IL-3 Receptor Expression on Activated Human Th Cells Is Regulated by IL-4, and IL-3 Synergizes with IL-4 to Enhance Th2 Cell Differentiation

Interleukin-3, a pleiotropic cytokine also known as multi-CSF, stimulates the proliferation, survival, and differentiation of multipotent hematopoietic stem cells. Unlike other CSFs, IL-3 has broader target range and ability to promote the growth of progenitors of myeloid lineage (1). IL-3 is mainly produced by activated T lymphocytes; however, other cells, such as NK cells, cells of myeloid lineage, astrocytes, thymic epithelial cells, and keratinocytes, are also known to secrete IL-3 (2–9). IL-3R is a membrane-bound heterodimer of two subunits, a cytokine-specific α-subunit, and a common β-subunit which is also shared by IL-5 and GM-CSF, and these three cytokines form a discrete group known as the β common–chain cytokine family. The α-subunit of IL-3R determines the ligand specificity and binds to IL-3 with low affinity, whereas the β common subunit does not bind to IL-3 itself, but coexpression of this subunit is critical for the formation of the high-affinity receptor for IL-3 (10). Most of the biological functions of IL-3 are mediated by the activation of downstream JAK2–STAT5 pathway (11).

In addition to hematopoiesis, the role of IL-3 has also been implicated in other biological processes, such as angiogenesis (12) and proliferation and survival of neuronal progenitor cells (13). Recent studies from our laboratory have demonstrated the novel role of IL-3 in regulation of bone remodeling. IL-3 inhibits mouse osteoclast differentiation and diverts the osteoclast precursors toward macrophage lineage (14). IL-3 also inhibits human osteoclast differentiation and diverts the cells toward dendritic cell lineage (15). Importantly, we have demonstrated that IL-3 has anti-inflammatory and immunomodulatory properties and protects bone and cartilage loss in vivo in arthritic mice by upregulating regulatory T (Treg) cells (16, 17). These results suggest that IL-3 has a regulatory role in Th cells.

Although activated T lymphocytes are the major source of IL-3, there is currently no convincing evidence to show that IL-3 has any direct or indirect influence on the development and functions of human T lymphocytes. IL-3 was initially purified from WEHI-3B leukemia cell line (1), and IL-3 showed its ability to induce 20-α hydroxyl-steroid-dehydrogenase (20-A-SDH) in Thy1.2⁺ and Lyt-1⁺, 2⁻ spleen cells in mice (18, 19). Earlier Thy1.2 and 20-A-SDH were considered as T lymphocyte–specific markers. However, this claim was refuted because it was established that Thy-1 Ag was not specific to T lymphocytes and its expression was also observed on macrophages and polymorphonuclear cells (20). In addition, ¹²⁵I-labeled IL-3 failed to bind to thymus or spleen lymphocytes, supporting the fact that it is unlikely for IL-3 to induce 20-A-SDH in T lymphocytes unless this is done through some ambiguous process (21).

We have recently shown that mice Treg cells express IL-3R, and IL-3 in the presence of TGF-β1 and IL-2 enhances the differentiation of naive Th cells into induced Treg cells in a STAT-5–dependent manner (17). All these observations were made in mice, and there are no reports on the role of IL-3 in development and functions of human Th cells. Sato et al. (22) analyzed human hematopoietic cells for IL-3R expression and found that CD3⁺ T cells lack IL-3R expression. The examination of specific cytokine receptor expression on the cell surface is crucial for understanding cytokine function. In the current study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in the effector functions of Th cells.

We found that resting human Th cells constitutively express IL-3R at transcript and intracellular protein levels but not on the surface. The surface expression of IL-3R was detected only after antigenic stimulation, which was significantly increased by IL-4. We further demonstrate that IL-3R⁺ Th cells show characteristics of Th2 cell lineage and IL-3 enhances their differentiation. Interestingly, we observed that IL-3R–expressing and IL-3–secreting Th cells were high in allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated mainly by IL-4, and IL-3 regulates the effector functions of Th2 cells. All these results suggest that IL-3 may play an important role in regulating allergic immune responses.

Peripheral blood samples from healthy donors and clinically proven allergic patients were collected by a professional phlebotomist after informed consent. All procedures involving the use of human samples were conducted with the approval of Institutional Ethics Committee of National Centre for Cell Science, Pune, India.

Anti-CD3 (HIT3a), anti-CD4 (OKT-4/SK3), anti-CD123 (9F5 and 6H6), anti-CD45RA (H100), anti-CCR4 (1G1), anti-CRTH2 (BM16), anti–p–STAT-5 (47/Stat5/pY694), anti–IL-2 (MQ1-17H12), anti–IL-4–purified and –tagged (MP4-25D2 and 8D4-8), anti–IFN-γ–purified and –tagged (B27), anti–IL-5 (TRFK-5), anti–IL-13 (JES10-5A2), and anti–IL-3–purified (BVD8-3G11) Abs were purchased from BD Biosciences. Anti–FOXP3 (259D), anti–IL-9 (MH9A4), anti–IL-3 (BVD3-1F9), anti–IL-17 (BL168), anti–IL-10 (JES3-9D7), purified anti-CD3 (HIT3a), and anti–T-bet (4B10) Abs were from BioLegend. Anti–GATA-3 (TWAJ) and anti-RORγt (AFKJS-9) Abs were from eBiosciences. For immunofluorescence staining, anti-CD123 (V-18) primary Ab was purchased from Santa Cruz Biotechnology and secondary TRITC-tagged Ab was from Bangalore Genei. Anti-GAPDH (rabbit polyclonal) Ab was from Sigma-Aldrich. Dynabeads Human T-Activator CD3/CD28 for T cell expansion and activation, CSFE, cell stimulation mixture, and FOXP3 staining buffers were obtained from Thermo Fisher Scientific. BD Cytofix/Cytoperm and Perm Buffer III, used for intracellular cytokines and p–STAT-5 staining, respectively, were obtained from BD Biosciences. Recombinant human (rh) cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, and TGF-β1, were purchased from R&D Systems. X-VIVO 15 serum-free media was from Lonza.

PBMCs were isolated using the Ficoll-Paque density gradient centrifugation method. Heparinized blood was mixed with an equal volume of Dulbecco’s PBS (Ca²⁺ and Mg²⁺ free) and carefully layered on the top of Ficoll in a 15-ml centrifuge tube. After centrifugation at 1800 rpm for 30 min, the upper layer, containing plasma and platelets, was discarded, and the mononuclear cell layer (buffy coat) was then carefully removed and transferred into a new centrifuge tube. Cells were washed, and CD4⁺ T cells were isolated using BD IMag Human CD4 T Lymphocyte Enrichment Set-DM as per manufacturer’s instructions.

Enriched CD4⁺ T cells were labeled with fluorochrome-tagged Abs against CD4, CD25, and CD45RA, and naive Th cells (CD4⁺CD25⁻CD45RA⁺) were sorted using a BD FACSAria SORP III flow cytometer. The purity of the naive Th cell fraction was routinely determined, and it was >95%. Naive Th cells in a 96-well plate were activated using human anti-CD3– and anti-CD28–coated beads in a ratio of 1:4 (cell/bead) and cultured in X-VIVO 15 serum-free medium for 4–6 d depending on experimental conditions.

For differentiation of Th1, activated Th cells were incubated with rhIL-12 (50 ng/ml) and anti–IL-4 Ab (10 μg/ml) for 4 d. For Th2 differentiation, activated Th cells were incubated in presence of rhIL-4 (50 ng/ml) and anti-human IFN-γ Ab (10 μg/ml). On day 3, activation beads were removed, and cells were replated with fresh media and factors and further cultured for 3 d. To polarize naive Th cells into Treg cells, rhTGF-β1 (5 ng/ml) and rhIL-2 (10 ng/ml) were added, and cells were cultured for 4 d. For differentiation of Th17 cells, naive Th cells were stimulated with plate-bound anti-CD3 (5 μg/ml) in presence of rhIL-1β (50 ng/ml), rhIL-23 (50 ng/ml), and anti–IL-4 (10 μg/ml) and anti–IFN-γ (10 μg/ml) Abs. On day 3, cells were washed and expanded under the same culture conditions without anti-CD3 for three more days. Cells were stimulated with eBioscience PMA/Ionomycin Cell Stimulation Cocktail for 6 h, and intracellular cytokine levels were determined by FACS.

For surface staining, cells were incubated with Abs against respective surface markers for 30 min at 4°C in staining buffer (Ca²⁺- and Mg²⁺_­-free Dulbecco’s PBS containing 2% FBS and 0.09% sodium azide), washed, and fixed in 1% paraformaldehyde for 20 min. For intracellular IL-3R and transcription factors staining, cells were first stained for surface markers followed by fixation and permeabilization using the eBioscience FOXP3 Staining Kit for 45 min and incubated with Abs against intracellular proteins for 45 min in Perm Buffer at 4°C. For intracellular cytokine staining, cells were stimulated with PMA/Ionomycin Cell Stimulation Cocktail for 6 h. Cells were harvested, surface stained wherever required, fixed, and permeabilized using BD Cytofix/Cytoperm Kit for 30 min followed by incubation with Abs against intracellular proteins for 45 min in Perm Buffer at 4°C. To check the phosphorylation status of STAT-5, cells were first stimulated with IL-2 or IL-3 for specific time points and fixed using 2% paraformaldehyde for 10 min at 37°C. Cells were washed and permeabilized using Perm Buffer III (BD Biosciences) for 30 min at 4°C. Cells were again washed twice with stain buffer and incubated with anti–p–STAT-5 Ab for 45 min at 4°C. For cell proliferation assay, cells were labeled with CFSE (2.5 μM) at 37°C for 20 min, followed by serum quenching and washing prior to cell culture. Stained cells were assessed by using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed by FlowJo X 10.0.7 software (Tree Star).

Cells were surface stained with primary Ab against IL-3R for 2 h in staining buffer on ice, followed by washing and incubation with TRITC-conjugated secondary Ab for 45 min. For intracellular receptor staining, cells were fixed and permeabilized with FOXP3 Staining Kit, washed, and stained with primary and secondary Abs. Cells were cytospun on glass slides, and coverslips were mounted using DAPI containing mounting media (Santa Cruz Biotechnology). Images were acquired using a confocal microscope (Leica Imaging Systems) at 63× magnification.

RNA was isolated using the TRIzol method (Invitrogen). For RT-PCR analysis, cDNA synthesis and PCR amplification was done using Superscript One-Step RT-PCR System (Invitrogen). A total of 1 μg RNA was mixed with Superscript II RT and Platinum Taq DNA Polymerase, 2× reaction mix, and gene-specific primers. cDNA was synthesized at 45°C for 30 min, and the target sequence was amplified for 35 cycles; each cycle consisted of denaturation at 94°C (30 s), annealing at 60°C (30 s), and extension at 72°C (30 s).

For real-time PCR, cDNA was synthesized using a cDNA Synthesis Kit (Invitrogen). A 10-μl reaction mixture containing SYBR Green and 10 pmol of each primer was set using the StepOnePlus system (Applied Biosystems). The PCR program consisted of one cycle of denaturation at 95°C for 10 min and 40 cycles of denaturation at 95°C for 15 s, primer annealing, and extension at 60°C for 60 s, followed by melt curve analysis. Each reaction was run in duplicates. Data were analyzed for fold difference using comparative 2^−ΔΔCT method. The primers used were as follows: IL-3R gene forward, 5′-ACGACAAACTTATCTGTG-3′ and reverse, 5′-TTGCCAACAGGCGTCAAC-3′; and GAPDH gene forward, 5′-CCTGCACCACCAACTGCTTAG-3′ and reverse 5′-TGAGTCCTTCCACGATACCAA-3′.

Cells were lysed using RIPA buffer containing protease inhibitor mixture, and protein concentration was measured using a Bicinchoninic Acid Protein Assay Kit. A total 30 μg of protein was subjected to 10% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane for immunoblot analysis. Blots were blocked for 1 h in nonfat dry milk in TBST buffer and incubated with primary Ab overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary Ab, and labeled proteins were detected using ECL reagents (Amersham Biosciences). Relative intensities of protein bands were analyzed by densitometry using ImageJ software.

Levels of IL-4 in culture supernatant was determined by fluorescent bead–based technology using Cytometric Bead Array (CBA) Flex Set as per manufacturer’s instructions (BD Biosciences). Fluorescent signals were measured on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using BD FCAP Array Software.

The skin prick test was performed at Sai Allergy Asthma Eye Children Hospital, Pune, India. Patients suffering from allergen-induced acute bronchitis, having shortness of breath, and chest tightness were screened for house dust mite allergen sensitivity using a skin prick test kit (CREDISOL Skin Test Allergens, Creative Diagnostic Medicare, Mumbai, India). Histamine (10 mg/ml) and normal saline were used as a positive control and negative control, respectively. Patients showing wheal size ≥6 mm were used in the study.

Results are represented as mean ± SEM. Statistical significance was calculated using the Student t test or one-way ANOVA with a subsequent post hoc Dunnett or Tukey test. The significance values were defined as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

To investigate whether human Th cells express IL-3R, we isolated PBMCs from healthy donors, and cells were stained for the expression of CD3, CD4, and IL-3R. Lymphocytes were gated, and expression of IL-3R was determined on CD3⁺CD4⁺ T cells by FACS. We found that human Th cells lack surface expression of IL-3R (Fig. 1A–C). In this experiment, monocytes were used as a positive control, which showed 53.9% positivity for IL-3R expression (Fig. 1D, 1E). Fig. 1F shows the average percentage of IL-3R⁺ cells in total monocytes and Th cells. These results suggest that human Th cells lack surface expression of IL-3R.

Because Th cells in PBMCs lack surface expression of IL-3R, we further analyzed the expression of the IL-3R transcript by RT-PCR and total protein by Western blotting. Contrary to the lack of surface expression, there was constitutive expression of IL-3R at mRNA (Fig. 2A, 2B) and protein levels (Fig. 2C) in peripheral blood–derived Th cells. FACS (Fig. 2D) and immunofluorescence staining (Fig. 2E) also confirmed the absence of surface expression of IL-3R on Th cells. However, IL-3R was clearly seen at intracellular levels (Fig. 2D, 2E).

Responsiveness of naive Th cells to various stimuli, such as cytokines and chemokines, is regulated by T cell activation signals. Therefore, we studied the expression of IL-3R in activated Th cells. To address this, naive Th cells were activated with anti-CD3 and anti-CD28, and IL-3R expression was evaluated at mRNA and protein levels. We observed that there was no change in both mRNA and total protein levels after TCR stimulation (Fig. 2F–H). However, TCR stimulation increased the percentage of IL-3R⁺ Th cells at 48 h (1.79%), and a significant increase in IL-3R was seen at 96 h (9.36%) as compared with inactivated Th cells (0.067%) (Fig. 2I, 2J). We further evaluated the median fluorescence intensity (MFI) and noted that IL-3R molecules on the cell surface were also increased at both 48 and 96 h postactivation, as shown by increase in MFI (Fig. 2K). These results indicate that IL-3R is constitutively expressed both at gene and intracellular protein levels in the resting Th cells and its surface expression is dependent on TCR stimulation.

Binding of IL-3 to its cognate receptor activates multiple downstream signaling pathways, including STAT-5 (11). We reasoned that if resting human Th cells do not express IL-3R on their surface and only activated Th cells do, then, in response to IL-3 treatment, activated Th cells would show STAT-5 phosphorylation, whereas resting Th cells would not respond to IL-3. To test this, resting Th cells were treated with IL-3 for 15, 30, and 45 min. IL-2, a known activator of STAT-5 in T lymphocytes, was used as a positive control (23). After IL-3 and IL-2 treatment, cells were intracellularly stained with anti–p–STAT-5 Ab. MFI of p–STAT-5 was calculated and normalized to MFI of p–STAT-5 in untreated cells. As expected, there was no STAT-5 phosphorylation in resting Th cells after IL-3 treatment, whereas IL-2 p–STAT-5 at all the time points (Fig. 3A, 3B). To dissect whether activated Th cells respond to IL-3, naive Th cells were activated for 4 d with anti-CD3 and anti-CD28 and treated with different concentrations of IL-3 for 15 min. As shown in Fig. 3C and 3D, activated Th cells with IL-3 stimulation significantly increased the percentage of Th cells with p–STAT-5 at 50 and 100 ng/ml concentrations, and MFI of p–STAT-5 was also upregulated at all the concentrations of IL-3 (Fig. 3E). These results suggest that TCR-mediated signaling controls the IL-3 responsiveness in human Th cells through STAT-5.

Cytokine-mediated regulation of IL-3R has previously been reported in other cells, such as endothelial cells, monocytes, and eosinophils (24–26). Therefore, we speculated whether various cytokines could influence the expression of IL-3R on Th cells. Accordingly, the effect of cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, and TGF-β1, on IL-3R expression was determined. The percentage of IL-3R⁺ cells in activated Th cells (6.21%) was significantly enhanced by IL-4 (44.5%) and IL-7 (18.4%) (Fig. 4A, 4B). All other cytokines showed no significant effect on IL-3R expression. IL-4 not only enhanced the percentage of IL-3R⁺ cells but also increased its MFI, whereas IL-7 enhanced only the percentage and showed no significant effect on MFI (Fig. 4C). IL-4 also increased IL-3R expression at total protein levels as confirmed by Western blotting (Fig. 4D). The increase in number of IL-3R–expressing Th cells after IL-4 and IL-7 treatments indicate that a functional link between IL-4, IL-7, and IL-3 might exist, and IL-4 and IL-7 may have a potential role in regulating IL-3–mediated adaptive immune responses in Th cells.

It is documented that IL-7–primed human T cells secrete IL-4 (27). This raises the possibility of IL-4–mediated cross-regulation of IL-3R in cells treated with IL-7. Therefore, to test whether IL-4 and IL-7 could cross-regulate each other’s secretion in activated Th cells, we neutralized IL-7 in IL-4–treated condition and vice versa. It was found that neutralization of IL-7 in IL-4–treated cells or neutralization of IL-4 in IL-7–treated cells had no effect on the proportion of IL-3R⁺ Th cells (Fig. 4E, 4F) and MFI (Fig. 4G). These results clearly show that both cytokines regulate IL-3R expression independently. To substantiate this finding, the IL-4 level was measured in activated as well as IL-7–treated culture conditions. In this experiment, naive Th cells activated in presence of exogenous rhIL-4 were used as a positive control. No IL-4 was detected in activated or IL-7–treated Th cells (Fig. 4H).

Our results indicated that IL-4 and IL-7 enhance IL-3R⁺ Th cells. Because these cytokines are known to be involved in the differentiation of Th2 cells, we speculated that IL-3R is expressed on Th2 cells. Also, the expression pattern of IL-3R on other human Th subsets is not yet defined. Therefore, we differentiated naive Th cells into different Th subsets, such as Th1, Th2, Treg, and Th17 cells, as described in 2Materials and Methods. As shown in Fig. 5A, the percentage of Th cells expressing IL-3R was indeed higher in Th2 cells (49.5%) as compared with other Th subsets (Th1, 1.48%; Treg cells, 3.44%; and Th17, 2.40%). To further explore the phenotype of these cells, we examined the effect of IL-4 and IL-7 on GATA-3 expression in IL-3R⁺ and IL-3R⁻ Th cells. It was observed that IL-4 increased GATA-3 expression in both IL-3R⁺ and IL-3R⁻ Th cells; however, its expression was higher in IL-3R⁺ Th cells (Fig. 5B, 5C). IL-7 alone or in combination with IL-4 had no effect on GATA-3 expression. We also examined the effect of IL-4 and IL-7 on proliferation of Th cells and found that IL-7 enhanced T cell proliferation, whereas IL-4 showed no effect (Supplemental Fig. 1). These results suggest that increase in the percentage of IL-3R⁺ Th cells by IL-7 is associated with its ability to enhance their proliferation. In addition to GATA-3 expression, we also examined the percentage of CCR4⁺CRTH2⁺ cells in IL-3R⁺ and IL-3R⁻ cell fractions in response to IL-4 and IL-7 stimulation. We observed that the percentage of CCR4⁺CRTH2⁺ cells were significantly higher in IL-3R⁺ cells than in their IL-3R⁻ counterpart (Fig. 5D, 5E). These results demonstrate that IL-3R⁺ cells belong to the Th2 lineage and express high GATA-3.

Because IL-3R⁺ cells showed Th2 cell phenotype, we further checked whether IL-3 could influence Th2 differentiation. Naive Th cells were polarized into Th2 cells with or without IL-3 (100 ng/ml), and the percentage of IL-4⁺–, IL-13⁺–, and IL-4⁺IL-13⁺–secreting cells was determined. We found that IL-3 significantly enhanced the percentage of IL-4⁺– and IL-4⁺IL-13⁺–secreting Th2 cells. IL-13⁺–secreting cells were slightly increased by IL-3 (Fig. 6A–D). In addition, IL-3 also enhanced the percentage of IL-5–secreting cells (Fig. 6E, 6F). It is known that IL-2, which is endogenously secreted by activated Th cells, plays a critical role in the polarization of naive CD4⁺ T cells into Th2 phenotype (28). Therefore, we investigated the effect of IL-3 on IL-2 secretion and found that IL-3 also enhanced the percentage of IL-2–secreting Th cells (Fig. 6E, 6G). Furthermore, we examined the effect of endogenously produced IL-3 on differentiation of Th2 cells by incubating naive Th cells under Th2 condition in the presence or absence of anti–IL-3 neutralizing Ab. It was observed that IL-3 neutralization significantly decreased the percentage of IL-4–secreting cells (Supplemental Fig. 2). All these results indicate that IL-3 regulates Th2 cells differentiation.

Th2 cytokines play an important role in the pathophysiology of allergic diseases, and the level of IL-4 is high in allergic patients (29). Because we observed that IL-4 regulates IL-3R expression and IL-3 in turn enhances Th2 differentiation, we next determined the levels of IL-3R and IL-3 expression in Th cells of house dust mite (Dermatophagoides)–sensitive allergic patients. These patients were also suffering from shortness of breath and chronic allergen-induced bronchitis. It was found that Th cells in the peripheral blood of allergic patients expressed IL-3R (4.08%) which was almost absent (0.62%) in healthy controls (Fig. 7A). Fig. 7B shows the percentage of IL-3R⁺ Th cells from 10 patients. To evaluate the levels of different cytokines, PBMCs were stimulated with PMA/ionomycin, and the levels of Th1, Th2, Treg, and Th17 cell–associated cytokines at single-cell levels were determined using intracellular cytokine staining. In this study, we demonstrate that not only the IL-3R–bearing Th cells but its ligand (IL-3) expressing Th cells were also significantly increased in allergic patients (Fig. 7C). Levels of Th2 cytokines, such as IL-4– (Fig. 7D), IL-15⁺IL-13⁺– (Fig. 7E), and IL-13–secreting (Fig. 7F) Th cells were significantly higher in allergic patients. However, in a majority of allergic patients, the levels of IL-9– (Fig. 7G), IL-10– (Fig. 7H), IFN-γ–(Fig. 7I) and IL-17–secreting (Fig. 7J) Th cells were unchanged. The representative FACS plots of all the above cytokines are shown in Supplemental Fig. 3. Taken together, these results suggest that, in addition to increased Th2 cytokines, the level of IL-3–secreting and also IL-3R–expressing Th cells were increased in allergic patients.

IL-3, a cytokine primarily secreted by Ag-stimulated T lymphocytes, acts as a functional link between the hematopoietic and immune systems. Among T lymphocytes, both Th1 and Th2 cells are known to produce IL-3 (30). Therefore, any effector Th cell response is always associated with consequent IL-3 production. Although activated T lymphocytes are the major source of IL-3, the role of IL-3 in development of human Th cells is not yet delineated. In the current study, we analyzed the regulation of IL-3R expression on human Th cells and also evaluated the role of IL-3 in effector functions of Th cells. Analysis of human peripheral blood Th cells revealed that these cells do not show surface expression of IL-3R. However, these cells in resting state constitutively expressed IL-3R at transcript and intracellular protein levels. These results indicate that lack of surface expression of IL-3R is not related to absence of its synthesis. We further revealed that the surface expression of IL-3R requires T cell activation by means of CD3 and CD28 engagement. A steady-state level of IL-3R is maintained in resting and activated Th cells, and its detection on cell surface after TCR stimulation suggests the translocation of a preformed IL-3R component to the surface. functions of IL-3 are mediated by the activation of the JAK2–STAT5 pathway (11). Activation-induced IL-3R surface expression in human Th cells is supported by the observation that activated but not resting Th cells respond to IL-3 and p–STAT-5.

In resting state, a majority of Th cells express IL-3R intracellularly; however, after activation, ∼10% of the cells were positive for surface expression. This suggests that surface expression of IL-3R may be present only on certain subtypes of Th cells. The cytokine microenvironment dictates many functional aspects of activated Th cells, such as proliferation and differentiation into different subsets. We next determined whether cytokines could influence the surface expression of IL-3R on Th cells. For this, different cytokines were selected, such as IL-12, which is required for Th1 cell differentiation (31, 32); IL-4 was selected for Th2 cells (33), and TGF-β1 was selected as a common regulator of Treg cells (34), Th17 (35), and Th9 cell differentiation (36). In addition, we also examined the effect of IL-2 and IL-7, which act as growth and survival factors for T lymphocytes (37). IL-4 and IL-7 significantly increased the percentage of IL-3R⁺ Th cells. However, expression of IL-3R at total protein level was increased by IL-4 and not by IL-7.

IL-4, a signature Th2 cytokine is required for differentiation of Th2 cells (38). IL-7 is also known to prime human naive Th cells for IL-4 secretion (27), and its role has been implicated in the differentiation of Th2 cells by the STAT-5 pathway (38). This raises the possibility that IL-7 can regulate IL-3R in activated Th cells through IL-4. However, after cross-neutralization of IL-4 or IL-7, there was no change in the percentage of IL-3R⁺ Th cells in either group. This was further supported by the fact that there was no IL-4 secretion in IL-7–treated cells. These observations strongly suggest that IL-7–mediated regulation of IL-3R is not dependent on IL-4. This result is contrary to the earlier study by Webb et al. (27), finding that IL-7 primes human naive CD4⁺ T cells for IL-4 secretion. This may be because of differences in the culture conditions. In their study, cord blood–derived CD4⁺ T cells were cultured with IL-7 for 7–10 d, and the IL-4 level was measured after 24 h restimulation with PMA/ionomycin. However, we used peripheral blood naive Th cells, and IL-4 was measured after 96 h without PMA/ionomycin stimulation. Thus, differences in the source of cells, culture durations, and lack of additional PMA/ionomycin stimulation could be the reasons for absence of IL-4 in our cultures. Furthermore, increase in the percentage of IL-3R⁺ cells by IL-7 could be because of its ability to enhance Th cell proliferation.

Regulation of IL-3R expression seems to be highly complex because many cytokines have been reported to regulate IL-3R expression differently in different cell types. We found that IL-10 and TGF-β1 has no effect on IL-3R expression on human Th cells; however, these cytokines are known to downregulate IL-3R in human monocytes (25). IL-3 and IL-5 showed no effect on IL-3R expression in human Th cells. However, these two cytokines enhance IL-3R expression in eosinophils, and IL-3–mediated upregulation of IL-3R was dependent on the PI3K (26). Thus, a particular cytokine may have a different effect on IL-3R expression, depending on the cell type.

Genes encoding for human IL-3R and GM-CSFR colocalize in the pseudoautosomal regions of the short arms of X and Y chromosomes (39). It was speculated that transcriptional regulation of these two genes is regulated by common factors. In hematopoietic cells, transcription factors PU.1 and C/EBPα direct the cell type–specific expression of the GM-CSFR gene (40). In the case of IL-3R, there are multiple putative transcription factor binding motifs reported in IL-3R promoter region, such as Sp1, PU.1, AP-2, AP-4, GATA-1, and C/EBPα or β (41). By using a series of 5′- deletion mutants of IL-3R promoter fused with a luciferase gene, Akagawa and coworkers (41) found that the region which contains a putative PU.1 binding site has no enhancing effect on IL-3R promoter activity. This suggests that different transcriptional activation mechanisms exist for the regulation of IL-3R and GM-CSFR genes. Moreover, by using an electrophoresis mobility shift assay, they revealed that Sp1 and unidentified proteins other than PU.1 binds to the enhancer region of the IL-3R promoter. Although transcription factor Sp1 seems to regulate IL-3R expressions, the signaling pathways that regulate constitutive IL-3R expression and its translocation to cell surface in human Th cells need more in-depth studies.

Next, we analyzed the expression of IL-3R in ex vivo–differentiated Th subsets to determine whether its expression is restricted to Th2 cells or if other Th subsets also express IL-3R. We observed that Th1, Treg, and Th17 cells show low expression of IL-3R; however, it was very high on Th2 cells. The increased number of Th2 cells expressing IL-3R indicate that IL-3 may play a role in regulating Th2 cell functions. The level of GATA-3 expression in Th2 cells determines its phenotypic stability, which is essential for sustained Th2 immune responses (42, 43). The enhanced expression of GATA-3 transcript in human Th2 cells correlates with increased IL-4 and decreased T-bet and IFN-γ expression (44). In addition to GATA-3, we also used chemokine receptor CCR4 and PG D2 receptor CRTH2 to identify human Th2 cells (45, 46). The high GATA-3 expression and CCR4⁺CRTH2⁺ cell number in the IL-3R⁺ fraction suggest a relatively stable Th2 phenotype with the ability to produce Th2 cytokines. Thus, our results clearly demonstrate that IL-3R⁺ Th cells belong to Th2 cell lineage.

IL-3 in presence of IL-4 diverts human monocytes into dendritic cells, which produce less IL-12 and skew the Th1/Th2 balance in favor of Th2 cells (47). IL-3 is also required for survival, proliferation, and differentiation of plasmacytoid dendritic cells (48). These cells express high IL-3R and induce Th2 responses in presence of IL-3 (49, 50). In addition, IL-3 plays an important role in the development of regulatory M2 macrophages (51). All these reports suggest that IL-3 influences innate immune cells to promote type 2 immunity. However, the role of IL-3 in Th cell–mediated adaptive immune responses is not yet known. Therefore, we evaluated the effect of IL-3 on regulation of Th2 cell effector functions. When naive Th cells were polarized into Th2 cells in presence of IL-3, we found that IL-3 significantly enhances the percentage of IL-4–secreting cells. This indicates that IL-4 amplifies its own expression by regulating the surface expression of IL-3R on Th2 cells. In addition to increasing the levels of Th2 cytokines, IL-3 enhanced the frequency of IL-2–secreting cells in Th2-polarizing conditions. IL-2–mediated activation of STAT-5 plays a crucial role in GATA-3–mediated expression of IL-4 in Th2 cells (38). An increase in the number of IL-2–secreting Th cells corroborates that IL-3 also creates a supportive cytokine microenvironment for Th2 polarization.

Th2 cytokines play a central role in the elicitation and maintenance of allergic responses (52). Because IL-3 enhances Th2 cells differentiation, we checked the levels of IL-3– and IL-3R–expressing Th cells in house dust mite–sensitive allergic patients. We found that the frequencies of IL-3R⁺ as well as IL-3–secreting Th cells were high in allergic patients as compared with healthy subjects. IL-3 expression in allergic inflammation has also been reported in atopic asthma patients, in which bronchoalveolar lavage cells expressing IL-3 along with IL-4 and IL-5 were increased as compared with healthy controls (53). Thus, it appears that selective modulation of IL-3– or IL-3R⁺ Th cells could be important in management of allergic diseases.

In conclusion, IL-3R expression in activated human Th cells is regulated by IL-4, and IL-3 synergizes with IL-4 to enhance Th2 cell differentiation. Increased IL-3– and IL-3R–expressing Th cells in house dust mite–sensitive allergic patients suggest that IL-3 may play an important role in regulating allergic immune responses.

We thank Dr. Vijay Warad from Sai Allergy Asthma Eye Children’s Hospital, Pune, India for providing blood samples of allergic patients.

The authors have no financial conflicts of interest.