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La Jolla Institute for Allergy and Immunology, Division of Immunochemistry, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2, or IL-4 and IFN- without
IL-2. Endogenous IFN- secreted over the initial 3 days of culture
was critical for generating Th0 cells, since neutralization allowed
IL-4 to induce differentiation into Th2-like cells. Successive
encounters with Ag were required for generating Th0 cells, and their
stability and persistence were governed by the balance of endogenous
IL-4 and IFN- secreted during the later stages of differentiation.
Studies blocking Fas-induced cell death showed that this process played
no role in Th0 cell generation, and differential death of committed Th1
or Th2 cells was not required for Th0 persistence. These data suggest
that Th0 cells can be as prevalent as Th1- or Th2-like cells after
naive CD4 activation, that the relative levels of autocrine IL-4 and
IFN- are important to the lack of commitment, and that not all cells
are predestined to the Th1 or Th2 phenotypes early in the response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and lymphotoxin
(LT)3-
, or Th2 cells
secreting IL-4, IL-5, IL-9, and IL-13, has received tremendous
attention. Although Th0 populations secreting combinations of Th1- and
Th2-type cytokines have been described, and Th0 clones have been
produced (5, 6, 7, 8), there has been a tendency recently to classify in vivo
or in vitro responses into Th1-like or Th2-like, purely based on the
predominance of one cytokine over another, rather than the absolute
presence or absence of particular cytokines. This has been particularly
surprising because, for many years, clones predominantly of the Th0
phenotype were produced from human subjects, and the existence of human
Th1 and Th2 cells was in doubt. In addition, the concept has been put
forward that naive CD4 cells become polarized into Th1 or Th2 cells
very shortly after activation and that, if they go through a Th0 stage,
it is only transitory (9, 10). Absolute commitment of these cells,
however, may not occur until many days or weeks later, if at all, as
shown by studies of reversal of Th1 and Th2 phenotypes (9, 11, 12, 13). The
techniques of intracellular cytokine staining and
immunohistochemical staining have also been used to promote the
idea that generally Th1 and Th2 subsets exist and that intermediate
subsets may be rare. Few studies using these methods have demonstrated
significant numbers of cells secreting cytokines of both types, with
isolated reports of what appear to be Th0 populations or clones,
suggesting that they may largely comprise individual Th1 and Th2 cells
(11, 13, 14, 15, 16, 17).
However, because many in vivo and in vitro studies of cytokine
secretion do not demonstrate absolute production of only Th1 or Th2
cytokines, it has recently been questioned as to how often polarized
subsets arise. Certainly, many intracellular and extracellular
infections have now been shown to consist of mixed responses, with
different cytokines apparently being required at different stages of
infection (18). Therefore, responses that may involve cells that could
be classified as Th0, but still produce varying levels of each
individual cytokine, are a distinct possibility. In the study here, we
have therefore utilized naive CD4 cells from TCR transgenic animals to
study whether Th0 cells can be generated reproducibly and how they are
regulated. We show that exogenous IL-4 in combination with endogenous
IFN- induces effector cells that make both Th1- and Th2-type
cytokines at high levels. These Th0 cells develop over 9 days in
culture and appear relatively stable during this time. Autocrine
production of IL-4 and IFN- is responsible in part for this
stability, in that effector populations can arise that secrete varying
levels of Th1 or Th2 cytokines if endogenous IL-4 or IFN- are
neutralized. However, the resultant effector cells still retain the Th0
phenotype, largely do not lose the ability to produce individual
cytokines, and do not become polarized Th1- or Th2-like cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AND mice, transgenic for the Vß3/V11 TCR, were bred on a
B10.BR background (H2k) as previously described
(19).
T cells and APC
Purified naive CD4+ T cells were isolated from the spleen of TCR transgenic mice (20) by passage over nylon columns, followed by complement depletion with Abs to CD8 (3.155), heat stable Ag (JIID), and class II MHC (M5/114 and CA-4.A12), cross-linked with mouse anti-rat k (MAR 18.5). Any residual APC and any in vivo-activated T cells were removed by isolating high density cells spun through a Percoll gradient (45, 53, 62, 80%) followed by adherence to plastic for 1 h. As previously described (19, 20), the resultant cells were resting (low FSc, IL-2R-) and >98% CD4+, and >95% of the CD4 cells possessed a naive phenotype (CD45RB+, CD62L+, CD44low).
APC were spleen cells depleted of T cells with Abs to CD4 (RL172.4), CD8 (3.155), and Thy1.2 (F7D5 and HO.13.4) and complement, and were treated with mitomycin (50 µg/ml, Sigma) for 30 min at 37°C before use.
Cell cultures
Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana,
CA) with penicillin, streptomycin, glutamine, 2-ME, sodium pyruvate,
and 7% FCS (HyClone Labs, Logan, UT; Sigma, St. Louis, MO). Effector
CD4 cells were generated from purified naive cells by stimulation at
2 x 105/ml over 3 to 9 days with a peptide of
pigeon cytochrome c (PCC 88–104 at 5 µM) presented by
1 x 106/ml T-depleted APC. Cultures were additionally
supplemented with recombinant IL-2 (10 ng/ml) during days 0 to 3 only
to maintain cell viability. Effectors of varying cytokine phenotypes
were generated by adding recombinant cytokines at the initiation of
culture. Th0-like effectors were routinely made with 10 ng/ml IL-4,
Th1-like effectors with 4 ng/ml IL-12 and 10 µg/ml anti-IL-4
(11B11, purified ascites), and Th2-like effectors with 20 ng/ml IL-4
and 10 µg/ml anti-IFN- (XMG1.2, purified ascites).
Several culture protocols were used depending on the experiment. In
general, a 9-day protocol was used in which effectors were generated
over 3 days with various cytokines, washed extensively, and then
recultured for an additional 3 days with PCC peptide and APC (identical
concentrations to the initial culture, i.e., 2 x
105/ml T cells, 1 x 106/ml APC, 5 µM
PCC) in the absence of added cytokines. This was repeated a further
time, so that cells at day 9 had been restimulated twice (day 3 and day
6). Other experiments involved generating effectors over 3 days only, 6
days, or 9 days without intermittent antigenic stimulation, or
stimulating for 6 days and restimulating for a further 3 days. In all
cases, the priming cytokines IL-2, IL-4, or IL-12 were added only on
day 0. Generally, cultures were set up in bulk 5-ml volumes in flasks
from day 0 to 3 and then in 1-ml volumes in 48-well plates (Costar,
Cambridge, MA) from day 3 to 9. At the end of the effector generation,
T cells were tested for cytokine secretion by restimulating again at
2 x 105/ml, with fivefold excess T-depleted APC and 5
µM PCC in 0.5- or 1-ml volumes, with supernatants collected after
24 h and intracellular cytokine staining performed at 5 h.
When modulation of Th0 effectors was performed, IL-4-primed cells were
taken at day 3, washed, and restimulated in the presence or absence of
either Abs to IL-4 (11B11), IFN- (XMG1.2), or Fas (PharMingen, San
Diego, CA) at 10 µg/ml, an Ig chimeric fusion protein of Fas (Fas.Fc,
5 µg/ml, kindly provided by Dr. Doug Green, La Jolla Institute for
Allergy and Immunology (LIAI), La Jolla, CA), or IL-2 (10 ng/ml)
combined with TGF-ß (1 ng/ml, R&D systems, Minneapolis, MN).
Cytokine secretion
IL-2 production was assessed as before (19) by titrating
supernatants onto NK.3 cells, in triplicate, in the presence of
anti-IL-4 (11B11). IL-4, IL-5, and IFN- were measured by ELISA
as in previous studies (19) using the Abs 11B11 and biotinylated
anti-IL4 (BVD6, PharMingen), TRFK5 and biotin-TRFK4, and R46A-2 and
biotin-XMG1.2, respectively. Standard curves were constructed with
purified IL-2, IL-4, IL-5, and IFN- (supernatants from the
respective X63.Ag. cell lines).
Intracellular cytokine staining
Cytokine staining was performed with protocols similar to those
described by Openshaw et al. (14). Identical cultures as described
above for cytokine determination were set up with 2 x
105/ml T cells, 1 x 106/ml APC, and
5 µM PCC peptide. Cells were incubated for 5 h at 37°C, with
Brefeldin A (10 µg/ml) added for the last 2 h. Immediately after
culture, cells were fixed with 2% paraformaldehyde for 20 min at room
temperature and then permeabilized with 0.5% saponin in PBS at 4°C
for 10 min. Staining was performed using standard procedures with
phycoerythrin-conjugated anti-IL-4, FITC-conjugated
anti-IFN- (both from Caltag Laboratories, South San Francisco,
CA), and phycoerythrin-conjugated anti-IL-2 (PharMingen) added at
5, 1, and 8 µg/ml, respectively, concentrations that produced maximal
staining. After 30 min, cells were washed free of saponin and stained
with Cychrome-conjugated anti-CD4 (PharMingen). Samples were
analyzed on a FACScan flow cytometer with Cellquest software (Becton
Dickinson, Mountain View, CA) after gating on CD4 positive cells.
Control Th1 and Th2 populations were set up for every experiment and
used to set quadrant gates for positive cytokine staining. In these
cases, cells were stained with only a single reagent, and negative
gates were set to correlate with this staining and with controls in
which only anti-CD4 was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Few studies have addressed how Th0-like populations may develop
and whether they consist of individual Th1- and Th2-like cells, or
cells making a range of cytokines of both subsets. We set up in vitro
cultures to address this and reasoned that either a moderate dose of
IL-4 or a mixture of IL-4 and IL-12 would result in T cell populations
that were not fully polarized either way. Naive CD4 cells were used
from AND TCR transgenic mice and stimulated with a peptide of PCC
presented on T-depleted APC. Recent data suggest that T cells may
become polarized to one pathway or the other after 2 to 3 days (10),
although commitment may not be evident for several weeks (11). Cultures
were thus set up over a 9-day period, rather than 3 to 4 days as we
have previously used (21), to assess stability over a period that is
reflective of the typical time course of a T cell response in vivo. T
cells were removed from culture, washed, and restimulated twice during
this 9-day culture (days 3 and 6), with priming cytokines added for the
first 3 days only, and not present between days 3 to 9. As shown in
Figure 1
, this procedure produces highly
differentiated effector populations that secrete massive levels of
cytokines.
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at high levels, with little or no detectable IL-4 or IL-5;
IL-12 priming (4 ng/ml) with a neutralizing Ab to IL-4 (10 µg/ml)
results in a population making huge amounts of IFN- with little
IL-2; and IL-4 priming (20 ng/ml) with a neutralizing Ab to block
endogenous IFN- (10 µg/ml) generates effectors making high levels
of IL-4 and IL-5 and lower levels of IFN- (Fig. 1
). In contrast,
priming with medium dose IL-4 alone (10 ng/ml) or a mixture of IL-4 and
IL-12 (4 ng/ml and 1 ng/ml, respectively) resulted in T cell
populations capable of producing high levels of both Th1 and Th2
cytokines. Since the Th0 population generated with IL-4 alone was
somewhat more consistently generated than that with IL-4 and IL-12,
further studies concentrated only on IL-4 priming.
To assess development of the Th0 population, we took cells after IL-4
priming at days 3, 6, and 9 and compared their cytokine profiles after
restimulation with Ag (Fig. 2). Generally
two patterns were seen, as shown in Examples 1 and
2, with Example 1 being prevalent during the
majority of experiments (70%). The production of cytokines in all
cases was much lower after 3 days than after 6 or 9 days, and generally
9-day effector populations secreted more of each cytokine on a per cell
basis than at earlier times. Because experiments were performed with
transgenic CD4 populations and under conditions of medium Ag (5 µM)
and high APC number, which allows each T cell to encounter an antigenic
stimulus (our unpublished observations), this effect was not due to
selection of Ag-specific cells or to the fact that different numbers of
T cells were stimulated. At day 3, T cell populations either produced
predominantly IL-4 and IL-5 with much lower, although easily
detectable, levels of IL-2 and IFN- (Fig. 2, upper
graph) or produced more equivalent levels of IL-4, IL-5,
and IFN- (Fig. 2, lower graph). In the latter
case, this pattern remained fairly constant through days 6 and 9,
whereas, in the former, roughly equivalent levels of IFN- became
evident by day 6, with this profile being stable through to day
9.
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To assess whether Th0 effectors consisted of Th0 cells or mixed
populations of cells secreting only Th1 or Th2 cytokines, we performed
intracellular cytokine staining. To confirm our ability to effectively
stain for cytokines, we analyzed polarized populations of Th1- and
Th2-like cells generated over 9 days with IL-12 and anti-IL-4, or
IL-4 and anti-IFN-, respectively (Fig. 3). As seen by other investigators, the
IL4 + anti-IFN--primed population contained mainly cells
secreting IL-4 with no detectable IFN-, and only a small percentage
(22%) costaining for IL-4 and low levels of IFN-. No cells
secreting IFN- alone were evident, and only a small number secreting
IL-2. We consistently failed to detect appreciable IL-5 staining, even
though these and other effector populations secreted high levels of
this cytokine. This was regardless of the time at which staining was
performed (up to 24 h). Not surprisingly, IL-12 +
anti-IL-4-primed populations consisted largely of cells secreting
IFN- (
74%), with a proportion of cells secreting IL-2 as well
(19%).
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contained a majority of cells that
costained strongly for IL-4 and IFN- together with IL-2 (68% in
the example in Figure 4A). Some cells also
appeared to produce only IL-4 or IL-2, and some cells produced IL-4 and
IFN- but without IL-2 (11%). This pattern of staining was
observed in three separate experiments. Generally, in the Th0
populations, we observed that the majority of IFN- was produced from
cells also making IL-4, whereas a significant number of cells did not
appear to additionally produce IL-2. Thus, IL-2 may not be intimately
associated with IFN-, and a Th0 cell may not necessarily make all
Th1 and Th2 cytokines. At day 3, intracytoplasmic staining paralleled
protein secretion (Fig. 2, Example 1) with the
majority of T cells staining strongly for IL-4 but weakly if at all for
IFN- and IL-2 (63%, Fig. 4A). Only a small
percentage (6%) were producing sufficient levels of IL-4, IFN-,
and IL-2 to be described as Th0-like. After 6 days, the percentage of
cells staining strongly for IL-4, IFN-, and IL-2 had increased to
33% in the example shown, with 21% apparently producing IL-4
and IFN- but not IL-2. Analyses of the intensity of cytokine
staining at each time point (Fig. 4B) suggested that
at least a proportion of cells in culture were differentiating such
that the amount of cytokine produced per cell increased with each
restimulation event.
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in generation and stability
of Th0 populations
Our previous studies have shown a role for autocrine IL-4 and
IFN- in regulating naive CD4 differentiation (20, 21). During the
initial 3 days of culture in the experiments here, it was also obvious
that endogenous IFN- played a role in the generation of Th0 cells
after IL-4 priming, since blocking IFN- was required to generate a
Th2-like population (Figs. 1 and 3). We should stress that we found no
role for IL-12 in this culture system, even though an APC population
was used that contained some dendritic cells and macrophages. Thus,
anti-IL-12 added at any time during the 9-day period had no
significant effect on the responses observed (data not shown). As
opposed to the initial days of naive T cell differentiation, we wished
to determine whether IL-4 and IFN- were active later in the response
and whether the balance between them was responsible for the
persistence of the Th0 phenotype.
Cultures were set up as before with IL-4 used during days 0 to 3 to
generate a Th0 population. T cells were washed and restimulated at days
3 and 6 but with anti-IL-4 or anti-IFN- present throughout
the later 6 day period. After 9 days, the T cells were again washed and
assayed for cytokine secretion by protein assay and intracellular
cytokine staining (Fig. 5 and Table I). Anti-IL-4 suppressed the generation
of an effector population secreting high levels of IL-4 (average 78%
inhibition, Fig. 5A and Table I), with concomitant increases
in IL-2 (mean 63% enhancement) and particularly IFN- (mean 395%).
Interestingly, effectors secreting IL-5 were suppressed only slightly
(20% inhibition), suggesting that, after 3 days of differentiation,
IL-5 production was no longer intimately associated with IL-4, as may
have been predicted. By intracellular staining (Fig. 5B), the population of cells generated in the
presence of anti-IL-4 was still largely Th0-like, with 70%
producing IL-4, IFN-, and IL-2, but with the intensity of staining
for IL-4 being lower and that for IFN- and IL-2 being higher in the
population overall, although a large range of intensities were seen in
individual cells. A small number of cells secreting IFN- in the
absence of IL-4 were seen (8%), and the number secreting IL-4 alone
decreased from 27% to 15%.
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strongly suppressed the production of
IFN- and IL-2 (mean inhibition of 82% and 65%, respectively, Fig. 5A and Table I) by the resultant effector population, while
concomitantly enhancing IL-5 production (average 70% increase) but not
affecting IL-4 production. Interestingly, again the number of Th0-like
cells secreting IL-4, IFN-, and IL-2 did not change appreciably
(67%). The intensity of staining for IFN-, and to a lesser
extent for IL-2, in the overall T cell population was lower after
anti-IFN- culture (Fig. 5B), although the
population was obviously heterogeneous in the levels of cytokines
produced by each cell.
Cell numbers over the 6-day treatment period were similar regardless of
anti-IL-4 or anti-IFN- treatment (Fig. 5C), suggesting that the effects seen were not due to
differential T cell growth rates. Significantly, cell expansion was
evident between 3 and 6 days, whereas, after reculture and
restimulation at day 6, there was little change in cell numbers. This
suggests that either the cells were turning over slowly, or,
alternatively, that there was a great deal of cell death in the
tertiary cultures, although we did not observe the latter by trypan
blue exclusion.
The role of cell death in generation and stability of Th0 populations
Several recent studies have suggested that Th1- and Th2-like cells (either clones or partially differentiated effector cells) may have different susceptibilities to Fas-induced death (22, 23), potentially promoting this as a mechanism for regulating cytokine phenotypes. Two methods for preventing Fas-induced death have been described, either directly blocking Fas/FasL interactions, or the use of a combination of IL-2 and TGF-ß (23, 24). We therefore determined whether cell death may have been involved in the generation or stability of Th0 cells using these methods.
Th0 populations were again induced with IL-4 over 3 days, and the
resultant cells recultured for an additional 6 days, with a second
restimulation after 3 days, in the presence or absence of either Fas.Fc
or IL-2 and TGF-ß. After 9 days, the T cell populations were
restimulated and analyzed for cytokine production. Reagents were used
at concentrations found to prevent Fas-induced death in other systems
(Refs. 24 and 25, and D. R. Green, unpublished observations).
Results similar to those with Fas.Fc were obtained with anti-Fas
and anti-FcR, which can also block apoptosis (data not shown).
Reagents blocking Fas/FasL interactions had minimal effect on
generation of the Th0 effector populations overall, with similar levels
of all cytokines secreted and similar staining patterns seen
intracellularly (Fig. 6, A and
B, and Table I). In contrast, IL-2 and TGF-ß strongly
skewed the T cell populations toward secreting IFN- (mean increase
of 485%, Table I), although secretion of IL-2, IL-4, and IL-5 were
largely unaffected (Fig. 6, A and B, and Table I). By intracellular staining, the only notable change observed was a
low percentage of T cells (estimated at <10%) that appeared to be
producing very high levels of IFN- in combination with IL-4 and IL-2
(Fig. 6B). The majority of cells in these experiments
were therefore still Th0 cells, although, as seen above, within this
population there was a continuum of cells secreting low through high
levels of IL-4, IFN-, and IL-2.
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secretion
within 24 h of addition on days 3 and 6 (data not shown), we favor
the notion that the overall effects of IL-2/TGF-ß on effector
differentiation were due to a direct effect of TGF-ß enhancing
IFN- production, as has been described by other investigators (26, 27), rather than any action preventing cell death or modulating cell
selection. This is substantiated by the fact that blocking IFN-
along with IL-2/TGF-ß treatment prevented the generation of effectors
secreting large quantities of this cytokine (average 70% inhibition,
Table I). Similarly anti-IL-4 still largely prevented induction of
effectors secreting IL-4 (78% inhibition) while enhancing the
generation of cells secreting IFN- (1300% increase). Similar
effects of anti-IL-4 and anti-IFN- were observed with Fas.Fc
or anti-Fas, although some anomalies were noted with IL-5 secretion
(Table I). The role of multiple encounters with Ag in Th0 development
Finally, we assessed whether several early or late stimulation
events were required for maintaining the Th0 phenotype (Fig. 7). T cells were primed with IL-4 as in
previous experiments at day 0 and differentiated over 6 or 9 days with
either no restimulations (6 days, 9 days, Fig. 7) or after reculturing
with Ag/APCs on day 3 (3 + 3 days) or day 6 (6 + 3 days), and
cytokine secretion from the resultant populations was compared with
that produced from the standard Th0 populations (3 + 3 + 3
days).
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phenotype (Fig. 7A), and a large percentage (41% in this example)
expressed high levels of both IL-4 and IFN- intracellularly (Fig. 7B). In contrast, without restimulation at day 3 (6
days), much less IFN- was secreted, and a lower percentage of cells
displayed the Th0 phenotype and with overall lower intensity staining
(32%). With multiple stimulations over 9 days (3 + 3 + 3),
nearly equivalent levels of IL-4 and IFN- were produced (average
ratio IL-4:IFN- of 0.6:1 from two experiments), and 57% of the T
cells displayed a strong Th0 phenotype by staining. A 9-day
differentiation, without serial encounters with Ag, produced a T cell
population much more biased toward IL-4, in that lower levels of
IFN- were produced (average ratio IL-4:IFN- 4.2:1), and
significantly fewer cells stained for both IL-4 and IFN- (24%).
Generally, the intensity of expression of intracellular IFN- in this
population was much lower than in the Th0 cells produced after multiple
stimulations (Fig. 7B). Encounter with Ag on day 6
(6 + 3 days) partially restored the Th0 phenotype, in that less
IL-4 and slightly more IFN- was produced (average ratio IL-4:IFN-
3.2:1), and a higher percentage of cells stained strongly for both IL-4
and IFN- (35% compared with 24%). However, this did not result in
a Th0 phenotype as good as effector T cells seeing Ag on day 3 (compare
to 3 + 3 days or 3 + 3 + 3 days). Therefore, multiple
encounters with Ag favored the generation and maintenance of the Th0
population, although they were not absolutely required. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-2, and probably also secreting IL-5, can readily be
generated from naive CD4 cells when exposed to a mixture of IL-4 and
IFN- (exogenous or endogenous) during the initial few days of
activation. In addition, the magnitude of Th1-like and Th2-like
cytokines produced from these cells is regulated by endogenous IL-4 and
IFN- secreted after Ag encounter, resulting in Th0 cells that differ
from one another in the ability to secrete varying levels of any of the
individual cytokines. These data question recent findings that have
suggested that Th0 cells are only rarely generated and may be
transitory in nature and suggest that the Th0 phenotype can represent
that of a fully differentiated subpopulation of cells.
Many studies have delineated that the cytokines IL-4, IFN-, and
IL-12 are primarily responsible for modulating CD4 differentiation to
distinct cytokine-secreting effector populations, with additional
cytokines such as TGF-ß, IL-10, and IFN--inducing factor (IGIF)
also influencing the final effector phenotype (28, 29, 30). Initial studies
speculated that, in the absence of these cytokines (derived from
exogenous sources such as mast cells, NK1.1 CD4 cells, conventional NK
cells, and macrophages), naive T cells would not differentiate beyond
the stage of synthesizing IL-2. However, several years ago we showed
that naive cells quickly gained the ability to secrete multiple
cytokines such as IL-4 and IFN- within 24 h of activation,
after initially synthesizing IL-2, and that this was independent of
exposure to sources of exogenous cytokines (20, 21). This was also seen
in other studies (31, 32) and has been referred to as a Th0 stage by
some investigators. We, in contrast, prefer the term multipotential
stage, since, this early in naive T cell differentiation, the levels of
IL-2 far exceed those of IL-4 and IFN-. Unlike this transition,
which is largely accepted now, the existence of what may be referred to
as conventional Th0 cells, which secrete high levels of both Th1- and
Th2-type cytokines, has been questioned recently. Several studies,
assessing cytokine production intracellularly in single cells, largely
found segregation into Th1 and Th2 phenotypes under the conditions
analyzed and only rarely (usually <10% of most T cell
populations) found cells that could be considered as Th0-like (11, 13, 14, 15, 16). Our studies here, however, show that it is relatively easy to
generate a population of effector cells with a Th0 cytokine profile and
that the population largely comprises cells that are Th0, in that high
levels of IL-4 and IFN- are produced simultaneously. The majority of
these cells also produce IL-2, and we suspect that they are also
synthesizing IL-5.
The number of cells secreting multiple cytokines was most pronounced
after 9 days in culture, and they appeared to be only a minority after
3 days, based on intracellular staining (Fig. 4). However, by protein
assay, we did generally see a mixed phenotype at the early stage,
although the levels of IL-2 and IFN- were often very low (Fig. 2).
Thus, it could be argued that the effector population was comprised
largely of Th0 cells even by day 3 and that we were unable to visualize
the cells as being Th0-like because of the insensitivity of staining
for IL-2 and IFN-. By day 6 and day 9, in contrast, the cells were
synthesizing sufficient levels of the latter cytokines to be readily
detectable by intracellular staining along with IL-4. The alternative
explanation, suggested by the staining results, is that Th0 cells are
generated only after a prolonged differentiation period and that this
involves passing through a stage where largely Th2 cytokines are made,
but without commitment to this pathway. This is somewhat reminiscent of
studies from Sornasse et al. (13) and Hu-Li et al. (33), who assessed
plasticity of Th1 and Th2 effector populations. In both cases, a
significant number of Th0-like cells were induced from 1- to 3-wk-old
IL-4-primed Th2 populations after exposure to IL-12. The fact that in
our experiments anti-IFN- had to be added with IL-4 to generate
significant numbers of Th2 cells (Figs. 1 and 3) suggests that
generation of Th0 cells is critically governed by the balance of IL-4
and IFN- available during the initial stages of T cell activation.
Lack of long-term commitment to a Th2 pathway therefore appears to
involve simultaneous exposure to sufficient levels of IFN-
(endogenous or exogenous), or IL-12, to counteract the effects of
IL-4.
Why were we able to generate a population consisting largely of Th0
cells while the majority of studies have not seen such a population?
There are several probable reasons. First, most reports that have
utilized intracellular cytokine staining used culture conditions that
have become known over the past few years as producing polarized Th1
and Th2 populations (e.g., high dose IL-4 or IL-12 with
anti-IFN- or anti-IL-4). In contrast, we purposely selected
conditions (moderate dose of IL-4 alone, or moderate doses of IL-4 with
IL-12) that would not lead to polarization and potential commitment to
one pathway or another. Thus, as discussed above, Th0 cells most likely
result when there is an equal balance between the three main polarizing
cytokines, IL-4, IFN-, and IL-12. Second, we used fairly neutral
conditions of Ag stimulation with moderate dose PCC (5 µM) and a
moderately stimulatory APC population (T-depleted spleen cells), in
contrast to using high or low dose Ag, or highly costimulatory APC,
which have been shown on occasion to skew responses to the Th1 or Th2
phenotypes. The APC population also probably did not contribute any
polarizing cytokines, since it was not previously activated, and we
confirmed this for IL-12 when a neutralizing Ab was seen to have no
effect on any responses elicited. Thus, any IL-12, IL-4, or IFN-
present in the cultures was most likely derived from the responding T
cells or our exogenous sources. Third, we used a protocol involving
serial stimulation events every 3 days for 9 days. As shown in Figure 7, without these repeated encounters with Ag, priming with moderate
dose IL-4 produced fewer Th0-like cells, and those that were induced
secreted lower levels of IFN-, compared with those elicited with
repetitive stimulation. This protocol contrasts sharply with other
published reports, in that most used long culture periods of 6 to 9
days before exposing T cells to stimuli again, implying that
polarization can take place even with suboptimal conditions, but only
over an extended period of time.
The blocking studies with anti-IL-4 and anti-IFN-
demonstrated that, even when a T cell population has already
differentiated over a period of time (3 days, Table I, and 6 days, not
shown), the balance of autocrine IL-4 vs IFN- produced still
dictates to a large extent the ultimate cytokine profile of the T cells
that differentiate further. The number of Th0-like cells in the
resultant populations generated after Ab blocking was essentially the
same, with the primary difference being a reduction in the number of
cells that stained strongly for the respective cytokines. We assume
that the intensity of staining is reasonably proportional to the
quantity of cytokines secreted per cell, although we cannot
definitively conclude this. These data therefore suggest that a Th0
cell can not only secrete multiple cytokines but can be regulated such
that it will secrete more or less of a particular cytokine. This was
most readily demonstrated in Figure 5 with Th0 populations derived
after 9 days in the presence of anti-IL-4. In this case, Th0 cells
were present that made high levels of IL-4 with high levels of IFN-,
high levels of IL-4 with low levels of IFN-, low levels of IL-4 with
high levels of IFN-, etc. It has been demonstrated in several
disease states that the relative level of IL-4 compared with IFN-
can have profound consequences regarding the overall direction of the
immune response; e.g., high levels of IFN- compared with IL-4 are
protective against several protozoans such as Leishmania and
Trypanosoma, whereas high levels of IL-4 compared with
IFN- are protective against some helminth infections such as
Trichuris and Heligmosomoides (34). The
results presented here therefore suggest that Th0 cells could be
capable of eliciting some of these extremely varied immune reactions
and that polarization to the Th1 and Th2 phenotypes may not have to
take place to alter the nature of the response.
A possibility existed that Th0 cells would be derived because of selection of a minor population of cells over time, or differential death of Th1 or Th2-like cells. However, analyses of cell recoveries suggested that efficient cell expansion occurred during the initial 6 days of culture, and then the T cells differentiated to a stage where they turned over very slowly. Blocking Fas/FasL interactions had no significant effect on generation of the Th0 population, suggesting that selection of a minor subset of cells over time was very unlikely to have been involved in the outgrowth of the Th0 cells.
In summary, we have demonstrated that Th0 cells secreting both Th1- and
Th2-type cytokines can easily be generated from naive CD4 cells and
that they arise under nonpolarizing conditions that are determined by
the balance of IL-4 and IFN- to which the responding cells are
exposed. Th0 cells represent a diverse population of cells with the
capabilities of secreting extremely different levels of any one of the
Th1- and Th2-type cytokines and thus could potentially represent
important mediators of many varied immune reactions. Finally, the
cultures used to induce the generation of Th0 cells provide a model
system in which the effects of exposure to varying levels of Ag and
various APC populations can be studied, and it is highly likely that
the Th0 cell represents a target for strategies aimed at immune
deviation.
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Acknowledgments |
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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: LT, lymphotoxin; PCC, pigeon cytochrome c.
Received for publication November 12, 1997. Accepted for publication January 30, 1998.
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