Download Immunity Cells Predominate in Type 1 and Type 2 Single

Single-Cytokine-Producing CD4 Memory
Cells Predominate in Type 1 and Type 2
Immunity
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of June 15, 2017.
Alexey Y. Karulin, Maike D. Hesse, Magdalena
Tary-Lehmann and Paul V. Lehmann
J Immunol 2000; 164:1862-1872; ;
doi: 10.4049/jimmunol.164.4.1862
http://www.jimmunol.org/content/164/4/1862
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References
Single-Cytokine-Producing CD4 Memory Cells Predominate in
Type 1 and Type 2 Immunity1
Alexey Y. Karulin, Maike D. Hesse, Magdalena Tary-Lehmann, and Paul V. Lehmann2
The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial
because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome
this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found
CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-␥ 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. The Journal of Immunology, 2000, 164: 1862–1872.
Department of Pathology, Case Western Reserve University, Cleveland, OH 44106
Received for publication August 19, 1999. Accepted for publication December
1, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by research grants to P.V.L. from the National Institutes
of Health (DK-48799, AI-42635, AI/DK-44484) and from the National Multiple Sclerosis Society (RG-2897). M.D.H. was supported by a fellowship of the Studienstiftung des Deutschen Volkes.
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: PVL2@PO.
CWRU.EDU
Copyright © 2000 by The American Association of Immunologists
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 duallabel cytokine hybridization also showed a high degree of heterogeneity in cytokine coexpression in cloned T cells (12–15). 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 –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, 23–25). 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
0022-1767/00/$02.00
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C
D4 memory cells are thought to occur in at least two
distinct subpopulations that can play opposing roles in
infectious and autoimmune diseases by producing different cytokines (1, 2). Considerable progress has been made in understanding how naive T cells differentiate into memory cells that
express either IFN-␥ 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 IL4/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– 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
The Journal of Immunology
1863
Materials and Methods
FIGURE 1. Coexpression of type 1 and type 2 cytokines in Th1 and Th2
clone as studied by intracytoplasmic staining and two-color ELISPOT
analysis. Th1 clone SH10 (A and B) and Th2 clone M33 (C and D) were
cultured with unirradiated, T cell-depleted, syngeneic LN cells from wildtype mice with and without Ag, and the induction of cytokine production
was measured in parallel by intracytoplasmic FACS analysis or two-color
ELISPOT analysis detecting IL-2 and IFN-␥ (A and B) and IL-4 and IL-5
(C and D). For intracytoplasmic staining, 3 ⫻ 105 T cells/ml were cultured
with 1 ⫻ 106 APC/ml with and without Ag: results are shown for the T
cell-gated population. Results are shown for the stimulated cultures with
the gates set using the nonstimulated cultures with relevant Abs and isotype
controls. For the ELISPOT assays, the indicated numbers of T cells were
plated per well with the numbers of APC constant at 1 ⫻ 106 cells per well.
The SD shown is from triplicate wells. The least square approximation of
the first order was used (Y ⫽ aX ⫹ b) to approximate these data. The
correlation coefficients for all graphs shown (r) were ⬎0.995 and b ⫽ 0 ⫾
2, showing that the graphs were linear and passed through the origin. The
results are from one experiment that is representative of four performed.
An example of two-color image analysis for B is provided in Fig. 2.
spectively (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
Animals and immunizations
scid
BALB/cByJ, BALB/cByJ Smn-Prkdc /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 ␮l, 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, re3
Abbreviations used in this paper: ELISPOT, enzyme-linked immunospot; LN,
lymph node.
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 ⫻
106 or 5 ⫻ 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 Lglutamine. 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)
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were seen, which fit, respectively, the classic Th1/Th2 paradigm
(26 –28), the stochastic model (29 –33), or a pattern of dissociated
cytokine-expression (34 –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.
1864
DISSOCIATED CYTOKINE EXPRESSION IN MEMORY CELLS
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 ␮g/ml), MP2-8F8 (5 ␮g/ml),
BVD4-1D11 (2 ␮g/ml), TRFK5 (5 ␮g/ml), and R46A2 (2.5 ␮g/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:XMG1.2HRP, MP2-43D11-biotin:XMG1.2-HRP, BVD4-24G2-biotin:TRFK4-HRP,
XMG1.2-biotin:TRFK4-HRP, BVD4-24G2-biotin:XMG1.2-HRP, and JES65H4-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
␮g/ml), MP2-43D11-biotin (2 ␮g/ml), BVD4-24G2-biotin (2.5 ␮g/ml),
TRFK4-HRP (2 ␮g/ml), and either XMG1.2-HRP (2 ␮g/ml) or XMG1.2biotin (2 ␮g/ml).
Computer-assisted ELISPOT image analysis
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.
Results
Two-color cytokine ELISPOT analysis of T cells has single-cell
resolution
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. 1A). 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 twocolor IFN-␥/IL-2 ELISPOT assays in serial dilutions keeping 1 ⫻
106 APC per well, similar results were obtained: an average of
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FIGURE 2. Computerized image analysis of twocolor ELISPOT assay. Enlarged image fragments of
IFN-␥/IL-2 assay wells from Fig. 1B are shown to illustrate single-positive spots and several shades of double-positive spots (IFN-␥ in red, IL-2 in blue). Unprocessed images of medium-control well (A) and of Agstimulated well (B) both containing 125 SH10 T cells
and 106 APC/well are shown. Consecutive steps of image analysis are shown (C–F) with the image analyzer
detecting red, blue, and double-colored spots separately
by three different threshold settings. C, With the blue
threshold active, single-color blue and all double-positive spots (which have blue as a part of their color composition) are detected and outlined, for better visibility
highlighted with arrows. Similarly, under the red threshold (D), only red single-color and double-color spots are
detected. E, The double-color threshold shown is a
mathematical intersection of the two single-color thresholds. F, The final step of analysis, with single-color and
double-color spots highlighted with artificial colors:
blue, red, and green, for single-blue, single-red, and
double-color spots, respectively. Counted spots are labeled with letter symbols of the corresponding color. To
eliminate the assessment of partially overlapping red
and blue spots as double-color spots, the image analyzer
counts only double-color spots that are formed by the
color mixture of concentric blue and red spots with single dense centers. Further detail on image analysis is
provided in Materials and Methods.
The Journal of Immunology
IL-2, IL-3, and IFN-␥ 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 ␮g/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
FIGURE 3. Spot size distribution of cloned T cells and freshly isolated
CD4 cells representing the spectrum of cytokine output per cell. A, Aginduced IFN-␥ spots produced by cloned SH10 T cells (F) and by purified
CD4 LN cells isolated from OVA:CFA-immunized BALB/c mice (䡺)
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-␥ staining of clone SH10 with and without added Ag,
as specified. The legend to Fig. 1 applies. Similar results were obtained in
assays of IL-2, IL-4, and IL-5 production by using both methods (data not
shown).
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 (singlepositive). 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 twocolor 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.
Neither unimmunized nor control-immunized mice produced
IL-2, IL-3, IL-4, IL-5, or IFN-␥ when challenged with OVA or
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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 ⫻ 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 singlepositive and double-positive cells linearly decrease with the number of CD4 cells plated, consistent with the assay having singlecell 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 twocolor 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.
1865
1866
DISSOCIATED CYTOKINE EXPRESSION IN MEMORY CELLS
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 adjuvantdriven CD4 cells differentiation in vivo, we immunized BALB/c
mice with doses of OVA323–339 peptide ranging from 0.01 to 100
␮g/mouse, in CFA, and performed recall assays on the memory T
cells induced, titrating the peptide dose from 0.01 to 40 ␮M. The
0.01-␮g immunization dose did not induce a detectable cytokine
recall response; it became detectable at 0.1 ␮g injected per mouse
and reached the plateau at 10 –100 ␮g/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).
The dose-response characteristics for the activation of cells producing IL-2, IL-3, and IFN-␥ 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
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FIGURE 4. Polarized and uninhibited production of IL-5 or IFN-␥ in freshly isolated spleen cells. BALB/c mice were immunized with either OVA:IFA
(A) or OVA:CFA (C) and their spleen cells were tested on day 21 in cultures containing either medium alone (the wells on the left) or OVA (all other wells)
in a two-color cytokine ELISPOT assay: IL-5 in red, IFN-␥ in blue. The cells from the primed mice were plated either undiluted, at 1 ⫻ 106 cells/well,
or in two-fold serial dilutions with unirradiated naive BALB/c spleen cells, thus keeping the total cell number constant at 1 ⫻ 106 cells/well. The cell
dilutions in A progress from the left to the right and, in C, from the right to the left. The number (⫻105) of cells derived from mice primed with either
OVA:IFA or OVA:CFA is shown, respectively, in the upper right or lower left corner of each panel showing a representative well. B, The spleen cells from
the mice primed with OVA:IFA and OVA:CFA were mixed such that the cell numbers correspond to the numbers shown in the panel above (A) and below
(C) the image. Beneath each panel, in the respective colors, the mean ⫾ SD of red or blue spots in quadruplicate wells is shown. The data are representative
of two experiments performed with unseparated spleen cells and one each with purified CD4 cells and Th1/Th2 clones.
The Journal of Immunology
1867
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 1polarized 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 cytokineproducing, 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).
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FIGURE 5. Dissociated production of IL-2, IL-3, and IFN-␥ in OVA-specific CD4 memory T cells from OVA:CFA-immunized mice. The recall
response to OVA323–339 by CD4 cells freshly isolated from the spleen of a BALB/c mouse immunized 21 days earlier with OVA:CFA is shown: A–C, IFN-␥
(red)/IL-2 (blue) assay; D–F, IFN-␥ (red)/IL-3 (blue) assay. CD4 cells mixed with irradiated, naive LN APC were tested in the absence (A and D) and the
presence of the OVA peptide (B and E). Two-color image analysis of B and E is shown in C and F, respectively. For image analysis, the legend to Fig.
2 applies. The wells shown have been selected from serial dilutions of CD4 cells for best visual representation of the data summarized in Table I and Figs.
6 and 8. The data are representative for three independent experiments in which 4 –24 mice per group were tested individually at days, 4, 10, 21, 42, and
91 after immunization.
1868
DISSOCIATED CYTOKINE EXPRESSION IN MEMORY CELLS
Table I. Nominal Ag-induced coexpression of IL-2, IL-4, IL-5, and IFN-␥ in freshly isolated, purified CD4 memory cells and in in vitro-cultured
DO11.10 cells
No. of Recall Ag-Induced Cytokine-Producing Cells/5 ⫻ 105 CD4 Cells Tested and %DP Cellsa
IFN-␥/IL-2 assay
IFN-␥
IL-2
DP
%DP
IL-4
OVA323–339/CFA/s.c.
2
4
7
10
10
21
42
91
LN
LN
LN
LN
SP
SP
SP
SP
1
20
64
95
72
55
62
35
9
38
80
112
65
48
34
41
0
3
9
11
6
3
4
2
0
4.9
5.9
5.0
4.2
2.8
4.0
2.6
2° DO11.10 cellsc
3° DO11.10 cells
21
42
—
—
33
69
38
18
8
16
OVA/IFA/i.p.d
21
91
SP
SP
2
4
37
45
OVA323–339/IFA/i.p.e
21
SP
34
L. major f
28
SP
89
b
IFN-␥/IL-4 assay
IL-5
DP
%DP
IFN-␥
IL-5
DP
%DP
IFN-␥
0
3
6
5
7
6
4
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
18
70
91
65
60
69
31
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
23
67
99
60
52
71
29
2
2
5
8
5
4
3
2
0
0
0
1
0
0
0
0
0
0
0
0.9
0
0
0
0
10.1
15.5
36
61
11
37
2
24
4.1
19.7
36
72
12
40
1
19
2.0
14.5
—
—
—
—
—
—
—
—
0
0
0
0
35
42
28
21
4
3
6.0
4.5
3
2
32
25
0
0
0
0
1
3
44
25
0
0
0
0
29
3
4.5
47
23
4
5.4
32
19
0
0
41
50
1
1.1
76
7
4.1
203
6
1
0.1
84
4
0
0
79
225
4
1.3
IL-4
DP %DP
a
DP, double positive.
b
BALB/c mice were immunized with OVA323–339 peptide (100 ␮g/mouse) in CFA, and their CD4 cells were tested at 5 ⫻ 105 cells per well with 5 ⫻ 105 APC (irradiated
BALB/c LN cells) for the recall response to OVA323–339 (10 ␮M) in two-color cytokine ELISPOT assays.
c
Spleen cells from DO11.10 mice were stimulated in vitro with irradiated BALB/c spleen cells and OVA323–339 without the addition or neutralization of cytokines (1°
stimulation). Half of the DO11.10 cells were tested after 21 days in culture in complete medium containing IL-2 (10 ng/ml) (2° stimulation): three-hundred DO11.10 cells and
3 ⫻ 105 unirradiated BALB/c LN cells per well with and without OVA323–339 peptide (5 ␮M) were tested in two-color cytokine ELISPOT assays. The other half of the DO11.10
cells were similarly restimulated for further cell culture and tested as above on day 42 (3° stimulation).
d
BALB/c mice were immunized with 100 ␮g/mouse OVA protein and their CD4 cells were tested as specified above.
e
BALB/c mice were immunized with 100 ␮g/mouse OVA323–339 peptide in IFA and their CD4 cells were tested as specified above.
f
BALB/c mice were infected with L. major, and purified CD4 spleen cells were tested 3 wk later for the soluble Leishmania Ag-specific recall response (5 ␮g/ml).
Data for individual mice representative of groups of 4 –24 mice tested per time point are shown. The numbers of single-positive and double-positive (DP) cells were measured
by image analysis. The %DP was calculated as the percentage of double-cytokine-producing cells from the total number of cells producing both cytokines: % DP ⫽ DP ⫻
100%/(DP ⫹ cytokine 1 only ⫹ cytokine 2 only). The mean spot numbers of three to four replicate wells are shown with SD ⬍ 10%. The SD for all % DP calculated was less than
% DP ⫾ 1%.
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-␥
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
In contrast to immunizations with CFA, which tend to induce the
classic aspects of polarized type 1 immunity (IFN-␥⫹, IL-2⫹, IL4⫺, 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.
In addition to the OVA:IFA-injected mice shown here, we observed the induction of high-frequency IL-2-producing memory
cells in the complete absence of a IFN-␥-producing memory cells
after injection of seven other protein Ags in IFA into six different
mouse strains (data not shown).
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-␥
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Organ
Immunization
IFN-␥/IL-5 assay
IL-4/IL-5 assay
Time
(days)
The Journal of Immunology
(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.
Discussion
The use of computer-assisted two-color cytokine ELISPOT overcame the limitation in measuring directly the Ag-induced cytokine
coexpressed by the low-frequency, in vivo-differentiated memory
cells from normal mice and avoided the pitfalls of in vivo propagation. As performed, two-color ELISPOT assays measured the
production of pairs of cytokines simultaneously during the first 24
or 48 h (based on the well-established kinetics for each cytokine)
after reencountering Ag, mimicking the initial effector phase after
memory cell activation and before T cells start to proliferate and
the memory resets itself by generating new daughter cells. By
comparing cytokine ELISPOT (24 or 48 h assay) and intracyto-
plasmic cytokine staining/FACS analysis (6 h assay) of cloned
cells (Fig. 1) 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 Agspecific 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 ⫻ 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 cytokineproducing CD4 cells. We also found that IL-4-producing memory
T cells had been engaged in the absence of IL-5 in Leishmaniainduced immunity and that IL-2 had been produced in the absence
of IFN-␥ in IFA-induced type 2 immunity (Table I).
The level of cytokine dissociation in individual cells that we
observed in vivo was striking as it approximates the precision of
allelic exclusion, e.g., 4% for TCR ␣-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
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. Dose-response characteristic of the type 1 cytokine response
of memory cells primed by different immunization doses. Groups of four
BALB/c mice were immunized with doses of 0.1–100 ␮g of OVA323–339 in
CFA, as indicated, and LN cells (1 ⫻ 106/well) were tested for the recall
response 21 days later. A, The production of the cytokine indicated was tested
at a 10-␮M recall dose of the peptide. The data are from individual mice
representative of three independent experiments. B–D, Dose response for the
indicated cytokines and cytokine pairs in mice immunized as specified in the
inserts. Because cells producing IL-4 and IL-5 were not detected in significant
numbers at any recall concentration, these data are not shown. SD for triplicate
wells were in the range of 5–7%. For each immunizing dose, the cytokine
recall response of a single representative mouse is shown. The data were reproduced in three experiments performed on day 21.
1869
1870
DISSOCIATED CYTOKINE EXPRESSION IN MEMORY CELLS
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
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FIGURE 7. Dissociated expression of IL-2, IL-4, and IL-5 in type-2 immunity. A–F, The OVA:IFA-induced response of BALB/c mice is shown. CD4
cells were isolated from the spleen 21 days after immunization and tested on naive, irradiated LN APC in IL-4(blue)/IL-5(red) assays (A–C) and
IL-2(blue)/IL-5(red) assay (D–F). G–I, Leishmania Ag-induced IL-4(blue)/IL-5(red) recall assay of CD4 cells from the spleen of a L. major-infected
BALB/c mouse on day 28. For figure layout and data representation, the legend to Fig. 5 applies. Numerical representation of this and additional
experiments is shown in Table I and Fig. 8.
The Journal of Immunology
from two (IFN-␥/IL-4, Th1/Th2) to many discrete types, even
within “type 1” and “type 2” immunity.
Acknowledgments
We thank R. Trezza for excellent technical assistance, Drs. P. Heeger and
H. Radeke for providing the T cell clones, Dr. F. P. Heinzel for providing
L. major-infected BALB/c mice and Leishmania Ag, and he and Drs.
R. Fairchild and D. Kaufman for valuable discussions. The data were presented at the 1997 AAI meeting (1997 J. Alergy Clin. Immunol. 99:1490).
We apologize to those authors whose work we could not cite due to limitation of space.
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FIGURE 8. Overall frequency of CD4 cells coexpressing select cytokine pairs. The cumulative data from multiple experiments are shown for
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