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Department of Pathology, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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to be rare
(0–10%). This cytokine segregation was seen in adjuvant-induced type
1, type 2, and mixed immunity to OVA, in Leishmania
infection regardless of the Ag dose used or how long after immunization
the assay was performed. The data suggest that type 1 and type 2
immunity in vivo is not mediated by classic Th1 or Th2 cells but by
single-cytokine-producing memory cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-4 (reviewed in Refs. 3 and 4).
Whether other cytokines are coexpressed by these memory cells when they
reencounter Ag in vivo and exactly which ones they are is not presently
known. Without knowing which cytokines are produced in a linked manner
in individual memory cells and which cytokines are expressed
independently, no conclusions can be drawn about how many distinct
types of memory T cells exist beyond the IL-4/IFN- dichotomy; thus,
it remains unclear how precise and versatile these memory cells are in
implementing the individual effector functions induced by the
individual cytokines. Because of the technical limitations that had
made it intractable to resolve this question by direct measurements,
several indirect approaches have been used, producing conflicting
results.
The first studies performed with long-term T cell clones suggested that
memory T cells express two sets of cytokines in a mutually exclusive
fashion; Th1 cells produce IL-2 and IFN-, among other cytokines,
while Th2 cells secrete IL-4 and IL-5 (1). Based on these
data, the mainstream model emerged, according to which naive T cells
(which do not produce cytokines or produce only IL-2) first
differentiate into Th0 cells that coexpress type 1 and type 2 cytokines
(5, 6, 7, 8) and then further differentiate into the polarized
Th1 or Th2 cells upon ongoing Ag stimulation. Subsequent studies of
short-term clones showed considerable heterogeneity in cytokine
profiles (9, 10), which suggested that T cells might not
occur in distinct Th1 or Th2 subsets and that individual T cells can
coexpress type 1 and type 2 cytokines in various combinations and
ratios, a view that gave rise to the stochastic model of cytokine gene
regulation (11). Studies performed later at the
single-cell level using intracytoplasmic cytokine staining and
dual-label cytokine hybridization also showed a high degree of
heterogeneity in cytokine coexpression in cloned T cells
(12, 13, 14, 15). While T cell clones have the advantage of
providing defined cell populations, the extent to which they are
representative of memory T cells in vivo is not known. During tissue
culture, T cells are continuously driven to cycle, they undergo changes
of chromatin structure, and their DNA (including their cytokine genes)
becomes demethylated (16, 17, 18). After 16–35 cell
divisions, T cells undergo replicative senescence in vitro, and cells
that survive in culture invariably reveal severe and multiple
chromosomal abnormalities (reviewed in Ref. 19), all of
which can affect their cytokine gene regulation. Therefore, it remains
unclear whether T cells generated under the conditions of an immune
response in vivo or during chronic T cell-mediated immune pathology
have cytokine expression patterns like those of in vitro-expanded
cells.
A different approach to the study of cytokine coexpression in memory
cells relies on TCR-transgenic models. TCR-transgenic mice themselves
show little immune competence (20); therefore, the priming
and subsequent differentiation of the transgenic T cells has been
primarily modeled in tissue culture. Unexpectedly, during the first 7
days in culture, the TCR-transgenic cells were found to express IL-2,
IL-4, IL-5, and IFN- in an almost completely dissociated fashion,
with each T cell expressing only one of these cytokine mRNAs. However,
after further propagation in vitro, this phenotype was lost, and the
transgenic cells started to coexpress these cytokine genes in
apparently random combinations (21, 22). When in
vitro-propagated TCR-transgenic cells were studied by intracytoplasmic
cytokine staining, various coexpression patterns were seen including a
high degree of "Th0-like" IFN-/IL-4 and IL-2/IL-4 coexpression
(13, 23, 24, 25). Does, therefore, the expression of only one
cytokine per cell characterize the initial phase of the T cell response
in vivo, while cytokine coexpression subsequently prevails, and, if so,
do the memory cells coexpress type 1 and type 2 cytokines in a mutually
exclusive fashion, or do they express them in random combinations?
Studies of freshly isolated, nontransgenic T cells have been primarily confined to polyclonal mitogen stimulation. Depending on the cell populations tested and activation/culturing conditions chosen, cytokine expression and coexpression patterns of various sorts were seen, which fit, respectively, the classic Th1/Th2 paradigm (26, 27, 28), the stochastic model (29, 30, 31, 32, 33), or a pattern of dissociated cytokine-expression (34, 35, 36). The reasons for these conflicting results might lie in the activation of T cells with different histories of Ag encounter and, perhaps more importantly, in the nonphysiological nature of the T cell-activating signal generated by the cross-linking of TCR by mitogens or by Abs (37, 38). During physiologic, MHC-restricted recognition of Ag, the TCR functions as a gauge for the strength of the signal (39), frequently interacting with only a few MHC-peptide complexes on the surface of the APC (40, 41); the number of TCRs engaged and the kinetics of the engagement translate into different intracellular signaling patterns (39). The strength of this signal has been shown to affect cytokine coexpression (14), and different signal strengths can induce different functions in memory T cells (42). Therefore, it remains unclear whether the cytokine expression patterns seen in mitogen-stimulated/signal-enhanced T cells will be the same as those after physiologic recognition of Ag, and how the signal strength affects the coexpression of cytokines in T cells.
An understanding of cytokine gene regulation might help to predict the coexpression of cytokines in individual T cells. The data in this field as well are still controversial. Depending on the model and the stimulation/culture conditions used, evidence supporting both stochastic gene regulation (25, 43) and precisely controlled cytokine gene activation including allelic exclusion (44) has been obtained. Although the bulk of emerging evidence seems to favor regulated expression of individual cytokine genes, predictions about the coexpression of specific type 1 and type 2 cytokines in individual T cells in vivo cannot presently be made.
To address this controversy around the cytokine signature of normal, in vivo-differentiated memory cells, we pursued the measurement of cytokine coexpression in freshly isolated CD4 cells that had been physiologically activated by the nominal Ag. Building on previous efforts (45), we have developed two-color cytokine enzyme-linked immunospot (ELISPOT)3 assays in conjunction with computer-assisted image analysis to this end. After validating this approach, we measured the coexpression of key type 1 and type 2 cytokines early and late in the course of the immune response, in polarized response types, and in chronic autoimmune stimulation. We found single-cytokine-expressing CD4 memory cells to predominate under all these conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cByJ, BALB/cByJ Smn-Prkdcscid/J, C57.BL/6J, and SJL/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at Case Western Reserve University under specific pathogen-free conditions. Female mice were injected at 6–10 wk of age. OVA was purchased from Sigma (St. Louis, MO). OVA peptide 323–339 was purchased from Princeton Biomolecules (Columbus, OH). IFA was purchased from Life Technologies (Grand Island, NY), and CFA was prepared by mixing inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI) at 1 mg/ml into IFA. Ags or peptides in PBS were mixed 1:1 with adjuvant, and the specified Ag dose was injected once in 100 µl, s.c. or i.p., as specified. For the Leishmania major model, infected BALB/c mice were provided by Dr. F. Heinzel (Case Western Reserve University). The care of mice was in accordance with institutional guidelines. TCR-transgenic DO11.10 mice that are OVA323–339 specific were obtained from Dr. M. K. Jenkins (University of Minnesota).
Intracytoplasmic cytokine staining
Intracytoplasmic staining was performed as described
(15). Dual-staining for IL-2:IFN- and IL-4:IL-5 was
achieved by combining JES6-5H4-PE/XMG1.2-FITC and
TRFK5-FITC/11B1-biotin with streptavidin-PE, respectively (PharMingen,
San Diego, CA). The isotype-matched control mAbs were obtained from
Becton Dickinson (San Jose, CA). The samples were analyzed on a FACScan
flow cytometer (Becton Dickinson). Th1 clone SH-10 (46)
and the Th2 clone M33.25.6 (47) were provided by Dr.
P. S. Heeger (Cleveland VA Medical Center, Cleveland,
OH).
Double-color cytokine ELISPOT assays
Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland,
OH) were coated overnight at 4°C with the cytokine-specific capture
Abs specified below. The plates were then blocked with 1% BSA in PBS
for 1 h at room temperature and washed four times with PBS.
Subsequently, irradiated lymph node (LN) APC from naive, syngeneic mice
were added (1 x 106 or 5 x
105/well, as specified). Cloned T cells (in
serial dilution, with the numbers specified in Fig. 1
) or freshly isolated CD4 cells
(obtained in >97% purity after separation on Mouse CD4 Subset
Columns; R&D Systems, Minneapolis, MN) were plated in serial dilution
in two to four replicate wells with or without the nominal Ag or
control Ags. When used, single-cell suspensions from spinal cords were
prepared according to the same procedure used for LN and spleen
(48). We used serum-free HL-1 medium (BioWhittaker,
Walkersville, MD) supplemented with 1 mM L-glutamine. After
24–48 h of cell culture in the incubator at 37°C, the cells were
removed by washing three times with PBS and four times with PBS
containing 0.05% Tween (PBST), and the two detection Abs were added
simultaneously and incubated at 4°C overnight. The plates were washed
three times with PBST. For the biotinylated detection mAbs, the
streptavidin-alkaline phosphatase conjugate (Dako, Carpenteria, CA) was
added (at 1:2000 dilution), incubated for 2 h at room temperature,
and removed by washing twice with PBST and twice with PBS. The
nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added
first, then, after washing twice with PBS, the 3-amino-9-ethylcarbazole
(AEC) substrate (Pierce, Rockford, IL) was added and left for
15–30 min for NBT/BCIP and 20–40 min for AEC. The following coating
mAbs were used for IL-2, IL-3, IL-4, IL-5, and IFN-: JES6-1A12 (5
µg/ml), MP2-8F8 (5 µg/ml), BVD4-1D11 (2 µg/ml), TRFK5 (5
µg/ml), and R46A2 (2.5 µg/ml). The combinations of detection Abs
for the IL-2:IFN-, IL-3:IFN-, IL-4:IL-5, IFN-:IL-5,
IL-4:IFN-, and IL-2:IL-5 assays were: JES6-5H4-biotin:XMG1.2-HRP,
MP2-43D11-biotin:XMG1.2-HRP, BVD4-24G2-biotin:TRFK4-HRP,
XMG1.2-biotin:TRFK4-HRP, BVD4-24G2-biotin:XMG1.2-HRP, and
JES6-5H4-biotin:TRFK4-HRP (all Abs were from PharMingen). HRP labeling
of Abs was performed according to the standard method. Unlabeled TRFK4
in combination with HRP-labeled mouse anti-rat IgG2a mAb (1:300
dilution; Zymed, San Francisco, CA) was also used for IFN-:IL-5 and
IL-4:IL-5 assays. The detection Ab concentrations were as follows:
JES6-5H4-biotin (2 µg/ml), MP2-43D11-biotin (2 µg/ml),
BVD4-24G2-biotin (2.5 µg/ml), TRFK4-HRP (2 µg/ml), and either
XMG1.2-HRP (2 µg/ml) or XMG1.2-biotin (2 µg/ml).
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We used an ImmunoSpot Image Analyzer (Cellular Technology
Limited) specifically designed for two-color ELISPOT analysis.
Digitized images were analyzed for the presence of areas in which color
density exceeds background by a factor set on the basis of the
comparison of control (containing T cells and APC without Ag) and
experimental wells (containing Ag, exemplified in Fig. 2, A vs B–F).
After separating spots that touch or partially overlap, additional
criteria of spot size and circularity are applied to gate out speckles
and noise caused by spontaneous substrate precipitation, nonspecific Ab
binding. Objects that do not meet these criteria are ignored and areas
that meet them are recognized as spots, counted, and highlighted.
Additionally, spot-size histograms were generated reflecting the
distribution of cells according to the cytokine output per cell (an
example is provided in Fig. 4). Two-color ELISPOT image analysis
follows the same principles except that the image analyzer detects red,
blue, and double-colored spots separately by using three different
threshold settings as specified in Fig. 2. Each color threshold is set
in RGB mode and consists of three numbers reflecting the threshold in
red, blue, and green channels. The red and blue thresholds are set by
using spots from single-color assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first set of experiments was done to validate the two-color
cytokine ELISPOT approach for measuring cytokine coexpressed by
individual T cells that occur in the low-frequency range
(1:1,000–1:1,000,000), wherein CD4 memory cells usually occur and
which is below the detection limit of FACS analysis. The sensitivity of
two-color ELISPOT assays was tested by subjecting T cell clones to
intracytoplasmic cytokine staining and ELISPOT analysis in parallel.
Intracytoplasmic IFN-/IL-2 staining of clone SH10 showed that 48%
of the cells expressed only IFN- and 2% only IL-2, with 23%
coexpressing these cytokines (Fig. 1
A). By not producing
either IL-4 or IL-5 (data not shown), SH10 qualifies as a Th1 clone.
When the same cells were tested in parallel by two-color IFN-/IL-2
ELISPOT assays in serial dilutions keeping 1 x
106 APC per well, similar results were obtained:
an average of 53% of the T cells was found to produce only IFN-,
4% to secrete only IL-2, and 19% to secrete both cytokines (Fig. 1B; the image analysis of such two-color spots is shown in
Fig. 2). The plot of the number of cloned cells plated per well against
the number of single-positive and double-positive spots was linear and
passed through the origin. Therefore, every cytokine-producing cell was
directly visualized, even in the presence of 1 x
106 bystander cells. This close correlation
between intracytoplasmic staining and ELISPOT assays was also seen in
similar experiments performed on the Th2 clone M33 (Fig. 1, C and D): IL-4 and IL-5 were coexpressed by
50% of the cloned cells. The numbers of single-positive and
double-positive cells linearly decrease with the number of CD4 cells
plated, consistent with the assay having single-cell resolution. In
addition, these data show that, as far as the detection of the
coexpression of IFN-/IL-2 and of IL-4/IL-5 in single T cells is
concerned, the sensitivities of intracytoplasmic staining/FACS analysis
and two-color ELISPOT analysis are comparable. The color resolution of
two-color ELISPOT assays based on AEC (the red substrate for HRP) and
NBT/BCIP (the blue substrate for AP) is at least as good as the color
resolution of FITC and PE staining in two-color FACS analysis. Last,
the use of two-color cytokine ELISPOT analysis confirmed that IFN-
and IL-2, and IL-4 and IL-5, are coexpressed in long-term cultured T
cells, an observation that led to the postulate that such Th1/Th2 cells
would also occur in vivo.
When we studied the size distribution of Ag-induced IFN- spots
generated by freshly isolated CD4 cells from the BALB/c mice primed
with OVA323–339 and those produced by cloned T
cells, we found them to be comparable: both types of cells produced a
wide spectrum of spots of various sizes with distributions close to
Gaussian and very similar to those obtained by intracytoplasmic
stainings (Fig. 3, A vs
B). Although the size of the spots depends on several
parameters including the kinetic of cytokine secretion, it is
proportional to the net cytokine output per cell over the culture
period (49). Therefore, such differences in spot sizes
(amount of cytokine produced) within clones and freshly isolated cells
reflects cell biology. (Similar results were obtained when comparing
IL-2, IL-4, and IL-5 produced by clones and freshly isolated CD4 cells,
data not shown.) Because cloned cells and freshly isolated T cells
produced comparable amount of cytokines, single-cell sensitivity also
seems to apply for freshly isolated memory T cells, which occurred at
frequencies too low for intracytoplasmic staining to measure, thereby
impeding direct comparison. The single-cell resolution of these
measurements was further supported by the linear decrease in spot
numbers seen when CD4 LN cells primed to produce either IFN- or IL-5
were serially diluted with unprimed spleen cells or were mixed in
different ratios (Fig. 4). These data
also showed that the cognate cytokine produced by type 1 or type 2
polarized memory T cells neither induced bystander cytokine production
in the APC population nor inhibited IFN-/IL-5 produced by the memory
cells.
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are produced by different CD4 cells in type
1 immunity
We chose to characterize more closely the immune response induced
in BALB/c mice with a well-defined Ag, OVA, and its immunodominant
peptide OVA323–339. First, we immunized mice
with the maximally immunogenic dose of
OVA323–339 peptide, 100 µg/mouse, in CFA, s.c.
and tested the peptide-induced recall response of CD4 cells purified
from the LN and spleens of these mice at various time points.
Representative data are shown in Fig. 5
and are summarized in Table I. A classic
type 1 (IFN-+, IL-2+,
IL-3+, IL-4-, and
IL-5-) recall response was seen. Image analysis
of data obtained in three independent experiments with 4–24 mice per
experiment in which cells from each mouse were tested in triplicate
wells and in serial dilutions (analyzing more than 600,000
peptide-induced cytokine spots) showed that 95 ± 4% of the spots
in the IFN-:IL-2 assay were either red or blue (single- positive).
Only about 5% of the spots appeared in various shades of purple
indicating that only a minor fraction of Ag-specific T cells produced
both cytokines simultaneously or switched cytokine production during
the assays’ 24–48 h duration. This frequency range of double-positive
spots was also seen when the primed T cells were tested in serial
dilutions (data not shown). We performed single-color IFN- and IL-2
assays in parallel to verify that we had detected all
double-cytokine-producing T cells. The frequencies of cells producing
IFN- and IL-2 in the single-color assays closely matched the sum of
the frequencies of the single producers and double producers of
IFN-/IL-2 detected in the two-color assay (within 5% error). These
additional single-color data prove that the two-color analysis did not
miss double-expressing cells. While we cannot exclude the possibility
that some cytokine is being coexpressed below the detection limit of
ELISPOT analysis, we can safely conclude that the secretion of the one
cytokine detected was highly polarized.
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when challenged with OVA or OVA peptide.
Although it has been suggested that naive cells can produce IL-2 (Refs.
50 and 51 ; a notion that is not unanimously
agreed upon, see Ref. 21), their frequency must have been
<1/1,000,000 the detection limit of the IL-2 ELISPOT assay as
performed. Only after immunization were cells producing IL-2, IL-3, or
IFN- detectable, suggesting that these cells have undergone
Ag-driven clonal expansion and differentiation. Moreover, we found that
these cytokine-producing T cells resided in the
L-selectin-, CD4 fraction (48),
which corresponds to a memory phenotype. Therefore, the
cytokine-producing cells that we detected after immunization appear to
be memory cells that acquired their cytokine phenotype during an
Ag-driven immune response in vivo.
Signal strength affects neither the induction of CD4 cell subsets
that selectively express IL-2/IL-3/IFN- nor the coexpression of
these cytokines
It has been suggested that the Ag dose (the density of MHC:nominal
peptide complexes on the APC defining the extent of TCR ligation and,
hence, the signal strength) affects both the postthymic differentiation
of naive T cells along the type 1/type 2 pathway and the pattern of
cytokine expressed by differentiated memory cells (42, 52, 53). To determine whether this also applies to adjuvant-driven
CD4 cells differentiation in vivo, we immunized BALB/c mice with doses
of OVA323–339 peptide ranging from 0.01 to 100
µg/mouse, in CFA, and performed recall assays on the memory T cells
induced, titrating the peptide dose from 0.01 to 40 µM. The 0.01-µg
immunization dose did not induce a detectable cytokine recall response;
it became detectable at 0.1 µg injected per mouse and reached the
plateau at 10–100 µg/mouse (Fig. 6A). The overall cytokine
signature of these recall responses was unaffected by the immunization
dose: induction of IL-2, IL-3, and IFN- was seen, with only marginal
IL-4 and no IL-5 being produced. No IL-5 and only marginal IL-4
production was seen over the full range of peptide concentrations
tested at recall (data not shown).
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were similar (Fig. 6, B–D). The
concentration of the OVA peptide at which 50% of the maximally
inducible cells become activated was 50 ± 15 nM for all three
cytokines; this defines the functional avidity of these T cells for
OVA323–339 (48). Whereas the
frequency of cytokine-producing cells increased with the peptide
concentration, the fraction of double-positive cells stayed largely
constant for given immunization doses varying between 1 and 8%. These
data, shown for the day 21 time point in Fig. 6, are representative for
all the time points tested (days 4, 10, 21, 42, and 91, data not
shown). Therefore, the dissociated production of IL-2, IL-3, and
IFN- was neither affected by the signal strength that induced the
differentiation of the naive T cells nor by the signal strength that
induced cytokine production in the memory cell. Similar results were
obtained when purified CD4 cells were tested on different types of APC
layers, irradiated or unirradiated spleen cells of
BALB/cSCID mice, or spleen, LN, or thymic cells
of naive BALB/c mice. Therefore, the presence or absence of B cells and
the variation of other class II-positive cell types in these organs had
no significant effect on the size and frequency of IFN-, IL-2, and
IL-3 spots detected (data not shown).
In type 1 immunity, CD4 cells directly assume the IL-2, IL-3, and
IFN- single-cytokine-expressing phenotype
It has been postulated that CD4 cells first differentiate into
memory cells that coexpress type 1 and type 2 cytokines (Th0-type
cells) in the course of the immune response and that only with chronic
stimulation would these cells further differentiate into type 1 or type
2 cytokine-expressing memory cells. According to this model, the
coexpression of type 1 and/or type 2 cytokines in individual T cells
should be observed in vivo early in the course of response, whereas the
cytokine expression pattern of the memory cells might become polarized
over the course of the immune response. To address this possibility, we
also studied OVA-peptide-induced CD4 memory cells soon after
immunization. By the earliest time that we could detect a specific
cytokine recall response, on day 4 after immunization, IL-2, IL-3, and
IFN- single-positive cells were detected and none producing IL-4 or
IL-5 were found. Therefore, at the population level, the cytokine
response was type 1- polarized early on, and, at the level of
individual memory cells, it was already mediated by
single-cytokine-producing cells. Memory cells seem to assume this
phenotype rapidly without first going through a prolonged state in
which they coexpress cytokines.
Loss of dissociated cytokine expression in tissue culture
Transient segregation of cytokine mRNA expression has been
reported for in vitro-primed D011.10 TCR-transgenic cells when using
dual-label, in situ hybridization (21, 22). Two-color
cytokine ELISPOT measurements yielded comparable results when the same
D011.10 cells were tested after the second cycle of in vitro
stimulation (Table I). We also observed that the frequency of
double-cytokine-producing cells increased with further restimulations
in vitro: after 42 days of cell culture, about 16% of the
cytokine-producing, transgenic cells became
IL-2+/IFN-+
double-positive, starting to approximate the phenotype of T cell clones
(Fig. 1). Moreover, also in confirmation of this previous report, we
found that, with prolonged culture, the transgenic cells started to
coexpress cytokine combinations like IFN- and IL-5 (close to 15%
double-producers detected on day 42, Table I) that we have not seen
directly ex vivo. This difference could be attributed to the continuous
Ag/IL-2-driven proliferation that the T cells undergo in cell culture.
Extensive cell cycling was shown to cause demethylation of cytokine
genes and changes in chromatin structure (16), which makes
cytokine genes more accessible and may favor cytokine coexpression. Our
data suggest that such changes in cytokine expression may not readily
occur in vivo, possibly because the T cells in vivo reach replicative
senescence after 17–35 cell divisions (19).
IL-4 and IL-5 produced by different CD4 cells in
adjuvant-induced, type 2 immunity; induction of CD4 subpopulations that
produce IL-2 but not IFN-
In contrast to immunizations with CFA, which tend to induce the
classic aspects of polarized type 1 immunity
(IFN-+, IL-2+,
IL-4-, IL-5- cytokine
recall, delayed-type hypersensitivity, production of specific IgG2a but
no IgE Abs), protein Ag injected in IFA typically results in polarized
type 2 immunity (IL-4+,
IL-5+, IFN--;
IgG1+, IgE+,
IgG2a-; no delayed-type hypersensitivity)
(48, 54). The prevalence of the single-cytokine-producing
T cell phenotype was also seen in the OVA:IFA-induced type 2 response
(Table I and Fig. 7, A–F):
95% of the Ag-specific CD4 cells produced IL-4 and IL-5 or IL-2 and
IL-5 mutually exclusively. Despite the vigorous Ag-specific IL-2 recall
response, there was no IFN- induced. This dissociation of IL-2,
IL-4, and IL-5, in the absence of IFN-, was seen over a wide range
of Ag concentrations used for immunization and recall and using APC
from different sources (LN, spleen, thymus of naive mice, or spleen of
SCID mice, data not shown). Therefore, the overall type 2-polarized
cytokine profile of the IFA-induced response was the result of
different T cells individually producing the individual cytokines
comprising this profile.
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-producing memory cells after injection of seven
other protein Ags in IFA into six different mouse strains (data not
shown).
IFA immunization with OVA323–339 peptide results in a
mixed cytokine profile in the absence of Th0 cells coexpressing IFN-
and IL-4 or IFN- and IL-5
Unlike the immunizations with OVA protein in IFA, injections of
BALB/c mice with OVA323–339 peptide in IFA
resulted in mixed "Th0-type" response with IL-2, IL-4, IL-5, and
IFN- production (48). Two-color assay performed on CD4
cells separated from spleens of these mice 3 wk after immunization
showed that each of these cytokines was produced by different cells
(Table I).
Different Leishmania Ag-specific CD4 cells produce
IFN-, IL-2, and IL-4 in immunity induced by
Leishmania infection; memory cells producing IL-4 but
not IL-5 are induced
To test whether dissociated cytokine expression is limited to
adjuvant-induced responses or is a more general feature of the CD4
memory cells, we characterized the CD4 cells that were primed during
the natural course of L. major infection in BALB/c mice
(Table I). Not only did these cells show dissociated Ag-induced
production of IL-2 and IFN-, but IL-4 production was seen in the
virtual absence of IL-5 (Fig. 7, G–I). The dissociation of
IL-4 and IL-5 was independent of the type of APC used; IL-4 and IFN-
were also produced by different cells (Table I). The production of IL-4
in the absence of IL-5 was also seen when culture supernatants were
studied by ELISA (data not shown). When Leishmania Ag was
injected with IFA, it induced the IL-5-producing memory cells in
addition to those secreting IL-2 and IL-4 but not IFN- (data not
shown). The absence of IL-5 in Leishmania-infected animals
cannot be attributed to an inhibiting effect of IFN-, because, based
on the data from immunizations of BALB/c mice with
OVA323–339 peptide in IFA, these two cytokines
are not expressed in a mutually exclusive manner on the population
level (although they were at the single-cell level) either at the stage
of priming (Table I) or at the stage of the recall (Fig. 4). Therefore,
the presence or absence of IL-5-producing memory cells was not dictated
by the nature of the Ag itself but seemed to be determined by the
mode/microenvironment of the induction of the immune response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or freshly isolated CD4 cells from
the LN after polyclonal stimulation with anti-CD3 mAbs or Con A
(data not shown), we showed that, indeed, this is the case, as within
this time frame proliferation did not occur. Moreover, by measuring
this early cytokine response, we wanted to ascertain that the cytokines
we measure were produced by the in vivo-differentiated memory cell, as
opposed to their daughter cells, which may or may not inherit the
cytokine commitment of the mother cell. Because differentiation of
naive cells into memory/effector cells requires longer than this 24–48
h, the cytokines measured were produced by in vivo-primed memory cells
(see above).
Unlike dual-label in situ hybridization, which measures mRNA levels in
the T cell at a single time point, these ELISPOT assays integrate the
production of both cytokines over the entire assay period (immediately
after the Ag was added to the cells), accounting for possible
differences in the kinetics of cytokine production, including switching
from the production of one cytokine to the another. Instead of
measuring mRNA (or measuring the accumulation of cytokine in the
cytoplasm of pharmacologically treated cells), the ELISPOT approach
detects the immunologically relevant secretion of the cytokine, thereby
accounting for posttranscriptional and posttranslational regulation.
The detection limit for the minimum amount of cytokine produced per
cell was found to be comparable in intracytoplasmic staining and in
ELISPOT (Fig. 1) for cloned T cells. Also, the distribution of cells
according to the amount of cytokine produced per cell was similar with
both methods (Fig. 3). However, when it came to detecting the few
Ag-specific cells within the majority of non-Ag-specific cells, only
the ELISPOT assay was suited for this purpose, as it proved to be
sensitive enough to detect one cytokine-producing cell within a million
non-cytokine-producing cells (Fig. 1). In contrast, when the frequency
of cytokine-producing cells fell below 1:1000, they became undetectable
by intracytoplasmic staining; therefore, ELISPOT was 1000 times more
sensitive to detect cytokine produced by rare Ag-specific cells in
freshly isolated populations.
Irrespective of how the immune response was induced (by infection or
immunization with adjuvant), the Ag doses used for priming and recall,
or the time point tested, the cytokines measured were mostly produced
by different CD4 memory cells; because very similar results were
obtained in all in vivo systems tested, the cumulative data obtained in
multiple independent experiments for all models studied are summarized
in Fig. 8 (they represent the frequency
of cytokine coexpression measured in over 1 x
106 individual CD4 cells). The dissociation of
IFN- from IL-5, and IL-4 and IL-3 from IL-5, was virtually complete.
The coexpression of the "type 1" cytokines IFN-, IL-2, and IL-3
or the "type 2" cytokines IL-2, IL-4, and IL-5 were confined to
4–6% of cytokine-producing CD4 cells. We also found that
IL-4-producing memory T cells had been engaged in the absence of IL-5
in Leishmania-induced immunity and that IL-2 had been
produced in the absence of IFN- in IFA-induced type 2 immunity
(Table I).
|
-chains (55); thus, it was
consistent with highly regulated cytokine gene regulation in T cells
physiologically stimulated by Ag. Our data can also be explained within
the framework of stochastic cytokine gene expression, provided that
only a small fraction of the activated memory T cells is actually
induced to produce cytokine. Moreover, one would have to postulate
additional very rapid and precise selection mechanisms
(11) to explain why there are essentially no
IL-5-producing memory cells present in Leishmania-induced
immunity and why the nearly complete polarization of IL-4/IFN- and
IL-5/IFN- is already seen at day 4 after immunization.
Alternatively, it is tempting to postulate that the differentiation and
subsequent expansion of each of the individual cytokine-producing
memory cell subpopulations is under independent instructive control. It
is well-established that IL-12/IL-18 and IL-4/IL-13 are differentiation
factors for the generation of memory cells expressing IFN- and IL-4
(3, 4). Recent evidence has emerged indicating that
differentiation into IL-5-producing memory cells may have different
differentiation requirements than IL-4 (56, 57), and this
may apply for the other type 2 and type 1 cytokine as well. It was also
demonstrated that the demethylation patterns of IFN- and IL-3 genes,
which define their ability to be expressed upon T cell activation
(16), are inherited in T cell lineages (17, 18). This observation might point toward the existence of memory
cell sublineages with inherited commitment for expressing certain
cytokine genes. Stringent control of the engagement and expansion of
these single-cytokine-expressing memory cell lineages during the
primary immune response would make the resulting composition of the
memory cell effector functions finely tunable. When and where the Ag is
reencountered for the second time, these memory cell lineages would
express the individual cytokines to which they are precommitted. On the
population level, they would create a cytokine microenvironment whose
exact quality (which individual cytokine is produced and which is not)
and magnitude (the population size of each single-cytokine-producing
population) precisely execute the required combination of effector
functions imprinted during the primary immune response.
Irrespective of the cytokine gene regulation mechanism underlying
dissociated cytokine expression, the implications of these data in
terms of immunobiology is that the different effector functions
associated with the individual cytokines are each independently
performed by the T cell system and, therefore, that cytokine-mediated
effector functions of T cells are much more versatile and precise than
anticipated. Dissociated expression of individual cytokines raises the
repertoire of CD4 memory response types from two (IFN-/IL-4,
Th1/Th2) to many discrete types, even within "type 1" and "type
2" immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul V. Lehmann, Department of Pathology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, BRB 929, Cleveland, OH 44106-4943. E-mail address:
3 Abbreviations used in this paper: ELISPOT, enzyme-linked immunospot; LN, lymph node.
Received for publication August 19, 1999. Accepted for publication December 1, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon, and interleukins 3 and 4 is random in murine alloreactive T-lymphocyte clones. Proc. Natl. Acad. Sci. USA 85:9189. (IFN-) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN- promoter demethylation and mRNA expression are heritable in CD44highCD8+ T cells. J. Exp. Med. 188:103. promoter demethylation in CD44highCD8+ T lymphocytes. J. Immunol. 162:5053.ß T cell receptor-transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene-specific antigen. Eur. J. Immunol. 27:1774.[Medline]
. J. Immunol. 160:5280. production by human T cells at a single-cell level. Eur. J. Immunol. 28:3154.[Medline]
by activated human blood lymphocytes. Eur. J. Immunol. 20:1591.[Medline]
and in interleukin-4-expressing cells. Eur. J. Immunol. 24:1097.[Medline]
and IL-2- producing cells in the CD8+ T cell subset. Eur. J. Immunol. 28:3630.[Medline]
phosphorylation. Science 281:572.This article has been cited by other articles:
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P. S. Heeger, T. Forsthuber, C. Shive, E. Biekert, C. Genain, H. H. Hofstetter, A. Karulin, and P. V. Lehmann Revisiting Tolerance Induced by Autoantigen in Incomplete Freund's Adjuvant J. Immunol., June 1, 2000; 164(11): 5771 - 5781. [Abstract] [Full Text] [PDF]
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) were categorized according to size.
The size histograms generated by the analyzer represent the
distribution of spots falling into 15 size categories (the symbols)
between 10-3 and 1 mm2. Spots of area less
than 10-2 mm2 (the hatched line, corresponding
to the background in the absence of the cognate Ag) were gated out.
Further specifics on morphometry in computerized ELISPOT image analysis
is given in Materials and Methods. B,
Intracytoplasmic IFN-
