Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4⁺ Lymphocytes with a Th2 Cytokine Profile

Experimental animals, infection procedure, and Ag preparation

IL-4Rα KO and Stat6 KO mice were generated, as previously described, by homologous recombination in BALB/c-I and 129-derived CCE embryonic stem cells, respectively (38, 5). The IL-4Rα KO animals were bred as homozygotes on the original BALB/c background, whereas the Stat6 KO mice were partially backcrossed to C57BL/6. Both mouse strains were bred and maintained at the American Association for the Accreditation of Laboratory Animal Care (AAALAC) accredited animal facility of National Institute of Allergy and Infectious Diseases (NIAID). Control BALB/cJ and C57BL/6 animals were purchased from The Jackson Laboratory (Bar Harbor, ME) and Division of Cancer Treatment (National Cancer Institute, Frederick, MD), respectively. C57BL/6 IL-4 KO mice (39) were obtained from Taconic Farms (Germantown, NY).

Age (8–12 wk)- and sex-matched mice were used in each experiment. Mice were percutaneously infected with 35 S. mansoni cercariae (NMRI strain) and sacrificed 8 wk later (36). A soluble extract of schistosome eggs (SEA) was prepared as described (40).

Cell preparations

Mesenteric LN were pooled from 8-wk schistosome-infected mice (n = 4–6/group), and single cell suspensions were prepared in tissue culture medium (RPMI 1640 with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 20 mM HEPES, and 50 μM 2-ME). CD4⁺ cells were purified from infected mice by staining LN cells with PE-labeled anti-CD4 mAb (RM4-5; PharMingen, La Jolla, CA), followed by sorting on a FACStar sorter (Becton Dickinson, San Jose, CA). The purity of sorted CD4⁺ populations was ≥ 97%.

Using the same approach, CD4⁺ cells were purified from splenocytes from uninfected wild-type (WT) and Stat6 KO mice, with the exception that RBC were lysed by osmotic treatment before staining. To isolate naive CD4⁺ cells, FITC-labeled anti-CD62L mAb (MEL-14; PharMingen) was added during the staining procedure and double-positive cells were collected. The purity of sorted CD4⁺CD62L^high populations was 95 ± 3%.

Spleen cells from uninfected mice were used as a source of APC. For primary cultures of sorted CD4⁺ cells from infected mice, splenocytes were depleted of T cells by anti-Thy-1.2 mAb (Cedarlane Laboratories, Hornby, Canada) and Low-Tox-M rabbit C (Cedarlane Laboratories) treatment (18). In subsequent stimulations, total splenocytes were used as APC. For anti-CD3 stimulation of CD4⁺ and CD4⁺CD62L^high cells from uninfected WT and Stat6 KO mice, splenocytes from IL-4 KO mice were used throughout the experiment. All preparations of APC were initially depleted of RBC by osmotic treatment and finally irradiated with 3000 rad.

Proliferation assay

Mesenteric LN cells from infected mice (3 × 10⁶/ml) were cultured in 0.2 ml in flat-bottom 96-well plates with medium alone or SEA (20 μg/ml). Anti-class II (M5/114, rat IgG2b) (41), anti-CD4 (GK1.5, rat IgG2b) (42), or anti-CD8 mAb (2.43, rat IgG2b) (43) were added to cultures 2 h before SEA. After 48 h, cultures were pulsed with [³H]TdR (0.5 μCi/well; New England Nuclear, Boston, MA; sp. act., 2 Ci/mmol) for 18 h, and incorporated isotype was measured.

Cytokine assays

Mesenteric LN cells from infected animals (3 × 10⁶/ml) were cultured in 24-well plates in 1 ml/well, while sorted CD4⁺ cells (2.5 × 10⁵/well) from the same LN populations were cultured with irradiated T-depleted syngeneic splenocytes as APC (7.5 × 10⁵/well) in flat-bottom 96-well plates in 0.2 ml/well in the presence or absence of SEA (20 μg/ml). Sorted CD4⁺ cells from uninfected WT and Stat6 KO mice (2 × 10⁵/well) were cultured with irradiated IL-4 KO splenocytes as APC (7.5 × 10⁵/well) in 0.2 ml/well in flat-bottom 96-well plates in the presence of anti-CD3 mAb (145-2C11; 1 μg/ml; PharMingen) with or without anti-IL-4R mAb (M1; 20 μg/ml; R&D Systems, Minneapolis, MN).

After 72 h, supernatants were collected for cytokine determinations. IFN-γ was measured by ELISA (44), while IL-4 was detected using a commercial ELISA kit from Endogen (Woburn, MA). The limit of detection was 125 pg/ml and 62 pg/ml for the IFN-γ and IL-4 assays, respectively.

Culture conditions before intracellular cytokine staining

Analyses of intracellular cytokine expression were performed on cells from the same SEA-stimulated cultures used in the cytokine secretion assays. Mesenteric LN cells from infected mice were incubated for an additional 18 h in fresh medium added to replace the culture supernatant collected at 72 h. In the case of sorted CD4⁺ cells, cultures were expanded by adding medium containing human rIL-2 (10 U/ml; Cetus Oncology, Chiron, Emeryville, CA) for a 7-day period, followed by restimulation with SEA (20 μg/ml) in the presence of irradiated splenocytes as APC. On day 3 of this second SEA stimulation, supernatants were collected and cells were cultured for an additional 4 days in medium with rIL-2 before intracellular staining. When indicated, rIL-4 (20 ng/ml; R&D Systems), rIL-12 (10 ng/ml; kindly provided by Hoffman-LaRoche, Nutley, NJ), anti-IL-4 (11B11; 20 μg/ml), anti-IL-4Rα (M1; 20 μg/ml; R&D Systems), anti-IL-12 (C17.8; 20 μg/ml) (45), or anti-IFN-γ (XMG-6; 20 μg/ml) (46) mAb were added to parallel SEA cultures during both Ag stimulations performed. Staining patterns were also compared after a third SEA stimulation done in the absence of exogenous cytokines or Ab.

CD4⁺CD62L^high cells from uninfected Stat6 KO mice were analyzed for intracellular cytokine expression 7 days after each of three consecutive anti-CD3 stimulations performed on days 0, 7, and 14. Following each stimulation, 72-h culture supernatant was replaced with medium containing rIL-2 (10 U/ml).

Intracellular cytokine staining and flow cytometry

For intracellular staining, cells were stimulated in 1-ml cultures in 24-well plates coated overnight with 2.5 μg/well of anti-CD3 mAb (145-2C11). Two hours later, brefeldin A (BFA; 10 μg/ml; Sigma, St. Louis, MO) was added, and after 5 h, cells were stained using a modified protocol based on that described by Prussin and Metcalfe (47). Briefly, cells were washed once in RPMI, stained with Cy-chrome-labeled anti-CD4 mAb (RM4-5; PharMingen), and fixed for 15 min in 2% paraformaldehyde (Sigma) at room temperature. After 30-min incubation in permeabilization buffer (PBS containing 0.1% Saponin (Calbiochem-Novabiochem, La Jolla, CA), 0.1% FCS, and 20 mM HEPES) with 10% normal mouse serum and anti-FcγRII/III mAb (2.4G2; 5 μg/ml; PharMingen) at 4°C, cells were stained for 30 min with different combinations of the following, pretitrated flourochrome-labeled mAb at 4°C: PE anti-IL-2 (JES6-5H4), PE anti-IL-4 (BVD4-1D11), FITC anti-IL-4 (BVD4-1D11) (all from BioSource International, Camarillo, CA), PE anti-IL-5 (TRFK5), FITC anti-IL-10 (JES5-16E3), and FITC anti-IFN-γ (XMG1.2) (all from PharMingen), washed twice with permeabilization buffer, and resuspended in PBS + 0.5% FCS. Cell fluorescence was measured using a FACScan flow cytometer and data were analyzed using CellQuest software (Becton Dickinson). The specificity of the cytokine staining was confirmed in initial experiments in which preincubation with corresponding unlabeled mAb completely inhibited the signal obtained with labeled anti-cytokine mAb (not shown).

To determine the Vβ8 usage of the IL-4⁺ CD4⁺ lymphocytes, cells were stained with Cy-chrome-labeled anti-CD4 and FITC-labeled anti-Vβ8 mAb (MR5-2; PharMingen), fixed, and subsequently stained with PE-labeled anti-IL-4, as described above.

Isolation of T cell clones

SEA-specific T cell clones from infected Stat6 KO mice were obtained by limiting dilution of CD4⁺ cells twice stimulated with SEA, as described for the intracellular cytokine-staining procedure. Briefly, 7 days after a second Ag stimulation, cells were cultured (1 or 0.3 cells/well) in 0.1 ml/well in round-bottom 96-well plates with 3 × 10⁵ irradiated syngeneic spleen cells and SEA (20 μg/ml). One day later, 0.1 ml/well of rIL-2 (20 U/ml) was added. After 2 wk, fresh medium with APC and Ag was added, followed by rIL-2 48 h later. During the fourth week, wells containing proliferative cells were identified visually and further expanded. Cloning efficiency was 12 and 4% for cultures with 1 and 0.3 cell/well, respectively. Cells from positive wells were characterized for their cytokine secretion profile by ELISA and by intracellular staining.

The IL-4-producing cells in helminth-infected IL-4Rα and Stat6 KO mice are conventional CD4⁺ T lymphocytes

We have previously shown that LN cells from IL-4Rα KO mice infected with S. mansoni develop diminished yet significant IL-4 responses when restimulated in vitro with parasite Ag (36). We began the present study by performing a series of experiments in both IL-4Rα- and Stat6-deficient mice to more precisely define the cell populations producing IL-4 in schistosome-infected mice of both types. Initial studies in infected IL-4Rα KO mice indicated that in vitro treatment with anti-CD4, but not anti-CD8 mAb abolished the residual Th2 cytokine production observed (not shown). Similarly, in vitro addition of anti-CD4 but not anti-CD8 mAb caused a dramatic reduction in the proliferative responses of mesenteric LN cells from 8-wk schistosome-infected IL-4Rα and Stat6 KO animals to soluble egg Ag (SEA) (Fig. 1⇓). Moreover, an even greater inhibition in blastogenesis was detected when anti-class II mAb was added to parallel cultures (Fig. 1⇓). Thus, the T cell responses to SEA in infected IL-4Rα and Stat6 KO mice are both CD4 and class II dependent.

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FIGURE 1.

Addition of anti-class II or anti-CD4, but not anti-CD8, mAb blocks SEA-specific proliferative responses of LN cells from S. mansoni-infected IL-4Rα and Stat6 KO mice. Mesenteric LN cells (3 × 10⁶/ml) from infected IL-4Rα KO, Stat6 KO, or the corresponding WT control mice were cultured in medium alone or with SEA (20 μg/ml). In the case of cells from KO animals, anti-CD4, anti-CD8, or anti-class II mAb were added to replicate cultures 2 h before the Ag. [³H]TdR was added after 48 h, and incorporation was measured 18 h later. Results shown are the mean (±SD) of duplicate cultures and are representative of two experiments performed.

To formally demonstrate that CD4⁺ cells are the source of the IL-4 produced, we purified CD4⁺ lymphocytes by cell sorting from LN of infected WT and IL-4Rα KO mice and compared their secretion of the cytokine in response to SEA. As shown in Fig. 2⇓, CD4⁺ cells isolated from KO animals retained the ability to secrete IL-4, albeit in lower amounts than equivalent populations from WT mice. In contrast, whereas LN cells from WT animals failed to produce appreciable amounts of IFN-γ, cells from IL-4Rα KO mice secreted high levels of the cytokine when stimulated with SEA. Again, this altered cytokine profile was retained in the CD4⁺ T cells purified from these populations (Fig. 2⇓). IL-4/IFN-γ cytokine secretion patterns similar to those described above in IL-4Rα KO mice were also obtained when purified CD4⁺ T lymphocytes from infected Stat6 KO animals were stimulated in vitro under the same conditions (data not shown).

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FIGURE 2.

Purified CD4⁺ cells from infected IL-4Rα KO mice secrete IL-4 in response to SEA. Total mesenteric LN cells (3 × 10⁶/ml) from 8-wk infected WT and IL-4Rα KO animals (left panels) or sorted CD4⁺ lymphocytes (1.25 × 10⁶/ml) from the same LN populations (right panels) were stimulated with SEA (20 μg/ml). The latter cultures were supplemented with irradiated T-depleted splenocytes from naive BALB/c mice as APC (3.75 × 10⁶/ml). Culture supernatants were collected after 72 h and assayed for IL-4 and IFN-γ. No cytokine was detected in supernatants from cultures stimulated with medium alone. Bars represent the mean (±SD) of ELISA values and are representative of three experiments performed.

To analyze the cytokine production capacity of CD4⁺ cells at the single cell level, intracellular staining was performed on LN cells from infected IL-4Rα and Stat6 KO mice. The frequency of cytokine-positive cells in ex vivo LN populations from both WT and KO animals was generally <1% (data not shown). To facilitate the detection of cytokines by this procedure, we stimulated the LN populations with SEA for 72 h in vitro before intracellular staining. As shown in Table I⇓, the cytokine production pattern of the CD4⁺ lymphocytes detected by flow cytometry was found to closely resemble that observed in supernatants from SEA-stimulated cultures (Fig. 2⇑) in that the majority of the Th cells in the WT animals stained positively for IL-4 and not IFN-γ, while in KO mice the reverse pattern was observed.

Because the percentage of cells producing IL-4 was low in cultures from KO animals, it reopened the question raised previously (38), that this population represents an NK T cell rather than a conventional CD4⁺ lineage. Nevertheless, when the IL-4⁺ cells were double stained for Vβ8, the TCR family predominantly expressed by NK T lymphocytes (48), the Vβ8⁺/Vβ8⁻ ratios observed (∼0.2) were comparable with those of the equivalent IL-4⁺ populations from WT mice (Table II⇓) and very different from the ratio (∼1.2) found among NK T cells (48). Thus, the IL-4-producing cells arising in the absence of IL-4R signaling appear to be conventional CD4⁺ T lymphocytes.

IL-4 and IFN-γ are produced by distinct CD4⁺ subpopulations in LN cell cultures from infected IL-4Rα and Stat6 KO animals

In the intracellular cytokine staining experiments described above we noticed that the small population of IL-4⁺ CD4⁺ T cells arising in the KO mice appeared to be negative for IFN-γ. To more accurately classify the phenotype of these cells, we expanded the sorted CD4⁺ population by performing an additional stimulation with SEA in vitro and repeated the intracellular staining for IL-4 and IFN-γ as well as IL-2 and IL-5. As expected, the frequency of cytokine-producing cells increased as a result of stimulation, but to the same degree in WT and KO (Stat6) cultures (Fig. 3⇓). Importantly, the majority of the IL-4⁺ cells from the KO mice was negative for both IL-2 and IFN-γ, while the IFN-γ⁺ and IL-2⁺ populations were also predominantly IL-4 negative (Fig. 3⇓, two top right panels). Only minor subpopulations (∼3%) were found to be double positive for IL-4 and either IL-2 or IFN-γ. The same segregation in phenotype was observed between IFN-γ- and IL-5-producing cells (Fig. 3⇓, third right panel). In contrast, IL-2 was heavily expressed by the IFN-γ⁺ population, supporting their classification as Th1 cells (Fig. 3⇓, bottom right panel). As expected, WT cells expressed a predominantly Th2 cytokine pattern (IL-4⁺, IL-5⁺, IFN-γ⁻ , IL-2⁻) (Fig. 3⇓, left panels), while the staining profile for CD4⁺ cells from infected IL-4Rα KO animals (data not shown) was similar to that found in Stat6 KO cells. Again, these CD4⁺ populations from infected KO mice were negative for NK1.1 and displayed Vβ8⁺/Vβ8⁻ ratios indistinguishable from WT cultures (data not shown).

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FIGURE 3.

Single cell analysis reveals segregation of CD4⁺ lymphocytes from infected Stat6 KO mice into cells producing either IL-4/IL-5 or IFN-γ. Sorted CD4⁺ cells from mesenteric LN from 8-wk infected WT and Stat6 KO animals were stimulated twice with SEA plus APC and expanded in rIL-2-containing medium. After the second Ag stimulation, cells were rechallenged with plate-bound anti-CD3 for 7 h, in the presence of BFA for the last 5 h. Each sample was stained for surface expression of CD4, fixed, permeabilized, and then stained with the indicated pairs of anti-cytokine mAb. The FACS dot plots shown are gated on CD4⁺ cells and are representative of three experiments performed on three independent groups of infected mice. The same staining patterns were obtained with CD4⁺ T cells isolated at 6 wk postinfection (data not shown).

The absence of cells with a mixed Th0 cytokine production profile in IL-4R/Stat6 KO mice was a striking finding. To rule out the possibility that this pattern represents an artifact due to differences in cytokine production kinetics, we repeated these experiments using cells obtained at later time points after activation before intracellular staining. As shown in Fig. 4⇓, although the percentage of CD4⁺ cells staining positive for IL-5 or IFN-γ increased over time, no increase in the proportion of cells with a mixed phenotype (IL-4⁺ IFN-γ⁺ or IL-5⁺ IFN-γ⁺) was observed in cultures from either Stat6 or IL-4Rα KO animals over an additional 14-h period.

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FIGURE 4.

The frequency of CD4⁺ lymphocytes coexpressing Th1/Th2 cytokines in cultures from infected Stat6 and IL-4Rα KO animals does not increase with extended anti-CD3 mAb stimulation. The SEA-specific CD4⁺ cells from infected Stat6 KO mice analyzed in Fig. 3⇑ as well as parallel cell populations obtained from infected IL-4Rα KO animals were stimulated with plate-bound anti-CD3 mAb for 7, 14, or 21 h, and BFA was added for the last 5 h. The cells were then stained with anti-CD4 mAb, followed by either anti-IL-4 and anti-IFN-γ or anti-IL-5 and anti-IFN-γ mAb. The data indicate the percentage of total CD4⁺ cells with each cytokine secretion phenotype, where filled, and open symbols represent single and double cytokine producers, respectively. The experiment shown is representative of two performed on independent groups of mice.

To further characterize the Th phenotype of the IL-4-producing subpopulation, we double stained the cells with either anti-IL-5 or anti-IL-10 mAb. As shown in Fig. 5⇓ (top panels), in both WT and Stat6 KO cultures, most of the IL-5-producing cells were also positive for IL-4, as expected for Th2 lymphocytes. In cultures from both WT and, to a lesser extent, KO mice, large numbers of IL-4⁺ IL-5⁻ cells were also observed, as expected from the delayed production of IL-5 vs IL-4 by CD4⁺ cells from both WT and KO cultures (data not shown and Fig. 4⇑). In contrast, while most of the IL-4⁺ cells from WT animals stained positively for IL-10, only 26% of the IL-4⁺ cells from Stat6 KO mice were positive for this second Th2 cytokine (Fig. 5⇓, lower panels), which was produced with the same kinetics as IL-4 (data not shown).

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FIGURE 5.

Production of IL-4, IL-5, and IL-10 by Th2 cells from infected WT and Stat6 KO mice. The SEA-specific CD4⁺ cells from infected WT and Stat6 KO mice described in Fig. 3⇑ were analyzed for coexpression of Th2 cytokines after 7 h of stimulation with plate-bound anti-CD3 mAb. This time point was chosen based on preliminary kinetics studies that showed maximal IL-10 expression at this incubation time. With longer stimulation, the frequency of IL-10⁺ cells decreases, while the percentage of IL-5⁺ cells increases to the levels observed for IL-4⁺ cells.

Phenotypic stability of Th2 cells generated in the absence of IL-4R signaling

T cell cloning was performed as a final step in the characterization of the Th2 cells arising in parasite-infected Stat6 KO mice. The sort-purified and restimulated CD4⁺ cells shown in Figs. 3–5⇑⇑⇑ were cloned by limiting dilution in the presence of SEA plus IL-2. Equal numbers of clones with a Th1 (IL-4⁻ IFN-γ⁺) or a Th2 (IL-4⁺, IL-5⁺, IL-13⁺, IFN-γ⁻) phenotype were obtained. Consistent with the intracellular staining results (Fig. 5⇑), 3 of 10 of these Th2 clones produced IL-10. The SEA-specific Stat6-negative Th2 cells derived have been established as long-term clones and have maintained their cytokine profile over a 3-mo period of testing.

To test the stability of the Th2 population generated in Stat6 KO mice, sorted CD4⁺ cells were stimulated twice with SEA in the presence of different cytokine and anti-cytokine reagents previously reported to influence the differentiation of Th cells in WT animals. Because the IL-4⁺ cells from Stat6 KO mice develop in the presence of significant amounts of IFN-γ, we first tested the effects of anti-IFN-γ or anti-IL-12 mAb on the distribution of cells staining positive for IL-4 or IFN-γ, and no significant changes were observed (data not shown). Similarly, addition of either rIL-4, anti-IL-4, or anti-IL-4R mAb failed to influence the frequency of the different Th cell populations in cultures from both Stat6 KO and WT mice (Fig. 6⇓ and data not shown). Consistent with previously published findings that Th2 lymphocytes can develop the capacity to produce IFN-γ (49, 50), addition of rIL-12 induced a fraction of the IL-4⁺ cells in WT cultures to start coexpressing IFN-γ⁺ (Fig. 6⇓). Interestingly, rIL-12 triggered a quite different change in the cultures obtained from Stat6 KO animals. Thus, while the frequency of IFN-γ⁺ CD4⁺ lymphocytes increased, these cells were not double IL-4⁺ IFN-γ⁺ producers (Fig. 6⇓). Moreover, the number of IL-4⁺ CD4⁺ lymphocytes was dramatically reduced. The same cultures also showed marked reductions in levels of IL-5, IL-10, and IL-13 (not shown), indicating a wholesale decrease in the expression of Th2 cytokines. Thus, in the absence of IL-4R/Stat6 signaling, Th2 cells appear to be more susceptible to the destabilizing effects of IL-12. Nevertheless, the expression of IFN-γ by IL-12-treated cells from both WT and Stat6-deficient mice was transient, returning to almost baseline after one restimulation in the absence of that cytokine (Fig. 6⇓, right panels). Importantly, removal of IL-12 also resulted in recovery of IL-4 synthesis in cultures from Stat6 KO mice, confirming the phenotypic identity of the Th2 population present.

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FIGURE 6.

Differential effects of rIL-12 on Th2 lymphocytes in LN cultures from infected WT vs Stat6 KO mice. Aliquots of sorted mesenteric LN CD4⁺ cells from infected WT and Stat6 KO mice were cultured in the absence or presence of exogenous rIL-4 (20 ng/ml), anti-IL-4R mAb (M1, 20 μg/ml), or rIL-12 (10 ng/ml) during two Ag stimulations performed. Three-color staining for CD4, IL-4, and IFN-γ was then conducted after 7 h of anti-CD3 stimulation in the absence of cytokine or Ab (eight left panels). Intracellular cytokine staining was also performed on the same cultures after an additional Ag stimulation in the absence of added cytokines or Ab. A change in staining pattern was only observed when rIL-12 was removed (right panels). The FACS dot plots shown are gated on CD4⁺ cells and are representative of two experiments performed.

CD4⁺ T cells with IL-4⁺ IFN-γ⁻ phenotype can also be derived from uninfected Stat6 KO mice

Since it could be argued that our observation of Th2 cells in IL-4Rα- or Stat6-deficient animals depends on stimulation by helminth infection, we attempted to generate cells with the same phenotype from noninfected KO donors. Splenic CD4⁺ T lymphocyte populations were purified from uninfected Stat6 KO or WT control mice and stimulated with anti-CD3 mAb in the presence of irradiated splenocytes from IL-4 KO donors as APC. As expected, IL-4 was not detected in 72-h supernatants from either WT or Stat6 KO anti-CD3-stimulated cultures. However, when anti-IL-4R-blocking mAb was added to parallel cultures to prevent IL-4 consumption, significant amounts of IL-4 were found in both WT and Stat6 KO supernatants (Table III⇓). Indeed, comparable levels of the cytokine were produced by cells from both strains, suggesting that the higher frequency of IL-4-producing CD4⁺ cells seen in vivo in helminth-infected WT vs Stat6 KO mice may reflect the enhancing effect of prolonged IL-4/IL-4R interaction on Th2 development. Consistent with their known Th1 bias, lymphocytes from Stat6 KO mice produced higher levels of IFN-γ than control cells from WT animals when stimulated with anti-CD3.

Intracellular staining was performed on cultures from uninfected Stat6 KO mice to confirm the existence of the IL-4-producing CD4⁺ cells as well as to define their Th phenotype. In these experiments, CD4⁺CD62L^high lymphocytes were employed to limit the involvement of memory and to exclude NK T cells. After a single round of anti-CD3 stimulation, generally less than 0.5% of the CD4⁺ cells stained positive for IL-4. Nevertheless, after a second and third stimulation, the numbers of IL-4⁺ CD4⁺ cells increased to significant levels (2.5% and 7.9%, respectively) (Fig. 7⇓). These cells were also found to produce IL-5 (data not shown). In agreement with the intracellular staining results obtained in the analysis of CD4⁺ lymphocytes from S. mansoni-infected IL-4Rα and Stat6 KO mice, only a very small proportion of the IL-4⁺ CD4⁺ cells coexpressed IFN-γ, confirming the segregation of differentiating cells into Th1 or Th2 in the absence of IL-4R signaling. As expected, when similar anti-CD3 experiments were performed with CD4⁺CD62L^high lymphocytes from WT animals, IL-4⁺ cells could be detected in significant proportion (1/3 cytokine-producing cells) even after a single round of stimulation (data not shown).

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FIGURE 7.

Single cell analysis reveals segregation of CD4⁺ lymphocytes from uninfected Stat6 KO mice into cells producing either IL-4 or IFN-γ. Sorted naive CD4⁺CD62L^high cells from uninfected Stat6 KO mice were stimulated once, twice, or three times for 3 days with soluble anti-CD3 mAb in the presence of irradiated splenocytes from IL-4 KO mice and in between expanded in rIL-2-containing medium. Three-color staining for CD4, IL-4, and IFN-γ was preformed on day 7 after each anti-CD3 stimulation. The FACS dot plots shown are gated on CD4⁺ cells and are representative of two experiments performed.