Abstract
The concept that IL-4 is the primary signal for Th2 lymphocyte differentiation has recently been put in doubt by studies in which the production of Th2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell analysis that CD4+ lymphocytes with a classical Th2 phenotype (IL-4+, IL-5+, IFN-γ−, IL-2−) develop in significant numbers in helminth-infected mice deficient in either IL-4R α-chain or Stat6. While an expanded population of Th1 (IL-4−, IL-5−, IFN-γ+, IL-2+) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokine pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected by in vitro neutralization of endogenous IL-4 or IFN-γ. Nevertheless, while addition of exogenous rIL-12 resulted in transitory IFN-γ production by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 synthesis was preserved in the former, but temporarily ablated in the latter cells. Importantly, IL-4+ IFN-γ− and IL-4− IFN-γ+ populations similar to those arising in helminth-infected Stat6-deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4+CD62Lhigh lymphocytes from uninfected mice of the same strain. Together, the results of these single cell analysis experiments demonstrate that IL-4R/Stat6 signaling, while influencing the final frequency of Th2 lymphocytes, is not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once they have emerged.
The differentiation of CD4+ T cells into Th1 and Th2 subsets is an important determinant of effector function in the immune system. A number of factors have been shown to control the direction of Th1/Th2 development, the most critical being the cytokine microenvironment during initial T cell priming (reviewed in Ref. 1). Thus, in the presence of IL-12, which triggers the Stat4 signaling pathway, CD4+ lymphocytes acquire a Th1 cytokine secretion profile (2, 3). Conversely, in the presence of IL-4, the Stat6 pathway is triggered, resulting in the development of cells with a Th2 cytokine phenotype (4, 5, 6).
As part of the above concept, it has been proposed that the early induction of IL-12 or IL-4 from a non-T cell source provides a key determinant of Th1/Th2 induction (reviewed by O’Garra (7)). Thus, the triggering of IL-12 from dendritic cells or macrophages has been shown to provide a strong Th1 bias to the CD4+ T cell response in both in vitro and in vivo systems (8, 9, 10). In the opposite direction, a number of different cell types, including mast cells (11), basophils (12), NK1.1+ T cells (13), γδ T cells (14), and eosinophils (15), can serve as a source of early IL-4. Nevertheless, none of these cell types appear to be obligatory for Th2 induction (16, 17, 18, 19, 20) and, in some cases, may even inhibit Th2 development (21). Instead, there is now abundant evidence that Th2 differentiation can be supported solely by IL-4 produced by Ag-primed (22, 23) or memory CD4+ lymphocytes (24). Furthermore, recent experiments using cytokine- or STAT-deficient mice indicate that Th1 or Th2 responses can emerge even in the absence of IL-12/IL-4 or STAT signaling. Thus, in a number of in vivo systems, significant Th2 cytokine production can be detected in mice genetically deficient in IL-4 (25, 26, 27, 28). In addition, while Stat4 knockout (KO)3 (1) mice are defective in Th1 cytokine production, double Stat4/Stat6 KO animals are able to mount significant Th1 responses in vitro (29), indicating the presence of a Stat4-independent pathway for Th1 development.
During this multistep process in which naive cells acquire either Th1 or Th2 phenotype, CD4+ T lymphocytes that produce a mixture of Th1 and Th2 cytokines also may arise (30, 31). However, it is not clear whether these cells of intermediate (Th0) phenotype represent an obligatory differentiation step (32) or are in themselves final effector stages (33). Moreover, if these cells do indeed play a role as transitional intermediates in Th1/Th2 development, the extent to which they are dependent and/or susceptible to IL-4/IL-12 differentiation signals is poorly defined.
We have been studying the regulation of Th2 development in an in vivo model involving infection with the helminth Schistosoma mansoni. At 5 wk postinfection, the parasite begins to deposit in host tissues large numbers of eggs that serve as a potent and persistent stimulus of CD4+ T cell activation. This response is dominated by a Th2 cytokine secretion profile characterized by high levels of IL-4, IL-5, IL-10, and IL-13, and importantly, fails to result in appreciable IL-12 production (34, 35). Previous studies revealed that the schistosome-induced Th2-type response is partially retained in IL-4-deficient mice (26), raising the possibility of IL-4-independent Th2 induction or the involvement of alternative IL-4R ligands. To distinguish between these two hypotheses, we recently studied Th2 cytokine production in S. mansoni-infected IL-4Rα KO animals. These mice displayed Th2 (IL-5, IL-10, and IL-13) as well as Th1 cytokine levels comparable with those found in IL-4 KO animals (36). In addition, in the data of Kaplan et al. (37) on S. mansoni-infected Stat6 KO mice, residual Th2 cytokine expression is apparent, although the significance of these low level responses was not determined.
In the present study, we have formally analyzed at the single cell level the source of the residual Th2 cytokine production occurring in schistosome-infected IL-4Rα as well as Stat6 KO animals. Our findings establish that the observed lymphokines originate from conventional CD4+ Th2 cells. Interestingly, these Th2 lymphocyte populations arise simultaneously with Th1 cells generated as a result of the absence of cross-regulation by IL-4. Nevertheless, lymphocytes with a mixed Th1/Th2 cytokine secretion profile are only rarely observed in the KO mice, suggesting that Th0 cells are not obligatory precursors in Th subset differentiation. Because similarly polarized Th2 and Th1 populations could be generated from uninfected Stat6 KO mice by repeated in vitro polyclonal stimulation of naive CD4+ cells, their occurrence is not dependent on stimulation by helminth exposure. Taken together, these observations are consistent with a model of Th1/Th2 differentiation in which the two subsets arise independently of each other and in which IL-4R/Stat6 signaling is not essential for Th2 development, but nevertheless plays a major role in determining the final frequency of cells that express a Th2 lymphokine secretion profile.
Materials and Methods
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+CD62Lhigh 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+CD62Lhigh 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
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+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+CD62Lhigh 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.
Results
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.
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).
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.
Distribution of IL-4 and IFN-γ-producing CD4+ cells in LN cultures from infected WT and KO micea
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.
Vβ8 usage is not increased in IL-4+ CD4+ populations in LN cultures from infected IL-4Rα and Stat6 KO micea
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).
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.
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).
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.
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.
CD4+ T cells from uninfected WT as well as Stat6 KO mice produce IL-4 in response to anti-CD3 stimulation in vitroa
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+CD62Lhigh 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+CD62Lhigh 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).
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+CD62Lhigh 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.
Discussion
The current paradigm for Th subset differentiation postulates that the balance of IL-4/IL-12 in the microenvironment during T cell priming determines by an instructional mechanism the outcome of CD4+ lymphocyte development. A number of recent observations suggest that this model may be oversimplified. An important discrepancy concerns the unexpected occurrence of Th2 cytokine responses in mice deficient in IL-4 or IL-4R signaling (19, 25, 38). In the present study, we have utilized single cell analysis to formally investigate the basis of this phenomenon in both in vivo and in vitro experimental systems involving helminth infection or polyclonal stimulation with anti-CD3, respectively. Our results establish that conventional Th2 cells can indeed develop in the absence of IL-4R signaling and are consistent with a model of CD4 differentiation in which a major function of this pathway is to both increase the frequency of and stabilize emerging Th2 populations.
Although Th2 cytokine production by both IL-4 and IL-4Rα KO mice has been observed in a number of experimental systems, it was possible that such responses originate from Th0 lymphocytes or unconventional CD4+ T cells (e.g., NK T cells) (25, 38). The data presented in this work demonstrate that the CD4+ IL-4-producing cells that arise in schistosome-infected IL-4Rα and Stat6 KO animals are conventional Th2 cells based on several criteria. First, these cells possess a pure Th2 (IL-4+, IL-5+, IFN-γ−, IL-2−) cytokine as opposed to mixed Th1/Th2 cytokine secretion profile of Th0 cells and NK T cells. Moreover, their response to schistosome Ag is class II restricted and they do not selectively display the TCR Vβ8 chain characteristically expressed by NK T cells. One unusual characteristic of this population, however, is the low frequency of cells expressing IL-10. This finding is, nevertheless, consistent with recent work demonstrating a role for IL-4 in the generation of IL-10-producing CD4+ cells (51).
Because the observed in vivo generation of CD4+ cells with a Th2 lymphokine pattern occurred in schistosome-infected IL-4Rα and Stat6 KO mice, it could be argued that the results obtained are uniquely dependent on this helminth stimulus. Nevertheless, it should be noted that residual Th2 cytokine synthesis in mice deficient in IL-4 or IL-4R signaling has been documented now in response to a variety of different helminth and protozoan infections, as well as to injected soluble protein Ag (25, 26, 27, 28, 52, 53, 54, 55). More importantly, as demonstrated in this work (Fig. 7⇑), CD4+ cells with a Th2 lymphokine profile can be generated in vitro from naive Th precursors from uninfected Stat6 KO mice by repeated polyclonal stimulation with anti-CD3. Thus, it is clear that Th2 development in the absence of IL-4R/Stat6 signaling is not an isolated phenomenon dependent on a particular form of T cell triggering.
In addition to low numbers of Th2 cells, the infected IL-4Rα and Stat6 KO mouse strains also developed high numbers of Th1 (IFN-γ+ IL-2+) cells, as predicted from the absence of IL-4R-dependent cross-regulation by IL-4 (29, 56, 57). Interestingly, however, this induction of Th1 cytokine activity occurs despite the absence of detectable IL-12 in vitro (<30 pg/ml of p40) and, moreover, has also been demonstrated to take place in schistosome-infected double IL-4/IL-12 KO animals (E. Patton, L. Rosa Brunet, and E. Pearce, personal communication). Nevertheless, we failed to detect significant numbers of cells with a mixed Th0 cytokine secretion pattern in the CD4+ populations from the same infected IL-4Rα or Stat6 KO mice from which both Th1 and Th2 cells were recovered. Thus, in this situation in which neither IL-4R signaling nor IL-12 production occurs, the potent T cell activation provided by S. mansoni infection leads directly to emergence of cells with a pure Th1 or Th2 phenotype. The above conclusion is supported by the results of Reiner and colleagues, who found that in the presence of pharmacologic epigenetic derepression, anti-CD3-stimulated CD4+ T cells from Stat6 KO mice develop into distinct populations expressing IFN-γ or IL-4 (58). As shown in the present study, a similar segregation into IL-4- or IFN-γ-producing cells can be achieved in the absence of pharmacologic derepression by repetitive anti-CD3 stimulation of CD4+CD62Lhigh cells from uninfected Stat6 KO mice (Fig. 7⇑).
The results of the present study have several implications for the mechanism of Th subset development. Most importantly, they formally confirm that generation of Th2 cells can indeed take place in the absence of IL-4R/Stat6 signaling, arguing against models in which this triggering event is essential for instructing naive cells to develop a Th2 cytokine secretion profile. Our findings are equally inconsistent with models of Th differentiation in which cytokine genes are expressed independently of each other at the single cell level, since we fail to detect significant numbers of cells with a mixed Th0 phenotype. Finally, the same observations imply that Th0 cells are not necessary precursors for Th1 and Th2 lymphocytes, and instead may represent a later stage of CD4+ cell differentiation. Although these conclusions are based on results obtained with mice deficient in IL-4R signaling, findings from previous studies in IL-4R/Stat6-sufficient animals suggest that they are generally applicable. For example, in analyses of cytokine gene expression at early time points during differentiation of transgenic CD4+ T lymphocytes, most cells were found to express either Th1 or Th2 cytokine mRNAs, with Th0 cells being rare (59). The generation of Th1 and Th2, in the absence of Th0 cells, has also been noted in related studies involving intracellular staining of in vitro differentiating transgenic CD4+ lymphocytes (60), as well as in polyclonally activated human peripheral blood T cells (61).
Nevertheless, stable clones with a mixed Th0 cytokine profile are frequently encountered in both mouse and human studies (30, 31, 62, 63). We speculate that such clones, while stable in vitro, do not persist in vivo due to the continuing influence of the cytokine microenvironment. Based on our finding that Th0 cells are rare in cultures from animals with defective IL-4R signaling, we argue that their development is also dependent on cytokine cross-talk, but that this step occurs after the initial generation of cells with a pure Th1 and Th2 phenotype.
While, as demonstrated in this study, IL-4R signaling is not essential for Th2 induction, this pathway is clearly involved in determining the outcome of CD4+ T cell differentiation in an IL-4R-sufficient setting. Thus, two key questions raised by our findings concern the role of IL-4 in the acquisition and maintenance of the Th2 phenotype and how Th2 cells are generated in its absence. Several possible, but not mutually exclusive, explanations exist. One alternative is that there is a small but significant probability that commitment to the Th2 phenotype may occur as a result of epigenetic derepression in a population of cells stimulated solely through their TCR. That probability may vary depending upon the conditions of such stimulation (e.g., high vs low Ag concentration (64, 65); level of B7/CD28 costimulation (66); presence of BCL-6 expression (67)), but is markedly augmented in the presence of IL-4. An additional mechanism by which IL-4 may increase the frequency of cells with a Th2 phenotype is by restricting Th1 development. This concept is supported by the observation of Kaplan et al. (29), that while Stat4 KO mice are defective in CD4-dependent IFN-γ production, such responses are restored in double Stat4/Stat6 KO animals in which IL-4R signaling is now ablated. Our observation of persistent Th1 cells in schistosome-infected IL-4Rα and Stat6 KO mice in the absence of significant IL-12 production (Fig. 3⇑) is also consistent with the latter hypothesis.
A second possible mechanism that would explain IL-4R-independent acquisition of the Th2 phenotype is that this process normally depends upon both TCR-mediated stimulation and IL-4Rα triggering, but that in its absence, other as yet undefined signals exist that can functionally substitute for that provided by IL-4. One important version of this hypothesis is that the absence of IL-12 in itself provides the critical secondary element required for Th2 differentiation. Indeed, lack of IL-12 induction is a common feature in all of the settings discussed above, in which residual Th2 cytokine synthesis was observed in mice deficient in IL-4 or IL-4R signaling.
In a third model of Th2 differentiation, TCR engagement is the major factor that leads to competence for the transcription of Th2 cytokines, but IL-4 acts after this initial event. That IL-4 acts purely as a selective agent and leads to the preferential growth or survival of cells that have already committed to the Th2 phenotype is highly unlikely. Although Th1 and Th2 cells do have different growth properties and different responsiveness to IL-12 and IL-4, one can employ tissue culture conditions in which there is no apparent growth advantage of the responding cells and still find that IL-4 favors the development of Th2 cells from naive precursors (68). Instead, we favor the hypothesis that autocrine IL-4R signaling plays a major role in stabilizing cells with a Th2 phenotype, protecting them against IL-12-mediated inhibition, as exemplified by the experiment shown in Fig. 6⇑. IL-4R signaling may also promote Th2 stabilization through its involvement in IL-10 induction (Fig. 5⇑), known to suppress Th1 cytokine expression (69). Indeed, priming Th1 or Th2 cells in the absence of their differentiative cytokines may render them generally more labile to subsequent alterations in the cytokine microenvironment because they have not been locked in by genetic remodeling. This system may be asymmetrical with paracrine IL-12 acting early in CD4+ differentiation to enhance Th1 generation, and in its absence, autocrine IL-4 acting at a later stage to promote and stabilize Th2 development.
Regardless of the precise mechanisms involved, it is clear that IL-4R/Stat6-independent Th2 development exists and may reflect a more general pathway for CD4 T cell subset diversification. Further studies on the regulatory effects of IL-4 and IL-12 on early Th1/Th2 populations generated from mice deficient for these two cytokines should offer a powerful approach for both delineating this process and assessing its overall contribution to CD4+ lymphocyte differentiation.
Acknowledgments
We thank S. Barbieri and C. Eigsti for assistance with FACS sorting, and C. Watson for maintaining the colony of Stat6 KO mice. We also thank Dr. D. Sacks for helpful discussion, and Drs. G. Milon, T. Nutman, D. Sacks, and G. Yap for critically reading the manuscript.
Footnotes
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↵1 D.J. and M.C.K. contributed equally to this work.
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↵2 Address correspondence and reprint requests to Dr. Dragana Jankovic, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 9000 Rockville Pike, MD 20892-0425. E-mail address: djankovic{at}niaid.nih.gov
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↵3 Abbreviations used in this paper: KO, knockout; BFA, brefeldin A; LN, lymph node; SEA, soluble egg Ag; WT, wild type.
- Received November 8, 1999.
- Accepted January 13, 2000.
- Copyright © 2000 by The American Association of Immunologists