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Either IL-2 or IL-12 Is Sufficient to Direct Th1 Differentiation by Nonobese Diabetic T Cells¹

Weisong Zhou^*, Feng Zhang^* and Thomas M. Aune^2,*,

^* Division of Rheumatology, Department of Medicine, and Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4⁺ T cells from nonobese diabetic mice spontaneously differentiate into IFN--producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4⁺ T cells. Its ability to direct Th1 differentiation of both naive and memory CD4⁺ T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN--producing autoreactive T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naïve CD4⁺ T cells produce IL-2 following stimulation with Ag and costimulatory molecules or with polyclonal activators such as anti-CD3 and anti-CD28 Abs (1, 2, 3). Signaling of IL-2 through its receptor is required for T cells to progress through the G₁/S restriction point of the cell cycle and to enter the phase of cell proliferation (4, 5). During the period of 4–5 days of extensive cell proliferation, T cells also differentiate into either effector Th1 or Th2 cells (6, 7), as characterized by their cytokine expression profiles: IFN- for Th1 cells, and IL-4 for Th2 cells. These effector T cells respond rapidly with higher levels of cytokine production to restimulation with Ag or anti-CD3 Ab.

The cytokine environment during primary T cell activation is the most critical factor that influences T cell differentiation (6, 7). IL-12 plays a key role in Th1 differentiation, while IL-4 is essential for Th2 development. IL-12 is produced by phagocytic APCs in response to inflammatory stimuli such as bacteria or intracellular pathogens (8). Engagement of IL-12R activates STAT4, which is critical for Th1 differentiation (9, 10, 11). The nuclear factor, T-bet, is also required for efficient Th1 differentiation and IFN- production (12, 13). The important role of IL-12 in Th1 development is underscored by studies of mice with IL-12, IL-12R, or STAT4 gene deficiencies. Mice with IL-12 p40 subunit, IL-12R 1 chain, or STAT4 deficiency exhibit impaired IFN- production (10, 11, 14). In addition to the direct effect on Th1 differentiation, IL-12 also increases expression of the IL-18R gene in T cells, and therefore enhances IL-18 responsiveness of T cells (15). Synergistically, IL-18 can function with IL-12 to increase the level of IFN- production (16).

The nonobese diabetic (NOD)³ mouse strain spontaneously develops autoimmune diabetes. The disease is characterized by insulitis of the pancreas, followed by selective destruction of cells in pancreatic islets (17). Evidence suggests that cell destruction is mediated, at least in part, by effector CD4⁺ T cells that preferentially secrete IFN- and TNF- (18, 19). NOD CD4⁺ T cells have been shown to have an increased propensity to become effector Th1 cells in tissue culture assays (20). We wanted to determine whether Th1 differentiation by NOD T cells had the same cytokine requirements as found in other strains. We found that IL-2 was necessary and sufficient to direct naive NOD T cells into the Th1 lineage.

	Materials and Methods

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Mice

Female NOD, NOD.B10 Idd9, nonobese diabetes-resistant (NOR), C57BL/6 (B6), C57BL/10 (B10), and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used at 6–8 wk of age.

Reagents

Complete RPMI 1640 medium supplemented with 10% FBS (Lot ALK 14837; HyClone Laboratories, Logan, UT), 100 U/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-glutamine, and 0.05 mM 2-ME (J. T. Baker, Phillipsburg, NJ) was used for cell culture. Human rIL-2 was a gift from Hoffmann-LaRoche (Nutley, NJ). Recombinant murine cytokines (IL-2, IL-4, and IL-12), purified mAbs (neutralizing anti-IL-12, C17.8; anti-CD8a, 53-6.7), labeled mAbs (FITC anti-CD4, APC anti-IFN-, APC anti-IL-2R -chain, PE anti-IL-2R -chain, PE-anti-IL-2R -chain), and GolgiPlug reagent were purchased from BD PharMingen (San Diego, CA). The following mAbs were purified from tissue culture supernatant of hybridoma cells purchased from American Type Culture Collection (Manassas, VA): anti-I-A (specific for I-A g7, k, r, f, or s haplotypes, 10-3.6.2; specific for d haplotype, 34-5-3S; or specific for b, d, q haplotypes, M5/114.15.2), anti-CD3 (145-2C11), anti-CD8 (2.43), and neutralizing anti-cytokine mAbs, including anti-IL-2 (S4B6-1), anti-IL-4 (11B11), and anti-IFN- (R4-6A2). CFSE was obtained from Molecular Probes (Eugene, OR).

Cell preparation and culture

T cells were purified by negative selection from pooled splenocytes of three to four mice, as previously described (21). Briefly, single cell suspensions were prepared from mouse spleen. RBCs were removed by hypotonic lysis. I-A-expressing cells and CD8⁺ T cells were removed by incubation with anti-I-A (10-3.6.2 for NOD, NOD.B10 Idd9, and NOR; M5/114.15.2 for B6 and B10; 34-5-3S for BALB/c) and anti-CD8 mAbs (53-6.7 for NOD, NOD.B10 Idd9, and NOR; 2.43 for B6, B10, and BALB/c) at 4°C for 30 min, washed, and incubated with goat anti-mouse and goat anti-rat IgG bound to magnetic beads (Polysciences, Warrington, PA) at room temperature for 30 min with rocking. Cells bound to the beads were removed with a magnet. The purity of CD4⁺ T cells was >90%, as determined by flow cytometry. RBC-depleted splenocytes were irradiated at 2000 rad from a ¹³⁷Ce source and used as APCs. Purified T cells (1 x 10⁶/ml) combined with irradiated splenocytes (1 x 10⁶/ml) were cultured in complete RPMI 1640 medium in 24- or 96-well tissue culture plates (1 or 0.2 ml/well, respectively) at 37°C in 5% CO₂ in air. CD4⁺CD44^low and CD4⁺CD44^high T cells (2 x 10⁵/ml) were purified by cell sorting and cultured with irradiated splenocytes (1 x 10⁶/ml) in 24-well tissue culture plates. In some experiments, CD4⁺ T cells were purified by positive selection using magnetic cell sorting (MACS) CD4 (L3T4) microbeads and MS separation columns (Miltenyi Biotec, Auburn, CA), according to manufacturer’s instructions. Briefly, spleen cells of three to four mice were pooled, and RBCs and tissue debris were removed. After labeling with MACS CD4 microbeads, the cells were applied to a separation column. The magnetically labeled CD4⁺ cells were retained in the column and then eluted as the positively selected cell fraction. The purity of the CD4⁺ T cells was >92%, as determined by flow cytometry.

A 7-day protocol was used to generate effector T cells. Purified T cells were cultured and stimulated with immobilized anti-CD3 plus irradiated splenocytes in the absence (neutral condition) or presence of IL-12 at 5 ng/ml plus anti-IL-4 at 10 µg/ml (Th1 condition) or IL-4 at 5 ng/ml plus anti-IFN- at 10 µg/ml (Th2 condition) for 5 days, washed twice, and restimulated with immobilized anti-CD3 for 2 days. To immobilize anti-CD3 on culture plates, anti-CD3 diluted to 10 µg/ml in 0.1 M sodium bicarbonate (pH 9.6) was added to the plates (0.5 and 0.1 ml/well for 24- and 96-well plates, respectively) and incubated at 37°C for 4–6 h or at 4°C overnight. The plates were washed thoroughly before use. Where indicated, neutralizing mAbs including anti-IL-2 (0.110 µg/ml), anti-IL-4 (10 µg/ml), anti-IL-12 (10 µg/ml), anti-IFN- (10 µg/ml), or anti-IL-2 plus either recombinant human IL-2 (50 U/ml) or mouse IL-12 (5 ng/ml) were included in the cultures during the 5 days of primary stimulation. Rat IgG2a (isotype matched with S4B-6) was used as a negative control in neutralization experiments with anti-IL-2 Ab. All priming cytokines and Abs were added on day 0 of the culture period.

Proliferation assay

Purified CD4⁺ T cells were cultured under neutral condition in the absence or presence of increasing levels of anti-IL-2 Ab for 3 days. [³H]Thymidine (5 µCi) was added during the last 8–12 h. [³H]Thymidine incorporation was determined with a beta scintillation counter.

Detection of cytokines

IFN-, IL-2, IL-4, IL-12, IL-18, and TNF- ELISAs were performed with mAbs from BD PharMingen, according to the manufacturer’s procedures. The concentration of cytokine was calculated from a curve constructed with a recombinant cytokine standard.

Flow cytometry

CFSE-labeled or unlabeled CD4⁺ T cells were stained for surface markers and intracellular cytokines. For CFSE labeling, 1–2 x 10⁷ cells/ml in PBS were incubated with 10 µM CFSE for 8 min at room temperature. After stopping the labeling reaction by addition of 1 vol FBS, the cells were washed three times and cultured under conditions, as indicated in the text, for 3 days. GolgiPlug containing 1 µg/ml of brefeldin A was added to cultures 4 h before harvest for intracellular IFN- staining. FITC-labeled anti-CD4 and APC-labeled anti-IFN- were used to stain cells according to BD PharMingen’s recommended method. Cells were analyzed by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA). In some experiments, cells cultured 2 days after restimulation were stained with FITC-labeled anti-CD4 and APC-labeled anti-IFN- for flow cytometric analysis. For separation of CD4⁺CD44^low and CD4⁺CD44^high populations, purified CD4⁺ T cells were stained with PE-labeled anti-CD44 Ab and sorted using a FACStar^Plus.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NOD CD4⁺ T cells differentiate into strong producers of IFN- in the absence of IL-12

Purified NOD T cells respond poorly to polyclonal TCR stimuli, as measured by the extent of proliferation and production of IL-2 and IL-4 (2). We wanted to determine whether NOD CD4⁺ T cells also produced low levels of IFN- in response to a polyclonal TCR stimulation. Purified CD4⁺ T cells from NOD, NOD.B10 Idd9, NOR, B6, B10, and BALB/c mice were stimulated with immobilized anti-CD3 plus irradiated splenocytes without (neutral conditions) or with either IL-12 plus anti-IL-4 (Th1 condition) or IL-4 plus anti-IFN- (Th2 condition) for 5 days and restimulated with anti-CD3 alone for 2 days. Culture fluids were harvested daily after the primary and secondary stimulation and analyzed for levels of IFN-. Surprisingly, NOD CD4⁺ T cells produced levels of IFN- under neutral conditions that were comparable to those of cells cultured under Th1 conditions (Fig. 1A). This was the case after both the primary and secondary stimulation. CD4⁺ T cells from NOD.B10 Idd9 and NOR mice also produced elevated levels of IFN- during culture under neutral conditions. The NOR strain is highly related to NOD, but is relatively free of autoimmune disease. The NOD.B10 Idd9 strain exhibits a reduced incidence of diabetes. These data indicate that the high level of IFN- production under neutral condition is a NOD genetic trait rather than a diabetes-related phenomena. As expected, IFN- production by B6, B10, and BALB/c T cells was highly dependent upon culture with IL-12.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. CD4+ T cells of NOD mice produce comparable levels of IFN- after activation under neutral or Th1 conditions. A, IFN- production by CD4+ T cells during the primary and secondary stimulation. CD4+ T cells were purified from spleens of NOD, NOD.B10 Idd9, NOR, B6, B10, and BALB/c mice and primarily (1o) stimulated with anti-CD3 (10 µg/ml) under neutral, Th1, or Th2 conditions. After 5 days, cultures were stimulated (2o) with anti-CD3 for 2 days. Levels of IFN- production from one of three representative experiments conducted in triplicate are shown (mean ± SD). B, Intracellular IFN- expression in CD4+ T cells. Purified CD4+ T cells were stimulated and restimulated, as described above. Cells were double stained with FITC-labeled anti-CD4 and APC-labeled anti-IFN- and analyzed by flow cytometry.

Elevated IFN- production and Th1 differentiation by NOD T cells require IL-2

A possible explanation for elevated IFN- expression after culture under neutral conditions may be the presence of Th1-promoting cytokines in the primary T cell cultures. To test this possibility, we examined levels of IL-2, IL-12, IL-18, TNF-, and IL-4 in primary cultures stimulated under neutral conditions. NOD and B6 CD4⁺ T cells produced comparable levels of IL-2 with a peak at day 2, whereas NOD cells produced less amounts of IL-4 than B6 cells (Fig. 2A). Cultures from both strains did not contain detectable levels of IL-12, IL-18, or TNF-. When neutralizing mAb against mouse IL-2, IL-12, IFN-, or IL-4 were added to the primary culture, IFN- production by NOD and B6 T cells in secondary culture was markedly inhibited by anti-IL-2, but not by the other Abs (Fig. 2B) or a control rat IgG2a (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. IL-2 is required for IFN- production by NOD T cells cultured under neutral conditions. A, Cytokine production by NOD and B6 CD4+ T cells during the primary stimulation. Purified CD4+ T cells were stimulated with anti-CD3 and irradiated splenocytes for 5 days. ELISA results of IL-2, IL-4, IL-12, and IFN- production in the culture supernatant from one of two representative experiments conducted in triplicate are shown (mean ± SD). B, Inhibition of IFN- production by anti-IL-2 mAb. Purified CD4+ T cells were stimulated with anti-CD3 plus irradiated splenocytes in the presence of 10 µg/ml of neutralizing anti-cytokine mAb. After 5 days, cultures were restimulated with anti-CD3 for 2 days. ELISA results of IFN- production in the culture supernatant after secondary stimulation from one of three representative experiments conducted in triplicate are shown (mean ± SD).

Segregation of IL-2-dependent IFN- expression from IL-2-dependent cell proliferation

Because IL-2 is a growth factor that stimulates T cells to undergo cell division, the suppression of IFN- production by NOD CD4⁺ T cells may result from inhibition of cell proliferation or differentiation. Surprisingly, inhibition of IFN- production by anti-IL-2 was readily dissociated from inhibition of cell proliferation based upon mAb concentration. Low concentrations of anti-IL-2 markedly inhibited IFN- production in cultures after primary stimulation (Fig. 3A) without a detectable effect on cell proliferation (Fig. 3B). Analysis of cell division and intracellular IFN- expression in primary cultures by flow cytometry showed comparable numbers of cell divisions in the presence or absence of anti-murine IL-2 mAb (1 µg/ml) (Fig. 3C), but marked inhibition of IFN- production in the presence of this amount of anti-IL-2 mAb (Fig. 3D). Similarly, we observed marked inhibition of IFN- production by NOR T cells by anti-IL-2 mAb (9.6 vs 0.9%). For the B6 strain, the percentage of IFN--producing cells without or with anti-IL-2 treatment was less than 1%. Because we wanted to directly compare the number of cell divisions with IFN- production on a per cell basis, these cultures were not restimulated with PMA and ionomycin or anti-CD3 before addition of inhibitors of protein export and flow cytometric analysis. These data indicate that IL-2-dependent IFN- expression and Th1 differentiation can be dissociated from IL-2-dependent T cell proliferation.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. IL-2-induced cell proliferation is uncoupled from IFN- production in NOD CD4+ T cells. A, Effect of anti-IL-2 on IFN- production. Cells were stimulated with anti-CD3 mAb for 5 days. ELISA results of IFN- production in the culture supernatant at day 5 from one of two representative experiments conducted in quadruplicate are shown (mean ± SD). B, Effect of anti-IL-2 mAb on cell proliferation. Purified CD4+ T cells were stimulated with anti-CD3 plus irradiated splenocytes under neutral condition in the presence of various amounts (0.3, 1, 3, or 10 µg/ml) of anti-IL-2. [3H]Thymidine was added to the cultures during the last 12 h before the cells were harvested at day 3. Results are from one of two representative experiments conducted in quadruplicate (mean ± SD). C and D, Segregation of IL-2-associated cell division and IFN- production. Purified CD4+ T cells were CFSE labeled and stimulated with anti-CD3 plus irradiated splenocytes in the absence or presence of anti-murine IL-2 (1 µg/ml) for 3 days. After blocking of cytokine secretion with brefeldin A for 4 h, the cells were stained and analyzed for cell division (C) and intracellular IFN- expression (D) by flow cytometry. Results are representative of three independent experiments.

Because IL-12 is a known Th1 differentiation factor, we compared IL-2 and IL-12 for their ability to direct CD4⁺ T cells from NOD, NOR, and B6 mice into Th1 lineage. MACS-purified CD4⁺ T cells were cultured under neutral conditions with anti-murine IL-2 mAb in the absence or presence of either human IL-2 or murine IL-12 during primary culture and IFN- examined at 2 days after restimulation. Supplementation with human IL-2 resulted in a marked increase in IFN- production by NOD and NOR T cells, but not by B6 T cells (Fig. 4). As expected, addition of IL-12 caused T cells from all the three strains to produce high levels of IFN- (Fig. 4). Therefore, IL-2 functioned as a potent Th1-driving cytokine as IL-12 did for NOD and NOR T cells, but not B6 T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. IL-2 and IL-12 are potent inducers of IFN- production by NOD T cells. Purified CD4+ T cells of NOD, NOR, and B6 mice were stimulated with anti-CD3 plus irradiated splenocytes under neutral conditions with anti-IL-2 mAb (10 µg/ml) and without (mock) or with cytokines (recombinant human IL-2 at 50 U/ml or mouse IL-12 at 5 ng/ml) for 5 days. ELISA results of IFN- levels in the culture supernatant at day 5 from one of three representative experiments conducted in triplicate are shown (mean ± SD).

IL-2-dependent IFN- production of NOD T cells is independent of previous activation status

To examine the responses of naive and memory CD4⁺ T cells to IL-2 and IL-12, CD4⁺CD44^low (naive) and CD4⁺CD44^high (memory) cells were purified by flow cytometry and stimulated with anti-CD3 and irradiated splenocytes plus either IL-2 or IL-12 for 5 days. IFN- production was determined 2 days after restimulation. Both naive and memory T cells of NOD mice differentiated into effector Th1 cells with comparable levels of IFN- in the presence of IL-2 or IL-12 (Fig. 5A). In contrast, only memory T cells of B6 mice responded with high levels of IFN- production to IL-2 or IL-12 stimulation (Fig. 5B). The optimal IFN- production by B6 naive T cells was IL-12 dependent (Fig. 5B).

View larger version (7K): [in this window] [in a new window]   FIGURE 5. IL-2 and IL-12 stimulate equivalent IFN- production by NOD CD4+CD44low and CD4+CD44high T cells. NOD and B6 CD4+CD44low and CD4+CD44high populations were isolated by flow cytometry. Cells were stimulated with anti-CD3 plus irradiated splenocytes in the presence of either murine IL-2 at 5 ng/ml or IL-12 at 5 ng/ml for 5 days, and then washed and restimulated with anti-CD3 for 2 days. ELISA results of IFN- production by NOD (A) and B6 (B) T cells after restimulation are from one of two representative experiments conducted in triplicate (mean ± SD).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Generally, IL-2 is thought to represent a Th1 cytokine. Its ability to drive Th1/Th2 differentiation has never been clearly delineated because of its critical role in promoting T cell division and survival. The requirement of IL-2 for development of Th2 effectors has been shown in vitro for T cells from BALB/c (22), B10.A (23), or B6 strains (22, 24). Neutralization of IL-2 (22, 23) or blockade of the IL-2R (24) during primary stimulation of CD4⁺ T cells results in diminished IL-4 expression. Our study extends the investigation of the function of IL-2 by showing that this cytokine can direct Th1 differentiation in the absence of IL-12 or IFN-. However, this ability is clearly strain dependent.

Our results support the hypothesized two-phase model for naive CD4⁺ T cell activation and differentiation. In this model, CD4⁺ T cells undergo an early phase of Ag-dependent cell activation and proliferation, followed by a late phase of cytokine-dependent effector cell differentiation (24, 25). In NOD T cells, these two events, cell proliferation and differentiation into effector cells, are both IL-2 dependent, but apparently require different levels of IL-2. This is indicated by suppression of cell proliferation and therefore IFN- production in the presence of high levels of anti-IL-2 mAb, but only inhibition of IFN- expression with low levels of anti-IL-2 mAb (Fig. 3, A and B). Uncoupling of IFN- production and cell proliferation in NOD T cells strongly argues that the elevated IFN- production by T cells cultured under neutral conditions (in the presence of autocrine IL-2) actually reflects Th1 differentiation.

In contrast to other strains, engagement of IL-2R or IL-12R is sufficient to direct Th1 differentiation by NOD T cells. IL-2R and IL-12R trigger distinct intracellular signaling pathways, e.g., STAT5 and STAT4 for IL-2 and IL-12, respectively (9, 26, 27). The important role of IL-12/STAT4 in Th1 development is underscored by studies of gene knockout mice (10, 11, 14). In contrast, STAT4-independent pathways of Th1 differentiation have also been demonstrated (28). T cells that exhibit both STAT4 and STAT6 gene deficiencies differentiate into IFN- producers or effector Th1 cells under neutral culture conditions. The IL-2-induced IFN- production and Th1 differentiation by NOD T cells, in vitro, support the existence of such a pathway. Consistent with this view, it has been shown that injection of human rIL-2 into DBA/1 mice, an animal model of rheumatoid arthritis, results in markedly increased IFN- production, in vivo (29). The comparable levels of IL-2 production and IL-2R expression by NOD and B6 CD4⁺ T cells suggest that signaling downstream of IL-2/IL-2R engagement plays a role in the IL-2-induced Th1 differentiation.

Normally, a rate-limiting factor in IFN- expression by T cells and Th1 differentiation, in vivo, is the requirement for both an antigenic stimulus and an inflammatory stimulus (6, 7, 8). The antigenic stimulus is required to stimulate expansion of T cells (30), and the inflammatory stimulus is required to stimulate IL-12 production by macrophages or dendritic cells (31). Presumably, these checks limit the ability of T cells to differentiate into Th1 cells in an inappropriate context, such as in response to self Ag. In principle, this restriction may serve as a safeguard against development of autoimmunity. In contrast, NOD T cells are able to bypass this potential safeguard and differentiate, in vitro, into IFN- producers using a simple autocrine pathway because T cells can produce their own differentiation factor, IL-2. These differences may contribute to the ability of NOD T cells to easily differentiate into IFN- producers that invade islets in the pancreas in the apparent absence of an inflammatory stimulus.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant DK 58765 and the Juvenile Diabetes Research Foundation.

² Address correspondence and reprint requests to Dr. Thomas M. Aune, MCN T3219, Vanderbilt University Medical Center, 21st and Garland Avenue, Nashville, TN 37232. E-mail address: Thomas.M.Aune{at}vanderbilt.edu

³ Abbreviations used in this paper: NOD, nonobese diabetic; NOR, nonobese diabetes-resistant.

Received for publication March 19, 2002. Accepted for publication November 11, 2002.

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