 cells 7 . It is difficult to
directly compare our system with the OVA model and other systems
because of the different genetic backgrounds of the mice. However, one
similarity in these systems is that the endogenous IL-4 that is
produced is absolutely critical for Th2 development at low doses of Ag
stimulation, since neutralizing Abs to IL-4 also abrogate this
phenotype in our system (data not shown) 7, 27 . By our
limiting-dilution experiments, we find that with Th1/Th2 the phenotype
choice is independent of Ag dose (Fig. 8), suggesting that the dose
effects are not due to direct alteration in the quality of signals
delivered to the T cells. Rather, this result is instead consistent
with the hypothesis that the dose effect is mediated by altering the
cytokine milieu. Kamogawa et al. have shown that upon T cell
activation, a naive cell expresses the IL-4 gene and gives rise to both
IL-4 and IFN--producing cells 28 . In our limiting-dilution assay,
we were able to detect both IL-4 producers and IFN- producers across
a wide spectrum of Ag dose. Together it seems that stochastic
differentiation of a T cell to produce IL-4 or IFN- is more
favorable than a preselected phenotype of Th1 vs Th2 cells. The
critical issue to address is the discrepancy between cytokines produced
at the single-cell level and those made by a bulk culture. Our
explanation is that, at low doses of Ag, fewer total cells are
activated in the primary stimulation, and thus the concentrations of T
cell-derived cytokines available in this case would be quite low. At
this stage, perhaps the level of IL-4 that is produced in the culture
is dominant over IFN- or the IL-12 from macrophages. The autocrine
effects of IL-4 could then induce a selective advantage for the
survival or development of Th2 cells. At higher doses, enough T cells
are activated that the level of IFN- is sufficient to overcome Th1
inhibition by IL-4 and induce Th1 development. We observed that by
neutralizing IFN-, cells lost the capacity to undergo Th1
development. However, neutralizing IFN- did not augment the ability
of cells to produce IL-4. Addition of exogenous IFN- promoted
acquisition of the Th1 phenotype even at low doses at which the Th2
phenotype had previously been observed. Thus, IFN- promotes the
ability of T cells to become Th1 cells and disfavors the development of
Th2 cells at every Ag dose. These data support the model that
increasing the number of IFN- producing T cells in the primary
culture promotes Th1 differentiation. We also envision that there may
exist a dose at which IL-4- and IFN--producing cells can exist
simultaneously (Fig. 6B).
An effect of APLs in influencing Th phenotype was previously reported for a T cell clone 26, 29 . Studies by Bottomly and colleagues have shown that, in the human collagen IV and moth cytochrome c systems, APLs can selectively skew the population toward either the Th1 or Th2 phenotype 9, 10, 30 . In these studies, the differential effects were attributed to the induction of qualitatively distinctive biochemical signals by APLs. If development of Th1 vs Th2 were dependent upon the qualitative nature of the signal delivered, then one would predict that the Th polarization effect would be unaltered across the entire range of doses of a given ligand. However, by altering the dose of any given APL, we are able to change the phenotype of the response. However, for extremely weak agonists such as D73, a sufficient dose for Th1 development is not attainable. Although qualitative effects of APLs cannot be completely excluded, previous observations with variant ligands may be explained by quantitative effects of Ag dose on the alteration of the cytokine milieu.
Our intracellular cytokine staining results correlate with previous
reports 31, 32 , in which Bucy and colleagues observed a higher
frequency of cytokine mRNA-expressing T cells with increasing Ag dose.
However, a difference in the intensity of the staining, using their
system of digoxigenin-labeled riboprobes, with the individual cells
across the range of antigenic doses was not seen. Accordingly, our
limiting-dilution assay shows that far fewer cells are activated at low
doses, but those that do respond give similar amounts of IL-4 or
IFN- and proliferation as individual cells from high Ag dose
stimulation. Thus, our conclusions support a simple model in which Th1
development in vitro requires a larger total number of cells making
IFN-, thus allowing the culture to reach a critical threshold for
cytokine production. Once the amount of cytokine in the medium passes
this threshold level, the bulk population is then polarized toward a
Th1 response.
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Acknowledgments |
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Footnotes |
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2 Kenneth M. Murphy is a Howard Hughes Medical Institute Investigator.
3 Address correspondence and reprint requests to Dr. Paul M. Allen, Department of Pathology, Campus Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
4 Abbreviations used in this paper: Hb, hemoglobin; APL, altered peptide ligand; Tg, transgenic.
Received for publication July 14, 1998. Accepted for publication October 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-transgenic model. J. Exp. Med. 182:1579.ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 75:985.[Medline]
by T cells. Proc. Natl. Acad. Sci. USA 92:9510.This article has been cited by other articles:
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  K. S. Weber, M. J. Miller, and P. M. Allen Th17 Cells Exhibit a Distinct Calcium Profile from Th1 and Th2 Cells and Have Th1-Like Motility and NF-AT Nuclear Localization J. Immunol., February 1, 2008; 180(3): 1442 - 1450. [Abstract] [Full Text] [PDF]
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 D. Jane-wit, C. Z. Altuntas, J. Monti, J. M. Johnson, T. G. Forsthuber, and V. K. Tuohy Sex-Defined T-Cell Responses to Cardiac Self Determine Differential Outcomes of Murine Dilated Cardiomyopathy Am. J. Pathol., January 1, 2008; 172(1): 11 - 21. [Abstract] [Full Text] [PDF]
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V. Sanchez-Guajardo, J. A. M. Borghans, M.-E. Marquez, S. Garcia, and A. A. Freitas Different Competitive Capacities of Stat4- and Stat6-Deficient CD4+ T Cells during Lymphophenia-Driven Proliferation J. Immunol., February 1, 2005; 174(3): 1178 - 1187. [Abstract] [Full Text] [PDF]
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L. Krymskaya, W.-H. Lee, L. Zhong, and C.-P. Liu Polarized Development of Memory Cell-Like IFN-{gamma}-Producing Cells in the Absence of TCR {zeta}-Chain J. Immunol., February 1, 2005; 174(3): 1188 - 1195. [Abstract] [Full Text] [PDF]
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M. L. Ford and B. D. Evavold Regulation of Polyclonal T Cell Responses by an MHC Anchor-Substituted Variant of Myelin Oligodendrocyte Glycoprotein 35-55 J. Immunol., August 1, 2003; 171(3): 1247 - 1254. [Abstract] [Full Text] [PDF]
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L. Zaliauskiene, S. Kang, K. Sparks, K. R. Zinn, L. M. Schwiebert, C. T. Weaver, and J. F. Collawn Enhancement of MHC Class II-Restricted Responses by Receptor-Mediated Uptake of Peptide Antigens J. Immunol., September 1, 2002; 169(5): 2337 - 2345. [Abstract] [Full Text] [PDF]
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 J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky Signals delivered through TCR instruct IL-12 receptor (IL-12R) expression: IL-12 and tumor necrosis factor-{alpha} synergize for IL-12R expression at low antigen dose Int. Immunol., November 1, 2001; 13(11): 1433 - 1442. [Abstract] [Full Text] [PDF]
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J. A. Woodfolk and T. A. E. Platts-Mills Diversity of the Human Allergen-Specific T Cell Repertoire Associated with Distinct Skin Test Reactions: Delayed-Type Hypersensitivity-Associated Major Epitopes Induce Th1- and Th2-Dominated Responses J. Immunol., November 1, 2001; 167(9): 5412 - 5419. [Abstract] [Full Text] [PDF]
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 S. Tangri, G. Y. Ishioka, X. Huang, J. Sidney, S. Southwood, J. Fikes, and A. Sette Structural Features of Peptide Analogs of Human Histocompatibility Leukocyte Antigen Class I Epitopes that Are More Potent and Immunogenic than Wild-Type Peptide J. Exp. Med., September 17, 2001; 194(6): 833 - 846. [Abstract] [Full Text] [PDF]
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J. A. Gebe, E. J. Novak, W. W. Kwok, A. G. Farr, G. T. Nepom, and J. H. Buckner T Cell Selection and Differential Activation on Structurally Related HLA-DR4 Ligands J. Immunol., September 15, 2001; 167(6): 3250 - 3256. [Abstract] [Full Text] [PDF]
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S. K. Joshi, P. R. Suresh, and V. S. Chauhan Flexibility in MHC and TCR Recognition: Degenerate Specificity at the T Cell Level in the Recognition of Promiscuous Th Epitopes Exhibiting No Primary Sequence Homology J. Immunol., June 1, 2001; 166(11): 6693 - 6703. [Abstract] [Full Text] [PDF]
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S.-s. J. Sung, C. E. Rose Jr., and S. M. Fu Intratracheal Priming with Ovalbumin- and Ovalbumin 323-339 Peptide-Pulsed Dendritic Cells Induces Airway Hyperresponsiveness, Lung Eosinophilia, Goblet Cell Hyperplasia, and Inflammation J. Immunol., January 15, 2001; 166(2): 1261 - 1271. [Abstract] [Full Text] [PDF]
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J. F. Panus, L. J. McHeyzer-Williams, and M. G. McHeyzer-Williams Antigen-specific T Helper Cell Function: Differential Cytokine Expression in Primary and Memory Responses J. Exp. Med., November 6, 2000; 192(9): 1301 - 1316. [Abstract] [Full Text] [PDF]
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 L. H. Glimcher and K. M. Murphy Lineage commitment in the immune system: the T helper lymphocyte grows up Genes & Dev., July 15, 2000; 14(14): 1693 - 1711. [Full Text]
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P. R. Rogers and M. Croft CD28, Ox-40, LFA-1, and CD4 Modulation of Th1/Th2 Differentiation Is Directly Dependent on the Dose of Antigen J. Immunol., March 15, 2000; 164(6): 2955 - 2963. [Abstract] [Full Text] [PDF] |
1 x 105) were restimulated
with 1 µM of Hb(64-76) in the presence of APCs for 48 h. Amounts
of IL-4 and IFN-
) depict Hb(64–76)
stimulation.