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. 2003 Sep;110(1):30–37. doi: 10.1046/j.1365-2567.2003.01701.x

Interleukin-12 and interleukin-2-induced invariant natural killer T-cell cytokine secretion and perforin expression independent of T-cell receptor activation

Runhua Hou *, Olga Goloubeva †, Donna S Neuberg †, Jack L Strominger *, S Brian Wilson *
PMCID: PMC1783023  PMID: 12941138

Abstract

Human invariant natural killer (iNK) T cells expressing an invariant Vα24-Jα15 T-cell receptor (TCR) are thought to be important regulators of autoimmunity and tumour surveillance. Two major subsets of iNK T cells, CD4+ or CD4− CD8− are known to exist, but the in vivo importance of CD4 expression is unclear. Since interleukin-12 (IL-12) is a key iNK T-cell-activating cytokine, the effect of IL-12 plus or minus the T-cell growth factor IL-2 on a large panel of CD4+ versus CD4− CD8− iNK T-cell clones was examined. Strikingly, IL-12 and IL-2 significantly activated iNK T cells to secrete IL-4, interferon-γ and granulocyte–macrophage colony-stimulating factor, and up-regulated perforin expression in the absence of TCR stimulation. Furthermore, IL-2 and IL-12 treatment resulted in a preferential increase in apoptosis of CD4− CD8− clones. Thus, independent of TCR activation, IL-2 and IL-12 can directly activate iNK T cells and provide a selective advantage to the CD4+ iNK T-cell population.

Introduction

Human invariant natural killer (iNK) T cells are a group of T cells that have a restricted usage of invariant Vα24-Jα15 (formerly JαQ) T-cell receptor (TCR) and express several cell-surface proteins found on NK cells.1,2 These T cells are restricted by the non-polymorphic class Ib molecule CD1d through presentation of glycolipid antigen,3,4 resulting in the burst secretion of several cytokines.5 Despite their function as members of the innate immune system,6–8 iNK T cells have been shown to control the metastases of several tumours9–11 and the development of type I diabetes, rheumatoid arthritis and other autoimmune diseases.12–15

Interleukin-12 (IL-12) can induce interferon-γ (IFN-γ) secretion in activated T, iNK T and NK cells, augment NK-cell mediated cytotoxicity, and facilitate the expansion of T helper type 1 (Th1) -biased CD4 and CD8 T effector cells.16,17 Dendritic cells are a major source of IL-12 and through intimate contact with T cells during initiation of a primary immune responses, dendritic cells are thought to be major regulators of T-cell Th1 or Th2 polarization.18–20 Interaction of dendritic cells and iNK T cells leads to the activation of iNK T cells, and secretion of many cytokines such as IL-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) that are important for the differentiation of myeloid dendritic cells.21–23 Collectively, these findings suggest that iNK T-cell and dendritic cell cross talk is critical for shaping the subsequent adaptive immune response. Thus the source and quantity of IL-12 is expected to have a significant impact on the activation of iNK T cells. To investigate the ability of IL-12 to activate iNK T cells in the absence of TCR stimulation, this cytokine was used to stimulate a large panel of CD4+ and CD4− CD8− iNK T cells. IL-12 treatment, when used together with IL-2, potently activated cytokine secretion by both CD4+ and CD4− CD8− iNK T cells, but resulted in a survival advantage for CD4+ iNK T cells.

Materials and methods

Cell culture and the generation of iNK T-cell clones

Invariant NK T-cell clones were generated according to established methods.15 Briefly, human peripheral blood mononuclear cells were isolated by Ficoll–Paque gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) and stained with different antibodies. Then, positive cells were single-cell sorted into 96-well plates by a MoFlow® (Becton Dickinson, Mountainview, CA). For the generation of iNK T-cell clones, cells were stained with Vα24/Vβ11, Vα24/6B11, or Vβ11/6B11 antibody pairs. 6B11 is an antibody that specifically recognizes invariant iNK T cells (Exley et al., manuscript in preparation). The sorted cells were initially stimulated with 1 μg/ml phytohaemagglutinin (Murex Biotech Ltd, Dartford, UK), 10 Units/ml IL-2 (Roche, Mannheim, Germany) and 10 Units/ml IL-7 (Roche) along with 1 × 105 irradiated allogenic feeder cells (5000 rads) in RPMI-1640 medium supplemented with 10% autologous serum, 100 Units/ml streptomycin/penicillin (Biowhittaker, Walkersville, MD), and 200 mm l-glutamine. Cells were fed every 2–3 days, with exchange of half of the volume with fresh RPMI-1640 complete medium supplemented with IL-2 and IL-7, as described above. The established iNK T-cell and other T-cell clones were stored in liquid nitrogen and re-stimulated once with 0·1 μg/ml anti-CD3 (Ancell, Bayport, MN) along with irradiated allogenic feeder cells (5000 rads) in the presence of 50 Units/ml of IL-2 2 weeks before, and rested overnight prior to the experiment.

Cytokine treatment

Two weeks after re-stimulation T-cell clones were cultured with 50 Units/ml IL-2 (Roche), 5 ng/ml IL-12 (R & D Systems, Minneapolis, MN), the combination of IL-2 and IL-12 at the above concentration, or medium alone in 96-well plates with 1 × 105 cells per well. In each experiment, triplicate wells for each cytokine treatment for each clone were prepared. The final value was the average of four independent experiments. At days 1, 3 and 5 cells were collected and used for fluorescence-activated cell sorter (FACS) analysis and their supernatants were used for cytokine analysis. In the same experiment, at days 1, 3 and 5, 1 μCi [3H]thymidine was also added into another set of plates for a proliferation assay. Proliferation was determined 18 hr after pulsing with thymidine.

Cell stimulation

The iNK T-cell clones were stimulated with purified 1 μg/ml anti-CD3 (Ancell) for 24 hr, then the supernatants were collected for enzyme-linked immunosorbent assay (ELISA). The same amount of isotype control (Sigma, St. Louis, MO) antibody was used at the same time.

Antibodies and FACS analysis

Fluorescein isothiocyanate (FITC) -labelled anti-CD3, cychrome™-labelled anti-CD3, anti-CD4, anti-CD8 were purchased from Pharmingen (San Diego, CA), FITC- or phycoerythrin- (PE) labelled anti-Vα24, PE-labelled anti-Vβ11 antibodies were obtained from Coulter-Immunotech (Miami, FL). A PE-labelled anti-perforin staining kit including isotype control antibody was purchased from Pharmingen. Purified IL-12R-β1 and IL-12R-β2 antibodies were kindly provided by Dr Jerome Ritz (Dana Farber Cancer Institute). Immunofluorescence staining was performed according to standard procedures. For intracellular staining, cells were fixed with 4% paraformaldehyde on ice for 30 min, washed twice, and then stained with labelled antibodies in phosphate-buffered saline (PBS) with 0·1% saponin for 30 min on ice. Afterwards, cells were washed with PBS buffer containing 0·1% saponin. Samples were analysed on a FACScan using the cellquest program (Becton Dickinson). Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide (Alexis Biochemicals, San Diego, CA) according to the manufacturer's instructions. The frequency of annexin-V-positive but propidium-iodide-negative cells under different treatments was used for comparison.

ELISA

Purified and biotinylated IL-4, IFN-γ and GM-CSF antibody pairs were purchased from Pharmingen and R & D Systems and used according to the manufacturer's instructions. At a 1 to 2000 dilution, DELFIA®, Eu-labelled streptavidin was used as the detection agent (Wallac, Turku, Finland). The results were obtained on a DELFIA® fluorometry plate reader (Wallac).

Statistical analysis

The Linear Mixed-Effects Models approach for replicated design was used to analyse the data.24 Random effects for clones, as well as for the interactions between clones and the cytokine type, were included in the model. The square root transformation of the original measurements was applied to the apoptosis data. The rest of the data were log-transformed (base 10) for the analyses to ensure induced normality in the data. Multiple comparisons between the groups' means were performed using Tukey–Kramer adjustment. The P-values reported are two-sided. A P-value less than 0·05 was assumed to indicate statistically significant difference between groups.

Results

IL-12 is a TCR-independent stimulus for cytokine secretion from iNK T cells

IL-12 is an important component of iNK T-cell–dendritic cell interaction during the early events of immune activation.23 This cytokine also potently biases the adaptive immune response towards a Th1 phenotype. To determine whether there is any difference between CD4+ and CD4− CD8− iNK T cells in response to IL-12 treatment, the amounts of secreted IL-4, IFN-γ and GM-CSF were measured after cell stimulation. The combination of IL-2 and IL-12 was the most potent treatment for all three of these cytokines. At days 1, 3, or 5 post-treatment, there was a significant increase in IFN-γ secretion when IL-2 and IL-12 were used in combination (P < 0·0001 for IFN-γ in all comparisons, Fig. 1a,b). On day 1, the IFN-γ secreted by untreated and IL-2/IL-12-treated CD4+ iNK T cells was 13·9 ± 3·6 and 1968·1 ± 205 pg/ml, respectively. CD4− CD8− iNK T cells treated with IL-2/IL-12 produced 1924·7 ± 101 pg/ml IFN-γ, compared to 19·1 ± 2·9 pg/ml by untreated cells. Interestingly, this combined stimulus was comparable to anti-CD3 stimulation (Fig. 2a). IL-12 combined with IL-2 also stimulated IL-4 secretion from both CD4+ and CD4− CD8− iNK T-cell clones (P < 0·01 for all different groups of comparisons, Fig. 1c,d) though the effect was not as potent as anti-CD3 activation (Fig. 2b). On day 1, the IL-4 secreted by untreated and IL-2/IL-12 treated CD4+ iNK T cells was 19·1 ± 1·91 and 140·6 ± 19·3 pg/ml, respectively. CD4− CD8− iNK T cells treated with IL-2/IL-12 had 160·6 ± 32·9 pg/ml IL-4 compared to 24·2 ± 2·8 pg/ml by untreated cells. Similar results were obtained on day 3 and day 5. IL-2 and IL-12 significantly stimulated GM-CSF secretion from both CD4+ and CD4− CD8− iNK T-cell clones as well (P < 0·01 for all different groups of comparisons, Fig. 1e,f) though slightly lower than anti-CD3 stimulation (Fig. 2c). On day 1, the GM-CSF secreted by untreated and IL-2/IL-12-treated CD4 + iNK T cells was 55·7 ± 10·9 and 752·8 ± 69·9 pg/ml, respectively. CD4− CD8− iNK T cells treated with IL-2/IL-12 produced 596·2 ± 89·2 pg/ml GM-CSF compared to 102·5 ± 7·3 pg/ml by untreated cells. Similar changes were observed on day 3 and day 5 but with much larger increases. Thus, in the presence of IL-2, IL-12 greatly stimulated the secretion of IL-4, IFN-γ and GM-CSF from quiescent iNK T-cell clones, even in the absence of TCR ligation.

Figure 1.

Figure 1

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Interleukin-2 and IL-12 synergistically promoted iNK T-cell clones to secrete IFN-γ, IL-4 and GM-CSF. CD4+ (a,c,e) and CD4− CD8− (b,d,f) iNK T-cell clones were treated with medium alone, IL-2, IL-12, or IL-2 plus IL-12 for 1, 3, and 5 days. At each time-point, supernatants were collected and IFN-γ (a,b), IL-4 (c,d), or GM-CSF (e,f) were measured by ELISA. Data shown were obtained from 10 CD4+ and 12 CD4− CD8− iNK T-cell clones. In each experiment, triplicate wells of cell culture were prepared for each clone with each cytokine treatment. The data shown were the final value of four independent experiments.

Figure 2.

Figure 2

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CD4+ and CD4− CD8− iNK T cells responded well to anti-CD3 stimulation. Resting CD4+ and CD4− CD8− iNK T-cell clones were treated with 1 μg/ml immunoglobulin G or anti-CD3 for 24 hr then supernatant was collected and analysed for IFN-γ (a), IL-4 (b) and GM-CSF (c). Data shown were obtained from the same groups of clones as in Fig. 1.

Compared to medium alone, IL-12 stimulated IFN-γ secretion by CD4− CD8− (P < 0·001 at 1, 3, or 5 days after cytokine treatment, Fig. 1b), and CD4+ iNK T-cell clones (P = 0·008, 0·006, 0·053 at 1, 3, or 5 days after cytokine treatment, Fig. 1a). Similarly, treatment with IL-2 stimulated IFN-γ secretion by CD4− CD8− iNK T-cell clones at all days tested (P < 0·01, Fig. 1b). However, IL-2 had only a marginal effect on IFN-γ secretion by CD4+ iNK T cells.

In contrast to the modest effects of IL-2 on IFN-γ and IL-4 secretion, treatment with IL-2 significantly enhanced GM-CSF secretion by CD4+ iNK T cells (P = 0·034, 0·0001, 0·0004 for day 1, 3, or 5, respectively, Fig. 1e) with only a modest effect on day 3 or day 5 for CD4− CD8− iNK T cells (P = 0·036, 0·021 for day 3 and 5 accordingly, Fig. 1f). Thus, in the presence of IL-2, IL-12 stimulated IFN-γ, IL-4 and GM-CSF, whereas IL-2 independently stimulated GM-CSF secretion by CD4+ clones.

Treatment with IL-12 selects for CD4+ iNK T cells

Next, we examined the effect of cytokine treatment on cytokine-induced rates of proliferation and apoptosis. IL-2 stimulated cell division in both CD4+ and CD4− CD8− clones (Fig. 3a,b). The combination of IL-2 and IL-12 significantly inhibited the proliferative response of CD4− CD8− iNK T cells relative to IL-2 alone (P < 0·01 on days 3 and 5, Fig. 3b). In contrast, the addition of IL-12 to IL-2 had only a modest inhibitory effect on the IL-2-driven proliferative response of CD4+ iNK T cells on day 5 (P < 0·05, Fig. 3a).

Figure 3.

Figure 3

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IL-2 preferentially promoted iNK T-cell proliferation. CD4+ (a) and CD4− CD8− (b) iNK T-cell clones were treated with medium alone, IL-2, IL-12, or IL-2 plus IL-12 for 1, 3, and 5 days. At each time-point, 1 μCi [3H]thymidine was added and proliferation was determined 18 hr later. Data shown were obtained from 10 CD4+ and 12 CD4− CD8− iNK T-cell clones. In each experiment, triplicate wells of cell culture were prepared for each clone with each cytokine treatment. The data shown was the final value of four independent experiments.

To investigate whether the inhibition of IL-2-induced proliferation in CD4− CD8− iNK T-cell clones by IL-12 was in part the result of a selective increase in cell death, the rate of apoptosis in response to IL-12 was determined for CD4+ and CD4− CD8− iNK T-cell clones. As expected, IL-2 treatment lead to the lowest apoptosis rate in both CD4+ and CD4− CD8− iNK T cells on day 3 and 5 (P < 0·01 for all days, Fig. 4a,b). Treatment with IL-2 and IL-12 decreased the survival of CD4− CD8− iNK T-cell clones when compared to IL-2 alone (P < 0·01 for all days, Fig. 4b). In contrast, treatment with IL-2 and IL-12 did not significantly increase the rate of apoptosis (Fig. 4a) of CD4+ iNK T-cell clones relative to IL-2 alone. When comparing CD4+ to CD4− CD8− iNK T cells treated with IL-2 and IL-12, CD4+ iNK T cells survived to a significantly greater extent than did CD4− CD8− iNK T cells (P = 0·001, 0·02 on day 3 and day 5, Fig. 4c,d). On day 3, the apoptosis rate was 7·7 ± 2·0% for CD4+ iNK T cells compared to 23·2 ± 3·7% for CD4− CD8− iNK T cells. On day 5, the apoptosis rate was 13·2 ± 3·2% for CD4+ and 28·6 ± 4·1% for CD4− CD8− iNK T cells. This effect could not be explained on the basis of differential IL-12 receptor β1 or β2 expression, as these receptors were equivalently expressed on CD4+ and CD4− CD8− iNK T cells (data not shown). Thus, CD4− CD8− iNK T cells are more susceptible to IL-12-induced apoptosis than CD4+ iNK T-cell clones in the presence of IL-2.

Figure 4.

Figure 4

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Combined treatment with IL-2 and IL-12 selectively induced apoptosis in CD4− CD8− iNK T cells. (a) Ten CD4+ iNK T cell clones were treated with medium, IL-2, IL-12, or IL-2 plus IL-12 for 1, 3 and 5 days. At each time-point, cells were collected and stained with annexin V and propidium iodide. The percentages of annexin-V-positive but PI-negative cells were determined. The data shown was the final value of four independent experiments. (b) Twelve CD4− CD8− iNK T cell clones were treated with cytokines and data were plotted as described in (a). The data shown were the final value of four independent experiments. (c) A direct comparison of 10 CD4+ and 12 CD4− CD8− iNK T-cell clones treated with IL-2 and IL-12 for 3 and 5 days is shown. (d) FACS profiles of representative clones treated with IL-2 and IL-12 on day 5 is shown. Apoptosis percentage was shown in RLQ.

CD4− CD8− iNK T-cell clones have a higher level of intracellular perforin expression

Both CD4+ and CD4− CD8− iNK T cells are able to kill target cells in a perforin-dependent fashion25,26 and exposure to IL-2, IL-12, or α-GalCer enhances the cytotoxicity of iNK T cells.23,27 To determine whether perforin expression was regulated in parallel to cytokine secretion, cytokine-induced changes in intracellular perforin were determined by FACS analysis. Treatment of CD4+ or CD4− CD8− iNK T clones with IL-2 or IL-12 alone significantly stimulated the intracellular perforin expression (P < 0·01 for all groups, Fig. 5). The stimulation of perforin expression was more pronounced in CD4− CD8− iNK T-cell clones (for IL-2 and IL-12 on day 3 and day 5 all P < 0·001, Fig. 5b) than for CD4+ (P < 0·01 for IL-2 and IL-12 on day 3 and day 5, Fig. 5a). The combined treatment synergistically increased perforin expression in CD4+ and CD4− CD8− iNK T cells at all time-points tested (P < 0·0001). However, both IL-2 and IL-12 induced perforin expression to an equivalent but modest extent when compared to the combination treatment of IL-2 and IL-12.

Figure 5.

Figure 5

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Treatment with either IL-2 or IL-12 independently induces perforin expression, but induction occurs to a significantly greater extent in CD4− CD8− iNK T-cell clones. Ten CD4+ (a) and 12 CD4− CD8− (b) iNK T-cell clones were treated with medium, IL-2, IL-12, or IL-2 plus IL-12 for 1, 3, and 5 days. At each time-point, cells were collected and stained for intracellular perforin. (c) Intracellular staining for perforin expression of representative CD4+ and CD4− CD8− iNK T-cell clones treated with IL-2 and IL-12 for 5 days is shown. The dotted line, the thin line and the thick line represent the isotype control, a CD4+ iNK T-cell clone and a CD4− CD8− iNK T-cell clone, respectively.

Irrespective of the stimulus, CD4− CD8− clones quantitatively expressed more perforin protein than CD4+ clones. Thus, treatment with IL-2 and IL-12 resulted in a greater NK-like profile for CD4− CD8− clones than for CD4+ clones (Fig. 5c).

Discussion

IL-12 is thought to be a key participant of in vivo iNK T-cell activation, and in the generation of Th1-like T cells.11,23 Despite its major role in Th1 biasing, there are studies demonstrating IL-12-induced Th2 responses.28–30 Whether IL-12 suppresses or enhances Th2 responses appears to depend both on the dose of cytokine and on the maturational state of the responding T cells.30 The combination of IL-12 and IL-18 has been shown to stimulate directly IFN-γ but not IL-4 secretion from IL-2-treated NK T-cell lines.31 Here we extended that observation to include GM-CSF from both CD4+ and CD4− CD8− clones. Previous reports suggest that CD4− CD8− clones were capable of secreting IL-4 in response to TCR stimulation.5,15,21,22,26,32–34 Moreover, it has been reported that when analysed ex vivo after a brief in vivo stimulation with α-GalCer both the CD4+ and CD4− CD8− murine liver iNK T-cell subsets had intracellular IL-4.35 In contrast, two recent publications reported that human CD4− CD8− cells stimulated with phorbol 12-myristate 13-acetate and ionomycin ex vivo did not secret IL-4 and that the secretion of Th2-like cytokines was limited to the CD4+ subset.36,37 The reason for this discrepancy is not clear and may be the result of the significant differences in experimental methodology used. IL-4 and GM-CSF are widely used to differentiate human monocytes into dendritic cells in vitro, they also enhance bioactive IL-12 production induced by bacterial and T-cell-derived stimuli from dendritic cells while inhibiting the antagonistic form of IL-12.38 Thus, the ability of iNK T cells to secrete IL-4 and GM-CSF after stimulation with IL-12 and IL-2 suggests that those T cells are capable of promoting the differentiation of dendritic cells in an antigen-independent fashion.

Major histocompatibility complex class I- or II-restricted T cells respond poorly, if at all, to IL-12 unless they are activated through ligation of the TCR–CD3 complex.39,40 Such a combined stimulus up-regulates the IL-12 receptor β1 and augments the responsiveness to IL-12.41,42 It is interesting that iNK T cells were activated without TCR ligation. The importance of this phenomenon might be related to the activation of innate immunity. There are now several reports that iNK T cells provide a protective role against intracellular parasites and this protective function was dependent on IFN-γ secreted by activated iNK T cells.6–8,43 Dendritic cells and macrophages secrete IL-12 after infection with intracellular parasites.43–49 Dendritic cells also secreted IL-2 after encounter with bacteria.43,45 Therefore the ability of iNK T cells to secrete cytokines such as IL-4, GM-CSF, and IFN-γ in the absence of CD3/TCR cross-linking may be critical to initiating the host response to infectious pathogens.

Mature T cells can proliferate in response to antigen, IL-2, or IL-12 stimulation.39,50,51 In the absence of TCR activation, IL-2 prevents iNK T-cell death. In contrast to conventional T cells where IL-12 prevented activation-induced cell death,52–55 IL-12 significantly enhanced apoptosis of CD4− CD8− iNK T-cell clones relative to CD4+ clones in the presence of IL-2. This effect of IL-12 in vitro correlates with the dramatic loss of iNK T cells in mice treated with IL-12 in vivo.56 Therefore, IL-12 reduced the rates of apoptosis but did not inhibit proliferation of CD4+ iNK T cells suggesting a potential mechanism for controlling the ratio of CD4+ to CD4− CD8− iNK T cells. Various animal models and in vitro experiments have demonstrated that IL-2 and IL-12 augmented the ability of NK cells and T cells to kill target cells.17,57 IL-2, IL-12, or the combination treatment significantly increased the perforin expression of CD4− CD8− relative to CD4+ iNK T cells, conferring, as was previously suggested, preferential cytotoxic roles for CD4− CD8− iNK T cells.37

Since CD1d expression markedly increased the susceptibility to cytotoxicity by iNK T cells and dendritic cells express CD1d,10,21,23,58 if CD4− CD8− iNK T cells dominate, their cytotoxicity towards dendritic cells may lead to the deletion of antigen-presenting cells. In contrast, if CD4+ iNK T cells dominate, the GM-CSF, IL-4 generated by CD4+ iNK T cells would mature more dendritic cells and enforce the cycle of cell interaction. Because there are strikingly high interindividual variations in terms of the total number of iNK T cells and the CD4+ : CD4− CD8− ratio from one donor to another,59 we speculate that the CD4+ : CD4− CD8− ratio influences the differentiation of conventional T cells and dendritic cells. Interaction of iNK T cells and dendritic cells in the presence of α-galactosylceramide stimulated the secretion of IL-12.23 Since IL-12 could bias iNK T cells into Th1 secretors, the outcome of the interaction between dendritic cells and iNK T cells in vivo may depend on the number of CD4+ or CD4− CD8− iNK T cells and the ratio of CD4+ : CD4− CD8− iNK T cells recruited, prior antigenic challenge and cytokines in the environment.

Acknowledgments

The authors would like to thank Drs Steven Balk and Mark Exley for critical reading of the manuscript. This work was supported by NIH grant R01 AI45051 (to S. B. Wilson).

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