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Department of Biology and University of California, San Diego Cancer Center, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) developed, except at very high doses of Ag, where there was a
striking reduction in IL-4 and IL-5 secretion. Our results imply that
Ag dose does not have a direct effect on Th1/Th2 polarization, except
under conditions that include a high level of TCR ligation and in the
presence of high levels of IL-2, where production of Th2 cytokines may
be down-regulated by a mechanism that is not yet
clear. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNFß, is
required for clearance or control of many viral and protozoan
infections, but the inflammatory reactions caused by these cytokines
may mediate tissue destruction in autoimmune diseases such as
rheumatoid arthritis (2) and multiple sclerosis (3). In contrast, the
Th2-associated cytokines, IL-4 and IL-5, may be required for protection
against helminth infections such as Nippostrongulus and
Trichuris (4, 5), but may exacerbate or mediate allergic
reactions. The roles of Th1 and Th2 responses in disease susceptibility
have been studied extensively in parasite and in leishmanial models (6, 7). In the leishmanial model, resistant mice developed a Th1 response
(mediated by IFN-), whereas susceptible mice developed an IL-4
dominated or Th2 response (6, 8). Importantly, susceptibility was
reversed if IL-4 was blocked during the initial response (9). The
generation of polarized Th1 and Th2 subsets is critical in determining
the balance of cell-mediated and humoral immunoresponses and may
determine the outcome of autoimmune reactions and infections by
pathogens.
Understanding the factors that promote preferential polarization of
naive T cells and differentiation into effectors that have the capacity
to be highly polarized (10) is important for understanding how the
appropriate class(es) of immune response can be elicited. It is
particularly clear that cytokines IL-4 and IFN-/IL-12 play critical
roles in achieving Th2 and Th1 polarization, respectively, but their in
situ source and which signals differentially regulate their
production are less clear. One clear-cut murine model indicated that
the bacterium Listeria monocytogenes, can interact
with host cells such as macrophages, resulting in production of high
levels of IL-12, which in turn drive a Th1-polarized response
(11, 12).
There have been a number of reports suggesting that the dose of Ag can determine the polarization of a CD4 response either in vivo (8, 13, 14) or in vitro (15, 16, 17, 18, 19). Low doses of Ag in vivo were reported to favor development of delayed-type hypersensitivity, presumably driven by Th1 polarization, while high doses favored Ab driven by Th2 polarization (8, 13). However, the strain of rat (13) or mouse (12, 19, 20) also had a major impact on polarization and could reverse the Ag dosage effect.
It has been suggested that the naive T cell can respond differentially to different extents of receptor ligation (15, 21), a concept fortified by the differential effects of distinct peptide analogues providing different avidity interactions on polarization (reviewed in 21 . The consensus paradigm that has emerged from the in vitro studies is that very low doses of priming Ag promote Th2 responses (15, 18), that intermediate to high doses promote Th1 responses (15, 18), and that very high doses promote Th2 polarization (18). However, several aspects of the in vitro studies reported argue against a direct effect. First, in one study, the dosage effect relied on the addition of exogenous IL-2 (15), while in the other it was shown that Th2 polarization required initial production of IL-4 during effector generation (18). Naive CD4 T cells do not produce detectable IL-4 when stimulated, unless they are stimulated at least twice over a period of several days (22, 23). These phenomena are compatible with an indirect effect mediated by cytokine production by cells that are Ag-experienced and/or respond directly to IL-2, as had been implied in some earlier studies (24, 25). Naive T cells require high Ag doses, whereas Ag-experienced effectors can be induced to secrete cytokines at much lower levels of TCR ligation (26) or peptide (10), so non-naive cells would contribute most at a low Ag dose. Finally, it is almost impossible to eliminate contaminating, non-naive T cells even in most TCR transgenic mice, because they are not of a reliably distinct phenotype (reviewed in 27 .
Therefore, we reexamined the mechanisms responsible for dose-dependent
selective polarization using a well-defined fibroblast APC population
and a TCR transgenic model (with a Vß3, V
ll receptor specific for
pigeon cytochrome c peptide 88–104
(PCCF)5 in which we could
substantially eliminate contamination of non-naive T cells by using
mice crossed to a RAG-2-/- background. We find that under
conditions in which contaminating T cells are minimized because no
endogenous receptor expression is present (RAG-2-/-) or
likely to grow (absence of exogenous IL-2), a Th population of cells
able to secrete high levels of IL-2 with only a low level of IL-4 or
IFN- is generated over a broad range of peptide doses. The
addition of exogenous IL-2 to effectors generated over a broad range of
peptide doses induced the generation of a Th0 population of cells able
to secrete both IL-4 and IFN- at substantial levels. Interestingly,
at very high doses of peptide, effectors developed that no longer
produced substantial levels of IL-4 and IL-5, suggesting a
down-regulation by some mechanism. From these studies, we suggest that
indirect mechanisms are largely responsible for the previously observed
effects of Ag dose in vitro and that such mechanisms may also
contribute to the strain differences in polarization and to in vivo
situations in which polarization is seen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H-2b/k and H-2k V11/Vß3 AND TCR
transgenic mice were bred in the animal facilities at the University of
California, San Diego (UCSD) or at the Trudeau Institute
(Saranac Lake, NY) and were used at 2 to 6 mo of age. Mice were
obtained on a C57BL/6 x 129 background (28) and were back-crossed
6 to 9 times to C57BL/6. Transgenic males (H-2b) were bred
to B10.BR females to produce transgenic H-2b/k offspring.
Transgenic H-2b/k mice were then bred four or more times to
B10.BR mice to obtain transgenic H-2k offspring. B10.BR and
(B10.BR x C57BL/6)F1 mice were bred in our facility
at UCSD. RAG-2-/- mice back-crossed on a C57BL/6
background (10 generations) were crossed to AND TCR transgenic mice
(ninth generation on C57BL/6 background). Offspring
(RAG-2+/- x TCR+/-) were mated, and TCR
transgenic RAG-2-/- (RAG/AND) mice were used in
experiments.
CD4+ T cell isolation
Spleen and lymph node cells from TCR transgenic mice were
isolated over nylon wool columns, treated with a panel of depleting Abs
and complement, and purified over Percoll gradients (Sigma, St. Louis,
MO) to obtain small resting CD4+ T cells (29, 30). Spleen
and lymph node cells were depleted with 3.155 (anti-CD8), CA4.2.12
and M5/114.15.2 (anti-class II), J11d (anti-HSA), 33D1
(anti-CD11c), and M1/70 (anti-CD11b, Mac-1). Rat Abs were
cross-linked with MAR18.5 (mouse anti-rat 
) and then incubated
with guinea pig complement, baby rabbit complement, and DNase I
(Sigma). High density resting CD4+ T cells were isolated
using discontinuous Percoll gradient centrifugation (4 layers: 40, 52,
63, and 80%). Cells at the 63/80% interface were collected and used
for T cell assays and for effector generation. Cells were routinely 90
to 99% CD4+Vß3+ and 85 to 95%
L-selectin+. Naive CD4 T cells were isolated, as described
(31), by magnetic separation (MACS; Miltenyi Biotech, Sunnyvale, CA)
based on L-selectin (CD62L) expression. T cells were sequentially
labeled with biotinylated rat anti-mouse L-selectin (MEL-14)
followed by FITC-streptavidin (Zymed, San Francisco, CA) and
biotinylated magnetic beads (Miltenyi Biotech). CD4+ T
cells, positively selected on magnetic beads, were at least 99%
L-selectin+. CD4+ T cells isolated from
transgenics on the RAG-2-/- background displayed a naive
phenotype (see Fig. 1
) and were not sorted with L-selectin. Inclusion
of anti-NK1.1 (PK136) in the depletion step or the use of
T-depleted spleen APCs did not alter the differentiation (IL-4 or
IFN- secretion) of T cells upon restimulation (data not shown).
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Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana,
CA) supplemented with penicillin, streptomycin, glutamine, 2-ME, HEPES,
and 7.5% FCS (HyClone Labs, Logan, UT). Cultures were set up in 2-ml
volumes in 24-well plates (Costar, Cambridge, MA). CD4+ T
cells (3 x 105/ml) were stimulated in the presence of
mitomycin-C-treated APC (1 x 105 DCEK.ICAM APC/ml
(32) (B7-1+, ICAM-1+,
I-Ek-expressing mouse fibroblast line) and various doses of
PCCF (0.001–100 µM) or with no PCCF, with or without exogenous IL-2
(10 ng/ml). In some experiments, APCs were pulsed with peptide for
2 h before incubation with T cells. Pulsing APCs with peptide is
10-fold less efficient in activating T cells compared with soluble
Ag, but reduces the ability of the few possible remaining APCs in the T
cell preparation from presenting Ag. We observed no significant
differences in Th1 or Th2 generation in pulsed vs soluble peptide
conditions. After 4 days, cells were harvested, counted, and washed
three times. Cell recovery was determined by trypan blue exclusion. For
restimulation, T cells were incubated at 3 x 105/ml
with DCEK.ICAM APCs (1.5 x 105/ml) and 5 to 10 µM
PCCF. Supernatant was collected after 24 and 48 h for cytokine
analysis.
Cytokine assays
Recombinant cytokines IL-2, IL-4, IFN-, and IL-5 were
obtained from culture supernatants of X63.Ag8-653 cells transfected
with murine cDNA for the respective cytokines (33). Recombinant murine
IL-12 was kindly provided by Dr. Stanley Wolf (Genetics Institute,
Cambridge, MA). The following anti-cytokine Abs were purified from
ascites or were prepared by Amicon (W. R. Grace, Beverly,
MA) concentration of hybridoma supernatants: 11B11 (anti-IL-4),
XMG1.2 and R46A2 (anti-IFN-), and TRFK4 and TRFK5
(anti-IL-5). IL-4, IFN-, and IL-5 were detected by ELISA using
11B11, R46A2, and TRFK5, respectively, as coating Abs, and biotinylated
rat anti-mouse IL-4 (PharMingen, San Diego, CA),
biotinylated-XMG1.2, and biotinylated-TRFK4, respectively, as
second-step reagents. The data were quantitated from standard curves
using recombinant cytokines and were expressed in ng/ml. One nanogram
per milliliter of IFN- equals 9 U/ml protein, and 1 ng/ml of IL-5
equals 8.5 U/ml protein. For effector T cells, IL-2 and IL-4 were
assayed from supernatants collected at 24 h, and IL-5 and IFN-
were assayed from supernatants collected at 48 h. IL-2 was
detected by bioassay as previously described (31, 34) by measuring
proliferation of the NK cell line in the presence of 11B11
(anti-IL-4). The data are expressed in ng/ml and are referenced to
murine rIL-2 (PharMingen). One nanogram per milliliter of IL-2 is equal
to 11 U/ml protein.
Antibodies
Anti-L-selectin (MEL-14), anti-CD44 (IM7), and mouse
anti-rat (RG7) were used as concentrated supernatants.
Phycoerythrin (PE)-labeled and FITC-labeled anti-CD4, PE-labeled
anti-Vß3 (KJ25), and isotype controls were purchased from
PharMingen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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11 TCR,
specific for the 88–104 fragment of PCCF, complexed to
I-Ek, is particularly good at excluding endogenous receptor
expression (28); among the CD4 T cell population of young TCR
transgenic mice, >90% of cells express the transgene and have a naive
phenotype (CD44lowCD62Lhigh) (35, 36). However,
there is some contamination (5–15%) with CD4 cells expressing low
levels of the TCR transgene, and many of these cells are not of naive
phenotype. As shown in Figure
1,
top, 14% of AND CD4 T cells were Vß3-
(nontransgenic) with 4% nontransgenic cells remaining after
purification (middle). In contrast, almost no nontransgenic
CD4 cells were detected in RAG/AND mice, either before (Fig. 1, bottom left) or after (bottom middle)
purification. Mice on the RAG/AND background also had few if any CD4 T
cells with a memory phenotype (CD44high) (Fig. 1, bottom right). As shown in Figure 1, 5.4% of CD4 cells in
AND mice were CD44high whereas <1% of CD4 cells were
CD44high in RAG/AND mice. Other studies have relied on
separation of naive cells with cell surface markers (CD45RB and CD62L);
however, some activated or memory cells have been shown to reexpress
the putative naive markers, so phenotype is not a completely reliable
indicator for distinguishing or separating naive from
Ag-experienced CD4 T cells (reviewed in 27 .
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99% TCRhigh (see Fig. 1). This is in contrast
to many other TCR transgenic animals in which the fraction of naive
phenotype cells is less and the TCRhigh CD4 cells may get
stimulated by Ag in the environment, leading to potential contamination
with non-naive CD4 T cells.
Two recent reports (15, 18) have shown that the dose of Ag added in
vitro to stimulate naive T cells played a role in development of Th1
and Th2 subsets. Both studies showed selective Th2 development (IL-4
secretion) at very low doses of Ag (
0.01 µM) and Th1 development
(IFN- secretion) at intermediate doses of Ag (0.1–10 µM). Th2
development was again seen at high doses of Ag (>10 µM) in one of
the studies (18). It seemed possible that the reported effects of Ag
were not mediated directly by Ag dose, but were mediated indirectly
through cytokines produced by contaminating T cells or by APC
populations, especially since exogenous IL-2 was used in one study
(15). Therefore, we examined whether Ag dose had a direct effect on
naive T cells in a transgenic model in which we could address the
effects of exogenous IL-2 and contaminating non-naive (effector and
memory) cells.
Effect of exogenous IL-2 on dose-dependent polarization (AND naive T cells)
We reasoned that addition of exogenous IL-2, as is routinely
practiced in generation of effectors, would lead to potential
Ag-independent outgrowth of contaminating non-naive CD4 T cells.
Therefore, we compared the effect of IL-2 on generation of effectors
from highly purified CD4 T cells derived from AND TCR transgenic mice.
The cell populations obtained were 90 to 98% CD4+ and
expressed high levels of Vß3 (see Figure 1). For APC, we used a
fibroblast cell line that was transfected with I-Ek and
ICAM-1 and expresses B7-1 at high levels (30). This APC, called
DCEK.ICAM, pulsed with optimum doses of PCCF is as efficient at
stimulating naive AND CD4 T cells as the combination of
plate-bound anti-CD3 and anti-CD28 (30) and is also
highly efficient at stimulating effector and memory CD4 T cells
(26, 37). The DCEK.ICAM line does not produce known polarizing
cytokines such as IL-4, IFN-, or IL-12 (Ref. 38; and X. Zhang, L.
Tsui, and S. L. Swain, unpublished data). This cell line
also does not synthesize IL-6 (detected by RNase protection, P.
Rogers and S. L. Swain, unpublished data). Moreover, we found that the
addition of exogenous IL-6 (up to 10 ng/ml) had only a marginal effect
on Th2 development (data not shown), increasing IL-4 secretion twofold
with low dose Ag in the presence of exogenous IL-2. With higher doses
of priming Ag, IL-6 had no effect on IL-4 secretion. As previously
published (39), IL-6 did enhance T cell proliferation and long term
cell survival, especially with high doses of priming Ag.
A wide range of doses of PCCF (0.001–100 µM plus no Ag) were
added to cultures of purified AND CD4 T cells and DCEK.ICAM
APCs. Parallel sets of cultures were set up with or without
exogenous IL-2. After 4 days, cultures were harvested and viable CD4
cells were determined by trypan blue exclusion. Samples were readjusted
to 3 x 105 T cells/ml for restimulation with a high
dose of peptide (10 µM) plus DCEK.ICAM APCs. Supernatants were
harvested after 24 or 48 h, and titers of IL-2, IL-4, and IFN-
were determined. Figures 2 and
3 illustrate a set of results
representative of those obtained in four such experiments. Results
without IL-2 are also shown on a reduced scale (Fig. 3, top
inset) so that the trends are obvious. Effector recovery (Fig. 2)
and IL-2 secretion (Fig. 3) upon restimulation (at day 4) increased
markedly with Ag dose (Fig. 2), but very few effectors were generated
at doses below 0.01 µM PCCF. When effectors were generated in the
presence of exogenous IL-2 (given on day 1), T cell recovery was
dramatically increased.
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). When the recovered effectors were
resuspended to a standard cell number and restimulated, the IL-2
secretion of effectors increased with dose in the absence of IL-2 (Fig. 3, top, inset). Little IL-4 or IFN- was produced by
effectors generated without exogenous IL-2 regardless of the priming Ag
dose (Fig. 3, middle and bottom), except for a
little IFN- at high peptide dose. This pattern of cytokine
production is rather like that of naive T cells (IL-2 without Th1 or
Th2 characteristic cytokines). Surprisingly, when exogenous IL-2 was
added, effector cytokine production increased greatly overall (up to
30-fold), and cytokines other than IL-2 were produced in substantial
amounts. IFN- was made in greater amounts than IL-4. With IL-2 added
to the priming culture, Ag doses below 0.001 µM led to effector
populations that when restimulated, secreted high levels of IFN-,
and significant levels of IL-4 (a Th0 pattern). IL-4 production
decreased with increased Ag dose, whereas IFN- remained high,
leading to a shift from a Th0 to Th1 pattern as the dose increased.
High levels of IL-2 and detectable IL-4 and IFN- were produced even
when no Ag was present. The fact that the Th0 pattern was also seen in
IL-2-supplemented cultures when no PCCF was added suggested that under
the conditions of low Ag dose, the few effectors present may not have
been derived from resting naive CD4 T cells, but from IL-2-responsive
cells. When exogenous IL-2 was present, there was also a gradual
decrease in the ability of effectors to secrete IL-2 with increasing Ag
doses.
In the absence of IL-2, the peak of IL-4 at lower doses of Ag was
always missing (four of four experiments), and IFN- production was
negligible (also four of four experiments), except at high doses of
PCCF. It is notable that even when IL-4 was produced at low doses of Ag
with exogenous IL-2, IFN- was also produced, so that the only
"polarized" pattern was a predominant Th1 pattern seen at high dose
with or without IL-2. This is consistent with many earlier reports,
which found a Th1 default polarization in mice of B10 background (12, 15, 40).
Effector generation in RAG-2-/- TCR transgenic mice
The above analysis suggested that IL-2 alone could cause limited effector generation and polarization of some CD4 T cells present in cultures even without Ag, but also that generation of a Th0 or Th1 pattern from naive CD4 cells was influenced by the amount of IL-2 in the culture or the activation state of the T cell. Moreover, the level of expansion of responding cells and effector generation was also directly related to the levels of IL-2. With IL-2 playing so many roles, interpreting the mechanisms involved when it is limiting is difficult. The most likely source of non-naive CD4 cells in this and in other TCR transgenic models is cells that express endogenous TCR chains as well as the transgenic TCR chains (41). These cells probably have responded to other Ags in the environment, but may still have the capacity to respond to the test Ag (in this model, PCCF). To evaluate the dosage effects on the generation of effectors in the absence of such cells, we bred AND mice onto a RAG-2-/- background (RAG/AND) and repeated the analysis of the effects of PCCF dose on effector polarization, again analyzing the development of effectors in the presence and absence of exogenous IL-2.
CD4 T cells from RAG/AND mice were 
98% Vß3+ (Fig. 1)
with homogeneously low expression of CD44 (Fig. 1) and high expression
of CD62L (not shown), supporting a lack of contaminating non-naive
cells. To minimize contributions from any contaminating APC, the
purified RAG/AND CD4 T cells were cultured with DCEK.ICAM APC prepulsed
with various doses of peptide, so that they were the sole APC. Two
parallel sets of cultures were set up, one with and one without
exogenous IL-2. As before, the cell recovery in effector cultures was
highly PCCF dose dependent, with rapidly increasing cell recoveries
from 0.1 to 30 µM PCCF in cultures both with and without IL-2 added
(data not shown).
There were several differences between effectors generated from naive
CD4 of RAG/AND vs AND mice (Fig. 4). In
the absence of exogenous IL-2, effectors generated from CD4 precursors
stimulated over a broad range of Ag doses of either
RAG-2-/- (RAG/AND) or wild-type (AND) backgrounds
developed no clear polarization to either Th1 or Th2 phenotypes (open
symbols). Effectors did make substantial levels of IL-2, and there was
an increase in IL-2 production and effector cell recovery with
increasing dose (not shown). However, as before, we did detect small
amounts of IFN- from AND T cells generated with very high doses of
Ag. With the addition of exogenous IL-2 to the naive CD4 population,
almost all doses of Ag induced a mixed (Th1 and Th2) or Th0 pattern in
cells from both mice. However, effectors from CD4 T cells on the
RAG-2-/- background consistently made more IL-4 and less
IFN- (three of three experiments) than AND-derived T cells. Thus,
the moderate Th1 polarization of effectors seen when AND naive CD4 were
stimulated was lost in favor of a balanced IL-4 and IFN- production
when the RAG-2-/- background was introduced.
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In both cases (RAG/AND + IL-2 and AND + IL-2), IL-4 secretion
dropped in effectors generated with high doses of PCCF, whereas IFN-
secretion remained high (Fig. 4). With AND CD4 precursors, this drop
was seen when peptide doses >1 µM were used for effector generation,
but with RAG/AND precursors, loss of IL-4 production was seen only at
doses 10 µM. Thus, the shift away from IL-4 production at higher
peptide dose may also depend, to some extent, on contaminating
non-naive T cells.
RAG/AND precursors produce a Th0 pattern over a broad dose range
We further analyzed the effectors that could be generated in the
presence and absence of IL-2 from RAG/AND precursors; results are shown
in Figure 5. In the absence of
exogenously added IL-2 (Fig. 5a), the effectors generated
from the naive RAG/AND cells gave results somewhat distinct from those
seen in earlier experiments. First, without PCCF, the few
"effectors" recovered did not secrete either IL-4 or IFN-. When
even as little as 0.001 µM PCCF was added, the effectors generated
produced small quantities of both IL-4 and IFN-. The dose response
profiles for IL-4 and IFN- were remarkably similar to generation of
effectors producing overall low quantities of both cytokines at all
doses. Very high levels of IL-2 were produced (not shown, but see Fig. 6). Addition of IL-2 (Fig. 5b)
led to generation of effectors with much enhanced production of
cytokines, but again the dose response curves were surprisingly flat,
and ratios of IL-4 and IFN- did not vary significantly for PCCF for
doses <1 µM. It should be noted again that the level of IL-4 was
substantially higher that that noted previously for AND mice. As
before, at the highest PCCF doses tested (1 and 10 µM), IL-4
production decreased (but only in cultures receiving exogenous IL-2).
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shows normalized data in which
the amount of cytokine production is multiplied by the recovery of CD4
T cells per culture, giving an estimate of the overall effect of Ag
dose and addition of IL-2. It is clear that the major effect of
increasing peptide density is an increase in the capacity of the
cultures to produce all cytokines. A similar effect is seen for each
cytokine at all doses in the absence of IL-2 and at all but the highest
dose in the presence of IL-2. It is also clear that supplementation of
the cultures with IL-2 results in a dramatic increase in the efficiency
of effector generation. Peak IL-4 production by effectors went from 3
ng for those generated without exogenous IL-2 to nearly 100 ng with
IL-2, while peak IFN- production rose from 15 to >1500 ng. IL-2
production by effectors is particularly high when RAG/AND precursors
are used. Thus, in the absence of polarizing cytokines produced by
contaminating non-naive cells or APCs, effectors with a mixed Th1 plus
Th2 (Th0) phenotype develop, regardless of peptide dose, except at very
high doses with IL-2, where production of Th2 characteristic cytokines,
IL-4 and IL-5, drops suddenly, supporting a down-regulatory mechanism
that merits further investigation. Of particular note is the lack of a
peak of Th2 cytokine production at low Ag dose and the lack of a
conversion to Th1 polarization except at very high peptide doses with
RAG/AND precursors. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for
Th1 and IL-4 for Th2). Recently, several groups have reported that the
dose of Ag in vivo or in vitro can influence Th1 and Th2 effector
polarization. Th2 polarization was favored by low Ag dose (15, 18), Th1
polarization by moderate to high doses (15, 18), and in one case, Th2
polarization at a very high dose (18). To analyze whether a similar
pattern of polarization related to Ag dose would be achieved when
non-naive CD4 contamination was minimized, we evaluated transgenic CD4
T cells from RAG-2-/- mice and T cells cultured without
exogenous IL-2. Using TCR transgenic mice on a RAG-2-/-
background (RAG/AND), we report that naive CD4 T cell populations
develop into effectors with a Th0 cytokine pattern over a broad range
of Ag doses. We also found that the addition of exogenous IL-2 promotes
expansion of responding cells and development of effectors secreting
high titers of cytokines. The pattern of cytokine production under
these circumstances did not change, although at very high doses of Ag a
Th1 phenotype was favored. We suggest that these data support the
hypothesis that Ag dose does not selectively favor Th1 or Th2
development except in the presence of IL-2 and strong TCR signaling. In
this case, a Th1 phenotype is favored.
Several factors led us to reexamine the mechanism of Ag dosage effects.
First, the Th2 polarization seen at low Ag doses was found to be
dependent on IL-4 when examined (18). Highly purified naive CD4 T cells
stimulated with either polyclonal activators or with APC and Ag secrete
IL-2 and little or no detectable IL-4 or IFN- (29, 30, 38),
especially with low doses of Ag. Naive T cells are unable to produce
significant quantities of cytokines other than IL-2 until 1 or 2 days
after initial stimulation (22, 23, 42) and then only with repeated
stimulation (22). Therefore, it is unlikely that at low Ag doses,
responding naive T cells would be a probable source of IL-4 to induce
autocrine Th2 polarization.
Second, maximal naive CD4 T cell response depends on high Ag dose and high levels of costimulation via more than one pathway (26, 30), while activated or memory CD4 can respond to lower doses of Ag and less costimulation (Ref. 26; and P.R.R. and S.L.S., manuscript in preparation). Thus at low Ag doses, the only cytokine production that would be expected is from Ag-experienced T cells or possibly other non-T cells.
Third, in the report by Constant et al., exogenous IL-2 was added to accomplish the polarization to Th2 at low Ag doses (15). IL-2 has many dramatic effects that could be required for development of effectors (the population expected to exhibit polarization), but there is also evidence that effector T cells (43), "resting" memory cells, and perhaps other non-T cells can directly proliferate in the presence of IL-2. Thus, at low Ag doses (and even with no Ag) the few non-naive T cells could potentially expand and contribute significantly either to the cytokine production upon restimulation or by secreting low levels of cytokines during culture. Indeed, the final effector recoveries at low Ag doses in the reported studies were very low. As the response of naive T cells increased with higher Ag doses, the naive response might well be expected to "swamp out" the response of contaminating cells.
In our study, the effects of IL-2 were examined over a broad range of
Ag doses and with naive CD4 cells highly purified (Fig. 1) from both
transgenic (AND) and RAG-2-/- transgenic (RAG/AND) mice.
In the absence of exogenous IL-2, effector cell recoveries after 4 days
were much lower than when IL-2 was added, especially with low doses of
priming Ag (Fig. 2). Although low doses of Ag (0.001–1 µM) activate
nearly all of the T cells, as assessed by early activation markers CD69
and IL-2R, at Ag doses <0.01 µM (.018 µg/ml), there is little
IL-2 secretion, little or no T cell proliferation, and low T cell
recoveries (44). Even with high doses of priming Ag, all of the IL-2 is
consumed by day 3 (L. Haynes and S. L. Swain, unpublished
observations). We analyzed T cell recoveries at day 4, rather than at
day 6 or 7 as previously published (18), because we found that T cells
consume their IL-2, especially at low doses, of Ag and start to undergo
programmed cell death (apoptosis) by day 4 to 5 (10, 45). This was
especially evident in cultures that received low doses of Ag and in
cultures that received no exogenous IL-2, consistent with the
hypothesis that IL-2 withdrawal is largely responsible for the cell
death. We have also found that peak cytokine production from
effector T cells generated after a single round of Ag stimulation
occurs on days 3 through 5 (Ref. 43; and P. R. Rogers, X. Zhang,
and S. L. Swain, unpublished observations). T cell recovery was
much higher on day 4 compared with day 6, and the cells recovered were
able to secrete much higher amounts of IL-2, IL-4, and IFN- (data
not shown), suggesting that the "activation" state of the T cell or
contributions (such as costimulation or cytokines) from the APC can
influence cytokine secretion. In support of this theory, T cell
recoveries with T-depleted spleen or activated B cell APCs were
generally higher than with fibroblast APCs with peak cytokine
production occurring on days 6 through 8 (data not shown).
IL-2 addition also has a second major impact, increasing cytokine
secretion potential per cell or effector (
Figs. 3–6). In the absence
of exogenous IL-2, low doses of Ag did not selectively induce
generation of Th2 cells from either transgenic (Fig. 3) or
transgenic/RAG-2-/- mice (
Figs. 4–6), but instead led to
a population of recovered cells with little capacity for producing
IL-4, IL-5, or IFN-. Thus, high levels of IL-2 seem to be required,
directly or indirectly, for efficient effector development and
polarization to either Th0, Th1 (IFN-), or Th2 (IL-4) subsets in
addition to the role of IL-2 in cell recovery. It should be
noted in evaluating these results that the cytokine production shown
was from an equal number of cells collected at each Ag dose (
Figs. 3–5), and with the very low cell recoveries at low doses of Ag
(especially in the absence of exogenous IL-2), IL-4/IFN- cytokine
production per initial cultured cell is extremely low (as shown in Fig. 6 with normalized data).
Another important point is that in the total absence of Ag,
adding IL-2 to AND transgenics led to the recovery of a population of T
cells that could produce cytokines upon restimulation (see especially
the results from Fig. 3). The level of non-Ag-driven effector
generation was less in RAG-2-/- background
experiments (Fig. 5). The induction of IFN--secreting T cells in the
absence of Ag but in the presence of exogenous IL-2 may be influenced
by the presence of NK cells in the T cell preparation; however,
we believe that this is unlikely. First, T cells, which were purified
over Percoll to obtain small resting cells, contained no detectable
NK1.1+ cells (data not shown). Second, depletion of
NK1.1+ cells with Ab and complement or the use of
T-depleted spleen APCs (which contain a small percentage of NK cells)
did not significantly alter the results shown in Figure 3. Thus, we
believe that it is the addition of exogenous IL-2 and the use of highly
costimulatory APCs that induce a small proportion of T cells to survive
and differentiate into IFN- (and IL-4)-secreting cells in the
absence of Ag. These observations provide support for the concept that
IL-2-induced outgrowth of non-naive cells may be occurring in TCR
transgenic models and could be responsible for the polarization
detected in some studies. However, interpretation of the requirement
for exogenous IL-2 is difficult because, although it is indeed likely
that it contributes to the outgrowth of cells other than naive CD4, it
also strongly promotes effector development as well as expansion, and
thus it cannot readily be removed from initial cultures.
In young TCR transgenic mice, a majority of CD4 T cells are naive
(90–95%) (46), but with increasing age there is an increase in the
fraction of T cells that express a memory phenotype (36). There is also
increased expression of endogenous TCR - and ß-chains along with
reduced expression of the transgenic TCR, both in the AND TCR
transgenic and in other TCR transgenic mice (36, 41, 47). These
non-naive CD4 T have indeed been shown to bias naive T cell responses
and subset polarization (24, 25). To reduce the possibility of
contaminating non-naive or nontransgenic cells, we tested the effects
of Ag dose on T cells derived from TCR transgenic mice on a
RAG-2-/- background. CD4 cells from RAG/AND mice have a
naive phenotype, and there is a reduction from 14 to 1% in
CD4+Tglow or Tg- cells
(before purification) and a reduction from 5 to <1% in
CD4+CD44high cells (after purification)
compared with AND transgenic cells on the conventional
(RAG-2+/+) background (Fig. 1).
In the absence of exogenous IL-2, T cells from mice on the
RAG-2-/- background developed a weak Th0 phenotype with
secretion of low amounts of both IL-4 and IFN-, regardless of Ag
dose (Figs. 4 and 5). There was no clear effect of Ag dose on Th1 or
Th2 development in T cells from RAG/AND mice and only a weak Th1
polarization at high Ag doses in T cells from AND mice. This is not a
classic Th1 phenotype, which is associated with high levels of IFN-
production, but instead may represent a partial or suboptimal Th1
polarization. When results are normalized to cell recovery, the effects
of increasing Ag dose on increasing production of cytokine-secreting
effectors is particularly obvious (Fig. 6).
In the presence of exogenous IL-2, low doses of priming Ag again
induced a Th0 phenotype with much higher levels of IL-2, IL-4, IL-5,
and IFN- secretion. When the amount of cytokine was adjusted for the
total number of T cells recovered, the results showed that there was a
dose-dependent increase in IL-2, IL-4, IL-5, and IFN- secretion but
again no clear effect of Ag dose on Th1 or Th2 polarization (Fig. 6). T
cells from mice on the RAG-2-/- background (RAG/AND)
secreted considerably more IL-4 and less IFN- than mice on the
RAG-2+/+ background (AND) (Fig. 4), especially when
effectors were generated in the presence of exogenous IL-2.
As Ag dose increased >0.01 µM, effectors generated from AND mice in
the presence of exogenous IL-2 gradually lost their ability to secrete
IL-4 (Fig. 2) (and IL-5, data not shown). Effectors generated from
RAG/AND mice produced IL-4 over a much broader range of Ag doses with
the fall in ability to produce IL-4 and IL-5 observed only at high
doses (>1 µM) (Figs. 5 and 6). We believe that this drop in the
level of IL-4 and IL-5 secretion is not due solely to cytokine uptake
but rather to a reduced ability of T cells to secrete these cytokines,
thus resulting in a preferential Th1 phenotype. Although we concede
that there may be some IL-4 uptake by T cells, it is not likely that
such high amounts of IL-4 and IL-5 can be absorbed by the T cells or
APCs, especially since IL-5R have not been described on T cells. In
addition, upon extended culture (using T-depleted spleen as the initial
APC), high doses of priming Ag still resulted in suppressed levels of
IL-4 and IL-5 upon restimulation (data not shown). Thus, we believe
that the reduced ability to secrete IL-4 and IL-5 is related to the
ability of high Ag dose and IL-2 to suppress Th2 generation. Additional
experiments using enzyme-linked immunospot (ELISPOT) and mRNA analysis
will confirm these observations.
In contrast to our results, Hosken et al. (18) reported Th2 development
with high doses of Ag (3–100 µM) (18). The difference in Th1 vs Th2
development at high Ag doses could also potentially be influenced by
the strain of mice, culture conditions, purity of naive T cells, and/or
the TCR affinity for the peptide/MHC. The Hosken et al. study used mice
on the BALB/c background in which the percentage of
transgene-expressing CD4+ cells is only 80%. Moreover,
it is known that the BALB/c strain has a predilection for production of
Th2 effectors (12). Although cells were sorted with CD4 and CD62L,
contamination with non-naive T cells could be at higher levels (since
CD62L expression may not be a confined to naive T cells). In addition,
Hosken et al. used lightly irradiated dendritic cells or activated B
cells to present an OVA peptide to CD4 and L-selectin-sorted T cells
for a 6-day culture period. These culture conditions raise two other
possibilities for the observations in this model. First, it is possible
that APC interacting with CD4 cells can produce polarizing cytokines.
Different APC preparations may thus influence the polarization of
developing effectors. By using a fibroblast cell line as APC, we have
minimized the (potential polarizing) cytokine contribution from APCs.
Another potential difference that could affect differentiation in these
systems is the differential expression of costimulatory molecules,
particularly B7 (CD80 and CD86), on the APC. The differences seen in
our study vs Hosken’s study, however, are not likely to stem from
differential expression of B7-1 or B7-2 on the APC because T-depleted
spleen and LPS-activated B cell APCs, which express high levels of B7-2
and low levels of B7-1, gave results similar (data not shown) to those
using the B7-1+ fibroblast APCs (Fig. 5a).
Second, our own recent studies indicate loss of Th1 (but not Th2)
effectors by programmed cell death and activation-induced cell
death (45), especially between day 4 and 6 of culture. If a
mixed population were present, culture after 4 days could lead to
selection of Th2 cells, and in a model system in which Th1 and Th2
effectors are mixed, does indeed lead to Th2 predominance (X. Zhang et
al., unpublished observations). Finally, the systems may differ
by virtue of different avidities between the TCR and its ligand (48).
It will be important to determine whether peptide affinity and APC
effects (i.e., costimulation or cytokine secretion) are involved in
other models in which Ag dose (8, 48) and peptide analogues with
differing avidities (21, 49, 50) have been reported to determine CD4
effector polarization.
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Acknowledgments |
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Footnotes |
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2 Current address: La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
3 Current address: Trudeau Institute, P.O. Box 59, 100 Algonquin Avenue, Saranac Lake, NY 12983.
4 Address correspondence and reprint requests to (current address) Dr. Susan L. Swain, Trudeau Institute, P.O. Box 59, 100 Algonquin Avenue, Saranac Lake, NY 12983.
5 Abbreviations used in this paper: PCCF, pigeon cytochrome c fragment 88–104; AND, V11/Vß3 TCR transgenic mice; RAG/AND, AND TCR transgenic mice on a RAG-2-/- background; DCEK.ICAM, B7-1+/ICAM-1+/I-Ek-expressing mouse fibroblast line; PE, phycoerythrin.
Received for publication January 16, 1998. Accepted for publication June 4, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or interleukin 4 during the resolution or progression of murine leishmaniasis. J. Exp. Med. 169:59.-independent mechanism. J. Exp. Med. 171:115.ß-transgenic model. J. Exp. Med. 182:1579.-producing CD4+ T cells following activation of naive CD4+ T cells. J. Immunol. 158:1085.[Abstract]
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production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188. in regulation of Th1 cell development. J. Immunol. 157:1350.[Abstract]
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