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Clinical and Vaccine Immunology, June 2009, p. 798-805, Vol. 16, No. 6
1071-412X/09/$08.00+0     doi:10.1128/CVI.00022-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interleukin-12 Is the Optimum Cytokine To Expand Human Th17 Cells In Vitro

Soad Nady, James Ignatz-Hoover, and Mohamed T. Shata^*

The University of Cincinnati College of Medicine, Division of Digestive Diseases, Viral Immunology Laboratory, Cincinnati, Ohio

Received 16 January 2009/ Returned for modification 29 January 2009/ Accepted 13 April 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, a new lineage of CD4⁺ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4⁺ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R⁺ cells from the CD4⁺ T cells. Functional studies revealed that CD4⁺ IL-23R⁺ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-). Furthermore, we expanded the CD4⁺ IL-23R⁺ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4⁺ IL-23R⁺ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN- secreted by IL-23R⁺ CD4⁺ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R⁺ CD4⁺ cells and the secretion of IFN-, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, a new subset of T helper cells have been identified and designated Th17 cells. Th17 cells preferentially produce interleukin-17 (IL-17) and express retinoid-related orphan receptor gamma T (13, 20, 42). IL-17A is specifically expressed in this subset of T cells, and it has become the hallmark cytokine of Th17 cells (20). Accumulating data suggest that Th17 cells are highly proinflammatory and that Th17 cells with specificity for self-antigens lead to severe autoimmunity in various animal models (4). While the function of these cell subtypes has not been elucidated completely, emerging data suggest that Th17 cells may play an important role in host defense against extracellular pathogens, which are not cleared efficiently by Th1-type or Th2-type immunity (39). Understanding the roles of different branches of T-effector responses may lead to the development of novel therapeutic strategies for pathological states characterized by the expansion of Th17 cells (6).

During the past 2 years, cytokines and signaling pathways involved in regulating this new lineage differentiation, as well as the importance of this differentiation in the pathogenesis of autoimmune disease, have been investigated intensively with various mouse models. Given the pathogenic relevance of IL-17, it is important to understand how this cytokine is controlled in human T cells and to define the conditions under which human naïve CD4⁺ T cells might become Th17 cells (7). Moreover, a recent study has indicated that some of these cells have the ability to produce gamma interferon (IFN-) (2).

Similarly to Th1 and Th2 cells, Th17 cells require specific cytokines and transcription factors for their differentiation (39). A limited amount of information about the trafficking receptors of Th17 cells is available (1, 2, 31). In the mouse, the differentiation pathway of Th17 cells has been linked to the presence of transforming growth factor β and IL-6, while the maintenance and expansion of these cells seem to be IL-23 dependent (4, 23, 42). Moreover, due to the lack of Th17-specific phenotypic markers, identification and analysis of these cells to date have relied largely on detection of IL-17 mRNA in tissues or measurement of IL-17 protein levels in biological fluids (12, 27). However, recent studies have described the expression of the IL-23 receptor (IL-23R) as a phenotypic marker of Th17 cells (1, 2, 44).

IL-23 consists of a heterodimer of a 40-kDa protein (p40), which is also a component of heterodimeric IL-12, and a protein termed p19. Human p19 and mouse p19 share 70% amino acid sequence identity and are the proteins most closely related to p35, the subunit of IL-12 not shared with IL-23 (28). In humans, IL-12 promotes proliferation of both naïve and memory human T cells; however, the proliferative effect of IL-23 is still restricted to memory T cells (11). Although IL-23 is not involved in Th17 differentiation, it plays an important role in maintaining Th17 effector function (38, 42). IL-23 uses the same Jak-Stat signaling molecules as IL-12. However, the compositions of DNA-binding Stat complexes induced by IL-12 and IL-23 exhibit potentially important differences. IL-12 induces a DNA-binding complex containing only Stat4, while IL-23 induces several complexes containing Stat3, Stat1, Stat4, and possibly Stat3/Stat4. These significant differences should be expected in the biological responses induced by IL-23 and IL-12 (29). Through the IL-23R, IL-23 activates STAT1, STAT3, STAT4, and STAT5 and can induce IFN-, IL-10, and IL-17, depending on the cell type (41).

It is well-known that IL-2 is a T-cell growth factor in vitro (7). Recently, IL-21 has emerged as a key modulator of transforming growth factor β signaling, leading to the reciprocal differentiation of regulatory T cells and Th17 cells (10). IL-21 is a type I cytokine that shares a common cytokine receptor gamma chain (36) with IL-2, IL-4, IL-7, IL-9, and IL-15. These cytokines are critically important for both the maintenance and the function of T and B cells (9). In addition, it was recently reported that IL-15 stimulates the expression of IL-17 by T cells (8).

Most of the information on Th17 cells has been generated using mouse models to understand the differentiation of Th17 cells from naïve Th0 cells. However, little information about the propagation of Th17 cells in vitro in humans is known (39). In this study, we optimized the propagation conditions of Th17 cells isolated from human peripheral blood in vitro and determined the optimum conditions for isolation of Th17 clones.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects. Discarded whole-blood units from unidentifiable healthy donors were purchased from Hoxworth Blood Center, University of Cincinnati (Cincinnati, OH). Peripheral blood mononuclear cells (PBMC) were separated using a standard density gradient centrifugation method (Ficoll-Paque; Amersham Pharmacia, Piscataway, NJ). PBMC were either used fresh or cryopreserved at –70°C in 90% fetal calf serum (FCS) (Gibco) and 10% dimethyl sulfoxide (Sigma, St. Louis, MO) until needed. For cryopreserved PBMC, cells were thawed, washed twice, counted, and incubated overnight at 37°C and 5% CO₂ before any assay was performed. No significant differences in data were observed with fresh or cryopreserved PBMC, except that the yield was better when fresh cells were used.

CD4⁺ T-cell enrichment. CD4⁺ T lymphocytes were negatively isolated from the PBMC by use of a Dynabead CD4 kit 2 (Invitrogen, Carlsbad, CA), with the addition of an antibody mixture containing mouse immunoglobulin G antibodies against human CD14, CD16 (specific for CD16a and CD16b), CD56, CDw123, CD36, CD8, CD19, and glycophorin A according to the manufacturer's instructions. Briefly, 100 µl of the cells (1 x 10⁷) was suspended in buffer 1 (consisting of phosphate-buffered saline [PBS] [without Ca²⁺ and Mg²⁺] with 0.1% bovine serum albumin and 2 mM EDTA) and mixed with 20 µl of heat-inactivated FCS (Invitrogen, Carlsbad, CA) and 20 µl of antibody mixture. Cells were mixed well, incubated for 20 to 30 min at 2°C to 8°C, and then washed by the addition of 2 ml buffer 1, mixed well, and centrifuged at 300 x g for 10 min at 2°C to 8°C. The supernatant was discarded, and the cells were resuspended in 900 µl buffer 1. Then, 100 µl of prewashed (by buffer 1) depletion Dynabeads was added to the cells and incubated for 15 min at 18°C to 25°C, with gentle tilting and rotation. The bead-bound cells were resuspended by vigorous pipetting with a 1,000-µl pipette 5 to 10 times, 1 ml buffer 1 was added, and the tube was placed in the magnet for 3 min. The supernatant was transferred to a new tube, and the bead-bound cells (non-CD4⁺ cells) and bead-unbound cells (CD4⁺ cells) were counted. Then, the unbound cells (CD4⁺ T cells) were used for Th17 separation.

CD4⁺ IL-23R⁺ cell enrichment. CD4⁺ IL-23R⁺ T cells were isolated from the negatively isolated CD4⁺ T cells with a biotinylated monoclonal antibody (MAb) against IL-23R and avidin beads by use of a Cellection biotin binder kit (Invitrogen, Carlsbad, CA). DNase buffer was used to release the cells from the beads, and the beads were removed by a magnet as described by the manufacturer. Briefly, 1 x 10⁷ cells/ml were suspended in buffer 1, IL-23R biotinylated antibody was added to the cell suspension (1 µg/10⁶ cells), and the suspension was mixed well and incubated for 30 min at 2°C to 8°C. The cells were washed twice by addition of buffer 1 and centrifuged at 300 x g for 10 min. The supernatant was discarded, and the cells were resuspended in buffer 1 at 1 x 10⁷ cells per ml. One hundred microliters of prewashed (by buffer 2, consisting of PBS, without Ca²⁺ and Mg²⁺, and 0.1% bovine serum albumin) Dynabeads was added to the antibody-labeled cells. Cells were incubated for 20 min at 2°C to 8°C, with gentle tilting and rotation. Then, the tube was placed in a magnet for 2 min. The supernatant was discarded, and the bead-bound cells were washed three times by resuspension in buffer 2 to the original sample volume and separated using a magnet. The bead-bound cells were resuspended in preheated (37°C) buffer 3 (consisting of RPMI 1640 with 1% FCS, 1 mM CaCl₂, and 4 mM MgCl₂). Then, 16 µl of the releasing buffer (DNase I) was added to the bead-bound cells, and the cells were incubated for 30 min at room temperature, with gentle tilting and rotation. Cells were pipetted vigorously to maximize cell release. Then, the tube was placed in a magnet for 2 min and the supernatant was transferred with released cells into a tube (precoated with buffer 3). The cells were resuspended in buffer 3, and this step was repeated five times. The number of the released cells was counted, and the percentage of IL-23R⁺ cells was calculated.

Flow cytometric analysis of the enriched cells. The IL-23R⁺ cells were resuspended in flow staining buffer (consisting of PBS, 2% AB human plasma (Sigma-Aldrich, St. Louis, MO), and 0.1% sodium azide) at a concentration of 1 x 10⁶ cells/ml. The cells were stained with biotinylated anti-IL-23R (R & D Systems, Inc., Minneapolis, MN), avidin-fluorescein isothiocyanate (FITC) (eBioscience, San Diego, CA), and 20 µl of anti-CD4-phycoerythrin (PE) (eBioscience), according to the manufacturers' instructions. Cells were incubated for 20 min at 4°C and then washed, resuspended in 500 µl PBS, and analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ). For surface staining of the IL-12 receptor (IL-12R), enriched CD4⁺ IL-23R⁺ cells were stained with 5 µl of biotinylated anti-IL-23R (R & D Systems, Inc.) for 20 min at 4°C and then washed and stained with avidin-FITC (eBioscience, San Diego, CA), according to manufacturer instructions. Isotype control antibodies were used as negative controls.

Expansion of CD4⁺ IL-23R⁺ cells. CD4⁺ IL-23R⁺ cells were expanded for 12 days in the presence of anti-CD3/CD28 beads (Invitrogen), irradiated autologous feeder cells, and IL-2 (100 IU/ml; Cell Sciences, Inc., Canton, MA), IL-7 (7 ng/ml; Millipore, Bedford, MA), IL-12 (2.5 ng/ml; R & D Systems, Inc.), IL-15 (7 ng/ml; Pierce Endogen, Rockford, IL), IL-23 (10 ng/ml; eBioscience), or a combination of cytokines according to the experimental protocol.

IFN- ELISPOT assay. An enzyme-linked immunospot (ELISPOT) assay was performed with an IFN- ELISPOT kit (catalog no. M34201-A; Mabtech, Sweden) according to the manufacturer's instructions and as described by Shata et al. (34). Briefly, 96-well nitrocellulose-bottomed plates (Millipore, Bedford, MA) were coated with murine anti-human IFN- MAb at a concentration of 15 µg/ml in PBS and incubated at 4°C. After 24 h, the plates were washed and blocked with RPMI with 10% fetal bovine serum (FBS). Then, PBMC were added at a concentration of 10⁵ cells/well to a 100-µl volume of complete medium (RPMI 1640 containing 10% FBS). For stimulation, we used anti-CD3/CD28 beads (Invitrogen). After an 18-h incubation at 37°C and 5% CO₂, supernatants were collected for an IL-17 enzyme-linked immunosorbent assay (ELISA), and the plates were washed five times with washing buffer (PBS containing 0.5% [vol/vol] Tween 20; Sigma, St. Louis, MO) using an automatic plate washer (Bio Tec Instruments, Inc.). Biotinylated anti-human IFN- MAb (clone 4S.B3) at a concentration of 1 µg/ml in dilution buffer (PBS containing 0.05% FBS) was added, and the plates were incubated at room temperature for 2 h. Plates were then washed again five times, streptavidin-horseradish peroxidase (1:1,000) in dilution buffer was added, and plates were incubated at room temperature for 1 h, followed by five washes with washing buffer and addition of tetramethylbenzidine substrate (MabTech). After the spots were developed for 10 to 15 min, the plates were washed with distilled water and air dried. The number of spots was enumerated using an automated ELISPOT 3B analyzer (CTL, Cleveland, OH).

IL-17 ELISA. IL-17 concentrations were measured using a human IL-17A ELISA kit (eBioscience) as described by the manufacturer. Briefly, Corning Immunlon 4 HBX 96-well ELISA plates (Fisher catalog no. 3855) were coated with 100 µl/well of capture antibody in coating buffer (diluted 1:250). The plates were sealed and incubated overnight at 4°C. The plates were washed five times with washing buffer (PBS with 0.02% Tween 20) using an automatic ELISA plate washer. The wells were blocked with 200 µl/well of 1x assay diluent, and the plates were incubated at room temperature for 1 h. By use of the assay diluent, standards were prepared according to the manufacturer's instructions and added to the appropriate wells to make the standard curve. Unknown samples were added as 100 µl/well, and the plates were incubated at room temperature for 2 h. After the plates were washed, detection antibody diluted in assay diluent (diluted 1:250) was added to each well and the plates were incubated at room temperature for 1 h. After the plates were washed, 100 µl/well of streptavidin-horseradish peroxidase diluted in assay diluent (diluted 1:250) was added to each well and the plates were incubated at room temperature for 30 min. After the plates were washed five times, 100 µl/well of tetramethylbenzidine substrate solution was added to each well and incubated at room temperature for 15 min. Then, 50 µl of stop solution (1 M H₃PO₄) was added to each well and results were read at an optical density at 450 nm.

Intracellular staining of IL-17 and IFN-. To measure levels of IL-17 and IFN- production by intracellular cytokine staining, enriched CD4⁺ IL-23R⁺ T cells were stimulated for 4 to 6 h in the presence of anti-CD3/CD28 beads or phytohemagglutinin (PHA) (5 mg/ml) and Golgi Stop (BD Biosciences), as described previously (40). Cells were surface stained with anti-CD4-peridinin chlorophyll protein (BD Biosciences catalog no. 550631) for 20 min and then washed. Cells were made permeable with Cytofix/Cytoperm reagents (BD Biosciences) according to the manufacturer's instructions. Cells were stained with FITC-conjugated anti-IL-17 (eBioscience catalog no. 11-7179), anti-IFN--PE (BD Biosciences catalog no. 340452), or isotype controls and analyzed by flow cytometry (FACScan; Becton Dickinson) by gating on CD4⁺ T cells.

Statistical analysis. Statistical analysis was performed by paired Student's t test using Graph Pad Prism software. Results with a P value of <0.05 were considered significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation and phenotypic analysis of CD4⁺ IL-23R⁺ T cells from human blood. Negative selection of CD4⁺ T cells yielded approximately 4 x 10⁶ out of 1 x 10⁷ PBMC with approximately 95% purity, as shown by flow cytometric analysis (Fig. 1A). Positive selection of IL-23R⁺ cells from CD4⁺ T cells yielded approximately 1 x 10⁴ out of 1 x 10⁶ CD4 T cells with approximately 90% purity, as shown by flow cytometric analysis (Fig. 1B). Fewer than 3% of CD4⁺ IL-23R^– cells were positive for the IL-23R (Fig. 1C). CD4⁺ IL-23R⁺ cells had a higher expression of CD4 on the surface than did CD4⁺ IL-23R^– cells (Fig. 1B and C). Additionally, we measured the expression of the IL-12R on the isolated CD4⁺ IL-23R⁺ cells. As shown in Fig. 1D, more than 90% of the cells expressed the IL-12R.

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FIG. 1. Flow cytometric analysis of the isolated cells. Cells at a concentration of 1 x 106 cells/ml were stained with biotinylated anti-IL-23R, avidin-FITC, and anti-CD4-PE according to the manufacturers' instructions (A, B, and C). For surface staining of IL-12R, enriched CD4+ IL-23R+ cells were stained with biotinylated anti-IL-23R for 20 min at 4°C and then washed and stained with avidin-FITC according to manufacturer instructions. Isotype control antibodies were used as negative controls. (A) Enriched CD4+ cells. (B) Enriched CD4+ IL-23R+ cells. (C) Remaining CD4+ IL-23R– cells. (D) Graph of the expression of the IL-12R on the isolated CD4+ IL-23R+ cells.

Functional characterization of CD4⁺ IL-23R⁺ cells. To evaluate the functional activity of the IL-23R⁺ cells, levels of IL-17 and IFN- secretion by these cells were evaluated after stimulation with anti-CD3/CD28 beads. Our results revealed that IL-17 secretion, as measured by ELISA, was significantly higher (P = 0.0245) in CD4⁺ IL-23R⁺ cells than in CD4⁺ IL-23R^– cells (257.3 ± 12 and 98 ± 17 pg/ml, respectively) (Fig. 2A). Additionally, the number of CD4⁺ IL-23R⁺ cells secreting IFN- was significantly lower (P = 0.0188) than the number of CD4⁺ cells (549.25 ± 69.6 and 2,485 ± 374.8 IFN--secreting cells [ISCs]/10⁶ cells, respectively) (Fig. 2B).

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FIG. 2. Functional analysis of the enriched CD4+ IL-23R+ cells. Levels of IFN- secretion of CD4+ T cells before and after IL-23R enrichment (IL-23R+ and IL-23R– cells) were examined by ELISPOT assay, as described in Materials and Methods. Cells were stimulated with anti-CD3/CD28 beads for 18 h, and the supernatants were collected before the development of the ELISPOT plate to measure IL-17 by ELISA. (A) Measurement of IL-17 by ELISA in the supernatants of the stimulated cells. (B) Measurement of IFN- secretion by ELISPOT assay. Data represent averages and standard deviations from three experiments. Statistical analysis was done using paired Student's t test. There is a significant difference (P < 0.05, marked with a star) between IL-23R+ and IL-23R– cells in levels of secretion of both IFN- and IL-17.

Optimization of the conditions required for propagation of CD4⁺ IL-23R⁺ cells. To optimize the conditions for propagation of CD4⁺ IL-23⁺ T cells, we expanded the cells for 2 weeks in the presence of anti-CD3/CD28 beads and different cytokines (IL-2, IL-7, IL-12, and IL-15). The growth curves of the cells under different conditions were analyzed, and the time needed for 50% growth was calculated. IL-12 was the optimum cytokine, with 7.74 days for 50% growth, versus 9.32 days for IL-2, 8.93 days for IL-7, and 8.75 days for IL-15. (In the absence of cytokines, no growth was observed, and 50% cell growth could not be calculated.) To evaluate the best combination of cytokines to propagate CD4⁺ IL-23R⁺ cells in the presence of anti-CD3/CD28 beads, we used the optimum concentration of IL-7, IL-15, IL-12, or combinations to determine the optimum conditions for growth. As shown in Fig. 3, addition of a single cytokine (IL-7, IL-12, or IL-15) increased the number of cells 63- to 88-fold. However, IL-7 and IL-15 are antagonistic to each other, and the combination increased the number of cells only 14-fold. This growth was lower than the growth observed with each cytokine alone. In contrast, IL-7 is synergistic with IL-12, and this combination was the optimum for the expansion of CD4⁺ IL-23R⁺ cells (99-fold increase).

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FIG. 3. Synergistic IL-12 and antagonistic IL-15 effects on CD4+ IL-23R+ cell propagation in vitro in the presence of IL-7 and anti-CD3/CD28 beads. CD4+ IL-23R+ cells were expanded for 12 days in the presence of anti-CD3/CD28 beads, irradiated autologous feeder cells, and IL-2 (100 IU/ml), IL-7 (7 ng/ml), IL-12 (2.5 ng/ml), IL-15 (7 ng/ml), or combinations of cytokines according to the experimental protocol. The numbers of expanded cells were measured in the absence and presence of cytokines after 12 days. Changes were calculated as the number of expanded cells in the presence of cytokines divided by the number of cells before the addition of cytokines (time zero). Data are representative of one of three experiments.

Functional characterization of the expanded CD4⁺ IL-23R⁺ cells. To evaluate the functional activity of the expanded IL-23R⁺ CD4⁺ cells, we measured the cytokine secretion of the expanded cells after stimulation with anti-CD3/CD28 beads or PHA. As shown in Fig. 4 and 5, IFN- secretion and IL-17 secretion were evaluated for the expanded CD4⁺ IL-23R⁺ cells by ELISPOT assay and ELISA, respectively. IL-2, IL-7, IL-12, IL-12 plus IL-7, and IL-23 significantly increased IFN- secretion compared to the level for nonstimulated cells (P = 0.0318, P = 0.0061, P = 0.0134, P = 0.0411, and P = 0.0188, respectively). Additionally, IL-7, IL-12, and IL-12 plus IL-7 significantly increased IL-17 secretion compared to the level for nonstimulated cells (P = 0.005, P = 0.0056, and P = 0.0315, respectively). However, the combination of IL-7 and IL-12 was the optimum for the expanded CD4⁺ IL-23R⁺ T cells for secretion of IFN-, while IL-12 was the optimum for the expanded CD4⁺ IL-23R⁺ T cells for secretion of IL-17.

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FIG. 4. Expansion of CD4+ IL-23R+ cells in the presence of IL-23. CD4+ IL-23R+ cells were expanded for 12 days in the presence of anti-CD3/CD28 beads, irradiated autologous feeder cells, and IL-23 (10 ng/ml). Functional analysis of the expanded cells was done as described in the legend for Fig. 2. The number of CD4+ IL-23R+ cells secreting IFN- was highly significant (P = 0.0156) after stimulation with anti-CD3/CD28 beads (34,130 ± 5,218 ISCs/106 cells) compared to the number for nonstimulated cells (4,820 ± 424 ISCs/106 cells), as measured by ELISPOT assay (A). Additionally, IL-17 secretion by CD4+ IL-23R+ cells stimulated with anti-CD3/CD28 beads (386.5 ± 49.7 ng/ml) was highly significant (P = 0.0029) in comparison to the level for nonstimulated cells (48 ± 12.5 ng/ml), as measured by ELISA (B). Data represent averages and standard deviations. Conc., concentration.

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FIG. 5. Response of CD4+ IL-23R+ cells stimulated with anti-CD3/CD28 in the presence of different cytokines. CD4+ IL-23R+ cells were expanded for 12 days in the presence of anti-CD3/CD28 beads, irradiated autologous feeder cells, and IL-2 (100 IU/ml), IL-7 (7 ng/ml), IL-12 (2.5 ng/ml), IL-15 (7 ng/ml), or combinations of cytokines according to the experimental protocol. Functional analysis of the expanded cells was done as described in the legend for Fig. 2. Data were calculated as the responses in the presence of cytokines and anti-CD3/CD28 beads divided by the responses in the absence of cytokines and anti-CD3/CD28 beads. IL-2, IL-7, IL-12, and IL-12 plus IL-7 significantly increased IFN- secretion compared to the level for nonstimulated cells (P = 0.0318, P = 0.0061, P = 0.0134, and P = 0.0411, respectively). Additionally, IL-7, IL-12, and IL-12 plus IL-7 significantly increased IL-17 secretion compared to the level for nonstimulated cells (P = 0.005, P = 0.0056, and P = 0.0315, respectively). Data represent averages and standard deviations.

To evaluate the functional activity of the IL-23R⁺ cells expanded with IL-23 alone, levels of IL-17 and IFN- secretion by these cells were evaluated after stimulation with anti-CD3/CD28 beads. The results revealed that the number of CD4⁺ IL-23R⁺ cells secreting IFN- was highly significant (P = 0.0156) after stimulation with anti-CD3/CD28 beads (34,130 ± 5,218 ISCs/10⁶ cells) compared to the number for nonstimulated cells (4,820 ± 424 ISCs/10⁶ cells), as measured by ELISPOT assay (Fig. 4A). Similarly, IL-17 secretion by CD4⁺ IL-23R⁺ cells stimulated with anti-CD3/CD28 beads (386.5 ± 49.7 ng/ml) was highly significant (P = 0.0029) in comparison to the level for nonstimulated cells (48 ± 12.5 ng/ml), as measured by ELISA (Fig. 4B).

To further characterize the expanded CD4⁺ IL-23R⁺ T cells, secretion of IL-17 and IFN- was evaluated by intracellular staining. As shown in Fig. 6, 9.7% of CD4⁺ IL-23R⁺ T cells stimulated by anti-CD3/CD28 produced IFN-, while 1.2% of the stimulated cells produced IL-17, in comparison to 0% and 0.2%, respectively, for the unstimulated cells. Very few cells (0.1%), if any, secreted both IFN- and IL-17 under these conditions. With PHA stimulation, the number of IL-17-secreting cells was dramatically higher (94%) (data not shown).

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FIG. 6. IL-17 and IFN- secretion by expanded CD4+ IL-23R+ cells. Enriched CD4+ IL-23R+ T cells were stimulated for 4 to 6 h in the presence of anti-CD3/CD28 beads and Golgi Stop. Cells were surface stained with anti-CD4-peridinin chlorophyll protein for 20 min. Then, cells were made permeable with Cytofix/Cytoperm reagents (BD Biosciences) according to the manufacturer's instructions. Cells were stained with FITC-conjugated anti-IL-17 and PE-conjugated anti-IFN- or isotype controls and analyzed by flow cytometry (FACScan; Becton Dickinson) after gating on CD4+ T cells.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Th17 cells have been described as a CD4⁺ T-cell lineage distinct from Th1, Th2, and regulatory CD4⁺ T cells (30). Recent data suggest that Th17 cells play an important role in regulation of the immune responses and induction of autoimmunity (4). To evaluate the role of Th17 cells, a system is needed to optimize the propagation of Th17 cell lines and clones in vitro. In the current study, we isolated differentiated Th17 cells from human PBMC and identified the optimum conditions for the propagation of these cells in vitro. Differentiated Th17 cells were isolated from the PBMC by using immunomagnetic beads to remove all non-CD4 cells. Then, positive selection of IL-23R⁺ CD4⁺ T cells was accomplished by using a MAb against IL-23R. It has been reported that Th17 cells express specifically IL-23R (1, 2, 44). The frequency of IL-23R⁺ T cells among CD4⁺ T cells is only 1%. The purity of the isolated, enriched CD4⁺ IL-23R⁺ cells was approximately 90%, as measured by flow cytometry. In addition, fewer than 3% of CD4^– cells were positive for the IL-23R, which indicates that other cell types, such as NK cells (28), macrophages, and dendritic cells, express the IL-23R (29).

We further evaluated the functional activity of the ex vivo-isolated CD4⁺ IL-23R⁺ cells by measuring the levels of IL-17 and IFN- secretion with an IL-17 ELISA and an IFN- ELISPOT assay, respectively. Our results indicate that IL-17 secretion is significantly higher in CD4⁺ IL-23R⁺ cells than in CD4⁺ IL-23R^– cells. The number of CD4⁺ IL-23R⁺ cells secreting IFN- is significantly lower than the number of CD4⁺ cells. These findings suggest that CD4⁺ IL-23R⁺ cells secrete IL-17 and IFN-, in accordance with previous studies which suggest that IFN-- and IL-17-producing CD4 T cells might represent a common lineage (5, 26). Recently, the existence of remarkable proportions of IL-17- and IFN--producing Th17 cells in the guts of subjects with Crohn's disease has been described (2). It was also demonstrated that IL-12 downregulates IL-17 expression but upregulates IFN- expression in Th17 cells. Thus, Th17 cells could secrete IL-17 or IFN- in response to the dominant cytokines present in the microenvironment or the receptors involved in the activation. However, other studies have indicated that IFN-- and IL-17-producing CD4 T cells are distinct populations of effector cells, each having a unique role in the adaptive immune system; however, the lineage relationships between the two phenotypes were unclear (14).

None of these studies clarified whether the Th17 cells are two subsets, one secreting IFN- and another secreting IL-17, or whether they are one type of cell that secretes IFN- and/or IL-17 under different maturation and microenvironmental conditions. To address this question, cloning of Th17 cells may be necessary. However, to clone Th17 cells, we need to understand the optimum conditions necessary for in vitro expansion of these cells. In this study, we characterized the optimum conditions necessary for the proliferation of Th17 cells. Isolated CD4⁺ IL-23R⁺ cells were expanded in vitro for 2 weeks in the presence of anti-CD3/CD28 beads and different cytokines (IL-2, IL-7, IL-12, and IL-15). The growth curves of the cells under different conditions were analyzed, and the time needed for 50% growth was calculated. We found that IL-12 was the optimum cytokine, with about 7.7 days for 50% growth. Moreover, our results revealed that the addition of a single cytokine (IL-2, IL-7, IL-12, or IL-15) increased the number of cells 63- to 88-fold.

It is well known that IL-2, IL-7, and IL-15 are members of the IL-21 family. Most cytokines in this family are critically important for both the maintenance and the function of T cells and B cells (9). Additionally, it has been reported that IL-21 drives Th17 differentiation (36). In our study, IL-2 had minimal stimulatory effects on human Th17 propagation, which is in agreement with other studies suggesting that IL-2 promotes regulatory T cells and inhibits Th17 differentiation (18, 21).

Furthermore, we examined the effect of combined cytokines on the proliferation of Th17 cells. Our data indicate that IL-7 and IL-15 are antagonistic to each other and that the combined usage of both cytokines limits the increase in the number of cells (14-fold) compared to the increase when each cytokine is used alone (87- to 88-fold). In contrast, IL-7 is synergistic with IL-12, and this combination is the optimum for the expansion of CD4⁺ IL-23R⁺ cells (99-fold increase).

To evaluate the functional activity of the expanded CD4⁺ IL-23R⁺ cells, we measured the cytokine secretion of the expanded cells after stimulation with anti-CD3/CD28 beads in the presence of different cytokines. Levels of IFN- and IL-17 secretion in the expanded CD4⁺ IL-23R⁺ cells were evaluated by ELISPOT assay and ELISA, respectively. We tested the effect of a single cytokine (IL-2, IL-7, IL-12, IL-15, or IL-23) as well as the effect of combined cytokines (IL-12 plus IL-7 and IL-15 plus IL-7) on the secretion of cytokines by Th17 cells.

Our data indicate that a single cytokine (IL-2, IL-7, IL-15, IL-12, or IL-23) significantly increases the level of IFN- secreted by CD4⁺ IL-23R⁺ cells in comparison to the level for nonstimulated cells, in agreement with others (19, 25, 37, 43). The effect of IL-7 and IL-15 on Th17 cells was surprising to us, since there is no evidence that IL-7 or IL-15 affects CD4⁺ T-cell functions, while all of the published data suggested that IL-7 and IL-15 are crucial for the survival and expansion of naïve (3, 16, 32) and memory (15, 33) CD8⁺ T cells. However, in most of these studies, all of the CD4⁺ T cells were examined, rather than CD4⁺ IL-23R⁺ cells, which represent only 1% of CD4⁺ T cells. Additionally, IL-15 and IL-7 are members of the common gamma chain family of cytokines, which have multiple effects on T-cell function (17). Our data suggest that both IL-7 and IL-15 affect CD4⁺ IL-23R⁺ cell development and function.

Regarding the secretion of IFN- by CD4⁺ IL-23R⁺ cells, our data suggest that IL-7 and IL-12 have a synergistic effect and significantly increase IFN- production, while IL-7 and IL-15 have an antagonistic effect and decrease IFN- secretion. These data are in agreement with our proliferation data. In addition, these results are in agreement with results from a previous study that reported that the combination of IL-7 and IL-12 in either fresh CD3⁺ T cells or an IL-2-dependent CD4⁺ T-cell line (35) synergistically enhanced the proliferation of these CD3⁺ T cells and enhanced the production of IFN- (24). One potential explanation for the differential effects of IL-7 plus IL-12 (synergistic) and IL-7 plus IL-15 (antagonistic) is the requirements for the common cytokine receptor gamma chain. It is known that the gamma chain is a critical component of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (22, 45). Therefore, IL-7 could compete for the gamma chain of IL-15R and lead to the antagonistic effects.

Regarding IL-17 secretion, individual IL-23, IL-7, and IL-12, as well as the combination of IL-12 plus IL-7, significantly increase IL-17 secretion in comparison to the level for nonstimulated cells. We further characterized the secretion of IL-17 by the expanded CD4⁺ IL-23R⁺ T cells by intracellular staining of IL-17 and IFN-. IL-17 was produced by 1.2% of the enriched CD4⁺ IL-23R⁺ T cells stimulated by anti-CD3/CD28, in comparison to 0.2% of the unstimulated cells. Our data suggest that unstimulated Th17 does not secrete IL-17 spontaneously but needs stimulation through other T-cell receptors to secret IL-17. The mechanism of stimulation of Th17 to secrete IL-17 and not IFN- is still unknown and needs further investigation, but our data suggest that PHA is more efficient in the stimulation of Th17 to secrete IL-17 than anti-CD3/CD28 (data not shown). Additionally, our intracellular staining data indicate that IFN-- and IL-17-secreting cells are distinct populations, because few (<0.01%) CD4⁺ IL-23R⁺ cells secrete both IFN- and IL-17.

In conclusion, our study provides the optimum conditions for isolation and propagation of human Th17 cells and clones in vitro. In addition, our results indicate that the combination of IL-7 and IL-12 is the optimum for the expansion of cells and that IL-12 preferentially stimulated IL-23R⁺ CD4⁺ cells to secrete predominately IL-17.

 
This work was supported by NIH grant R21AI067868 (M. T. Shata), the University of Cincinnati ROSE program (J. Ignatz-Hoover), and Egyptian Ministry of High Education postdoctoral fellowship program SAB 1501 (S. Nady).

 
* Corresponding author. Mailing address: Viral Immunology Laboratory, MSB 6360, Department of Internal Medicine, Division of Digestive Diseases, 231 Albert B. Sabin Way, University of Cincinnati Medical Center, P.O. Box 670595, Cincinnati, OH 45267-0595. Phone: (513) 558-6110. Fax: (513) 558-1744. E-mail: mohamed.shata{at}uc.edu

Published ahead of print on 22 April 2009.

Present address: Zoology Department, Faculty of Science, Helwan University, Helwan, Egypt.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Clinical and Vaccine Immunology, June 2009, p. 798-805, Vol. 16, No. 6
1071-412X/09/$08.00+0     doi:10.1128/CVI.00022-09
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