IL-15 is a cytokine playing a crucial role in the function of immune cells, including NK and CD8 T cells. In this study, we demonstrated that in vivo, in mice, IL-15–prestimulated NK cells were no longer able to respond to a second cycle of IL-15 stimulation. This was illustrated by defects in cell maturation, proliferation, and activation, seemingly linked to the environment surrounding NK cells but not related to the presence of CD4 regulatory T cells, TGF-β, or IL-10. Moreover, NK cells from immunodeficient mice could respond to two cycles of IL-15 stimulation, whereas an adoptive transfer of CD44+CD8+ cells impaired their responsiveness to the second cycle. Conversely, in immunocompetent mice, NK cell responsiveness to a second IL-15 stimulation was restored by the depletion of CD8+ cells. These biological findings refine our understanding of the complex mode of action of NK cells in vivo, and they should be taken into consideration for IL-15–based therapy.

This article is featured in In This Issue, p.323

Immunotherapy aims at exploiting our natural immune defense mechanisms to either boost the clearance of infected or tumor cells, or dampen inappropriate or unwanted activation of the immune system. Immunotherapy brings a wild array of new strategies that complement more classical treatments. Indeed, whereas chemotherapies preferentially target cancer cells, immunotherapies aim at restoring the immune system’s ability to effectively fight against cancer cells. Among the targeted immune cells, NK cells are of particular interest, representing one of the major cell types involved in the elimination of intracellular pathogens, virally infected cells, and tumor cells. They are able to kill tumor cells while sparing normal cells by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-12, IL-18, and IL-15 (1). The main cytotoxic pathway involves the release of cytotoxic proteins such as perforin and granzymes. In addition to their cytotoxic abilities, NK cells also rapidly produce cytokines, including IFN-γ and TNF-α. Given these properties, diverse therapeutic approaches are explored to enhance their efficiency. Three major NK cell–based therapies have been developed, including adoptive transfer of wild-type (wt) or genetically modified NK cells, as well as cytokine infusion (2). The cytokine infusion approach consists in the administration of cytokines known to induce activation and proliferation of NK cells. Among these cytokines, IL-2 (proleukin) has been approved since 1992 by the Food and Drug Administration for the treatment of renal carcinomas and metastatic melanomas. However, IL-2–based treatment presents major side effects such as vascular leak syndrome and regulatory T cell expansion, limiting its use.

More than two decades ago, IL-15 was discovered as a T cell growth factor and was assigned to the IL-2 family (3). The two cytokines share particular functions, including the generation of cytotoxic T lymphocytes and NK cells and their proliferation (410), mainly due to the fact that they share a heterodimeric receptor formed by the IL-2/15Rβ (CD122) and common γ (CD132) chains. IL-15 or IL-2 binding to the CD122/CD132 receptor induces activation of the JAK/STAT pathway leading to the recruitment, phosphorylation, and activation of Jak1/Jak3 and Stat5/Stat3 (11). Moreover, Stat5 phosphorylation is pivotal to maintain NK and CD8 T cell survival at the periphery (12, 13). In addition to these similarities, IL-2 and IL-15 specificities are conferred by their respective α receptor chains, IL-2Rα and IL-15Rα respectively. Whereas IL-2 and IL-15 can act as soluble entities, the dominant mechanism by which IL-15 exerts its action is trans-presentation, a mechanism by which a membrane-bound IL-15Rα/IL-15 complex expressed on a producing cell is presented in trans to a neighboring cell expressing the CD122/CD132 heterodimeric receptor (14, 15). In contrast to IL-2, IL-15 has no marked effects on regulatory T cells, and it inhibits the activation-induced cell death induced by IL-2 (14, 16, 17). These properties, in addition to its ability to stimulate both the innate and adaptive systems, have brought IL-15 to become one of the most promising molecules in antitumor immunotherapy (18). A first in-human study conducted by Waldmann and colleagues (19) has shown that IL-15 could be safely administered to patients and increased their circulating levels of NK, CD8, and γδ T cells. Given the IL-15 potential as an immunotherapeutic drug, several groups have developed IL-15 agonists, mostly based on the IL-15/IL-15Rα complex that mimics trans-presentation. Two of these complexes are composed of a human IL-15, either wt or mutant (whereby the amino acid asparagine at position 72 is substituted by an aspartic acid, leading to an increased binding to the CD122 chain receptor), complexed to an IL-15Rα-chain and further associated with an Fc fragment from a human IgG1 (20, 21). Another complex consists of a human IL-15 fused via a linker to a soluble IL-15Rα sushi domain (22). These three complexes display improved pharmacokinetics and enhanced capacities to activate immune cells, and they are currently under evaluation for the treatment of cancer.

In cancer immunotherapy, treatment schedules are usually organized in multiple injection cycles (23), raising the question of how immune cells respond after such repeated cycles. In our study, we have tested whether the abilities of IL-15 or IL-15 agonists to upregulate functional NK cells would be maintained along repeated injections. We show that although NK cells respond to a single stimulation cycle with IL-15 or IL-15/IL-15Rα complexes, they become hyporesponsive and fail to expand after a second cycle of stimulation. This study further investigates the cellular and molecular basis of this NK cell hyporesponsiveness.

Recombinant IL-2 (Proleukin) was purchased from Chiron. Recombinant human IL-15 was purchased from Miltenyi Biotec. IL-15/IL-15Rα fusion protein (IL-15clpx) was described previously (22). The fusion protein linking IL-15clpx to the COOH terminus of an Fc fragment from a human IgG1 (Fc-15clpx) was produced by Evitria (Schlieren, Switzerland).

Abs raised against NKp46 (clone 29A1.4), NK1.1 (clone PK136), CD3 (clone 145-2C11), CD11b (M1/70), CD27 (clone LG.3A10), KLRG1 (clone 2F1), CD8a (clone 53-6.7), CD44 (clone IM7), CD62L (clone MEL-14), CD122 (clone TM-β1), CD132 (clone TUGm2), Ly49D (clone 4E5), NKG2D (clone CX5), CD69 (clone H1.2F3), IFN-γ (clone XMG1.2), p-Stat5 (pY694), Ki67 (clone SolA15), CD4 (clone H192.19), and CD25 (clone 7D4) were purchased from BD Biosciences, eBioscience, and Miltenyi Biotec.

Murine B16F10 melanoma cells were obtained from American Type Culture Collection and maintained in DMEM, modified to contain 4500 mg/l glucose, 1 mM sodium pyruvate, and 1500 mg/l sodium bicarbonate and supplemented with 2 mM l-glutamine, 100 IU/ml penicillin/streptomycin, and 10% FBS. B16F10 cells were harvested using trypsin, and for in vivo experiments, cells were resuspended in PBS alone. Cells were cultured in a humidified incubator with 5% CO2 at 37°C. Single-cell suspensions from spleens were obtained and cultured in R10 medium consisting of RPMI 1640 medium supplemented with 1 mM sodium/pyruvate, 1 mM nonessential amino acids, 100 IU/ml penicillin/streptomycin, 2 mM l-glutamine, and 10% FBS (Life Technologies, Saint Aubin, France) and 10 μM 2-ME.

Female C57BL/6, NMRI, and NMRI-nude mice were purchased from Janvier Labs (Le Genest St. Isle, France) and used between 8 and 13 wk of age. Each strain was cohoused during the experiments. All experiments were carried out in accordance with French and European laws and regulations and approved by the French Animal Experimentation Ethics Committee (no. 6).

To test whether NK and CD44+ CD8 T cells could be restimulated in vivo, we designed a model where mice were first injected with PBS, IL-15clpx, or Fc-15clpx at day 1 and day 3 (first cycle). A resting period of either 10, 20 or 50 d was respected. Then, mice were restimulated with another 2 d of injections 1 d apart with PBS, IL-2, IL-15, IL-15clpx, or Fc-15clpx (second cycle). Unless otherwise indicated, mice received an i.p. injection of 5 μg of IL-2, 10 μg of IL-15, 2 μg of IL-15clpx, or 3 μg of Fc-15clpx. When LPS (L6529; Sigma-Aldrich) was used at the first or the second cycle, one injection of 70 μg per mouse was performed. Mice were sacrificed 24 h following the last injection, 48 h when LPS was injected, or after the adapted resting period. For CD69 and p-Stat5 staining, mice were sacrificed 2 h after the last injection instead of 24 h later.

For adoptive transfer of splenocytes, cells from donor mice were isolated as a single-cell suspension, cleared for RBCs, labeled with BD Horizon violet proliferation dye 450 (VPD-450; BD Biosciences), and i.v. injected (12 × 106 cells) into recipient mice.

For adoptive transfer of T lymphocytes, splenocytes from donor mice were purified using a pan–T cell isolation kit II (Miltenyi Biotec) and i.v. injected (3 × 106 cells) into recipient mice, 24 h before the second cycle of stimulation.

For adoptive transfer of CD44CD8+ cells or CD44+CD8+ cells, splenocytes were first purified using CD8 (Ly-2) MicroBeads (Miltenyi Biotec). After CD8 enrichment, CD44 and CD44+ cells were sorted using an EasySep biotin positive selection kit (Stemcell Technologies) and anti-CD44 biotin Ab (clone IM7; BD Biosciences). Purity was confirmed by flow cytometry with anti-CD44 (clone KM114). Each recipient mouse received 1.5 × 106 enriched cells 24 h before the second cycle of stimulation.

To deplete either CD8+, CD4+, or CD25+ cells, blocking Abs were injected i.p. into mice 6 d before the second stimulation for 3 consecutive days with 100 μg per injection of anti-mouse CD8a mAb (clone 2.43; Bio X Cell), anti-mouse CD4 mAb (clone GK1.5; Bio X Cell), or anti-mouse CD25 mAb (clone PC-61.5.3; Bio X Cell), respectively. After another 3 d without injection, treatment with each anti-mouse Ab was prosecuted with one injection per day, 2 d apart until sacrifice. Cellular depletions were confirmed by flow cytometry with anti-CD8a (clone 53-6.7), anti-CD4 (clone H129.19), and anti-CD25 (clone 7D4).

To neutralize TGF-β, 250 μg per injection of anti–TGF-β mAb (clone 1D11; Bio X Cell) (24) or its isotype control (clone MOPC-1; Bio X Cell) were i.p. administered every other day for 6 d, 4 d before the second cycle of stimulation.

To neutralize the action of IL-10, 500 μg per injection of anti–IL-10R Ab (clone 1B1.3A; Bio X Cell) (25) or its isotype control (clone HRPN; Bio X Cell) was i.p. administered twice to mice. Each injection was performed 24 h before PBS or cytokine treatment.

A first cycle of IL-15clpx was administered to C57BL/6 mice at day 1 and day 3; then, at day 21, 1 × 105 B16F10 melanoma cells were i.v. injected. The graft was followed with another cycle of PBS or IL-15clpx at day 22 and day 24. Mice were sacrificed at day 42. For each cycle, 2 μg of IL-15clpx per injection was i.p. administered to mice.

Single-cell suspensions from spleen and blood cleared from RBCs, bone marrow, lungs, and liver were obtained and stained. Before staining, Fc receptors were blocked using anti-CD16/32 Ab (BD Biosciences). Then, cells were incubated with Abs for cell surface staining. For p-Stat5 staining, cells were fixed and permeabilized after cell surface staining according to the manufacturer using Lyse/Fix Buffer and Perm Buffer II (BD Biosciences). For IFN-γ staining, cells were fixed and permeabilized after cell surface staining according to the manufacturer using Cytofix/Cytoperm (BD Biosciences). For Ki67 staining, cells were fixed and permeabilized after cell surface staining according to the Anti-mouse/Rat Foxp3 Staining Set protocol (eBioscience). Samples were acquired on FACSCalibur or FACSCanto II. Analyses were performed using FlowJo software 9.9 (Beckman Coulter).

Functional assays. For IFN-γ staining, splenocytes were harvested from mice treated in vivo as indicated, cleared for RBCs, seeded at 4 × 106 cells per well in a 96-well plate previously coated overnight with purified anti-NKp46 (clone 29A1.4), cultured for 4 h at 37°C, and then stained for IFN-γ. For the ex vivo cytotoxicity assay, B16F10 melanoma cells were labeled with CFSE, seeded at 2 × 105 cells per well in a 24-well plate. Four million splenocytes, cleared from RBCs from mice treated in vivo as indicated, were then added. Splenocytes and tumor cells were cocultured for 18 h at 37°C. Percentages of dead cells were measured with the expression of the viability using the Viobility 405/520 fixable dye (Miltenyi Biotec) among CFSE+ cells.

For the proliferation assay, C57BL/6 mice were either untreated or in vivo prestimulated with IL-15clpx at day 1 and day 3 and splenocytes were harvested at day 10. A single-cell suspension of splenocytes, cleared from RBCs, was labeled with VPD-450. Then, cells were seeded at 2 × 106 cells in 1 ml of R10 per well in a 24-well plate and cultured with 1 nM IL-15clpx for 5 d.

Statistical analyses were performed using a Kruskal–Wallis test (GraphPad Software). Data are expressed as mean ± SEM. A p value < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

To test whether repeated treatments with IL-15 or IL-15 agonists would induce similar repeated increased levels of functional NK cells, we used two kinds of IL-15/IL-15Rα complexes: a fusion molecule between IL-15 and its receptor IL-15Rα, that we called IL-15cplx, and its conjugated form with a Fc fragment from a human IgG1, that we termed Fc-15clpx.

First, NK cell expansion was examined in the spleen of C57BL/6 mice after one or two cycles of stimulation with IL-15clpx. The two cycles were separated by either 10, 20, or 50 d of rest (Fig. 1A). As expected, the numbers of splenic NK cells were significantly increased in mice after a first cycle of stimulation as compared with the untreated mice, and then returned to the baseline 10, 20, or 50 d afterward. Surprisingly, upon mice rechallenge (two cycles separated by either 10, 20, or 50 d), NK cell expansion was lower compared with the one obtained after a single cycle (Fig. 1B). This result suggests that NK cells were refractory to a second cycle of IL-15clpx independently of the duration of the resting period between the two cycles.

FIGURE 1.

NK cells fail to respond to a second IL-15 stimulation cycle. (A) Experimental design. One or two cycles of stimulation with IL-15clpx, separated by a resting period of 10, 20, or 50 d, were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed the day following the last injection or after the adapted resting period for analysis. (B) Graphs represent the numbers of splenic CD3NK1.1+ NK cells after one cycle, one cycle followed by the indicated time of rest, or two cycles separated by the indicated resting periods. (C) Experimental design. One or two cycles of stimulation, separated by a 3-wk resting period, with IL-15, IL-15clpx, or Fc-15clpx, were administered to C57BL/6 mice. Mice were sacrificed the day following the last injection or after the resting period for analysis. (D) Graphs represent the numbers of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells after one cycle (●), one cycle followed by a 3-wk resting period (▴), or two cycles separated by a 3-wk resting period (●) performed with indicated molecules. Dotted lines represent the numbers of NK and CD44+ CD8 T cells in mice injected with PBS. (Statistical analyses are comparing one cycle and two cycles of stimulation). (E) Flow cytometric analysis of CD3NK1.1+ NK cells in blood, bone marrow, lungs, and liver of C57BL/6 mice after one cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period. The untreated (UT) group was injected with PBS. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

NK cells fail to respond to a second IL-15 stimulation cycle. (A) Experimental design. One or two cycles of stimulation with IL-15clpx, separated by a resting period of 10, 20, or 50 d, were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed the day following the last injection or after the adapted resting period for analysis. (B) Graphs represent the numbers of splenic CD3NK1.1+ NK cells after one cycle, one cycle followed by the indicated time of rest, or two cycles separated by the indicated resting periods. (C) Experimental design. One or two cycles of stimulation, separated by a 3-wk resting period, with IL-15, IL-15clpx, or Fc-15clpx, were administered to C57BL/6 mice. Mice were sacrificed the day following the last injection or after the resting period for analysis. (D) Graphs represent the numbers of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells after one cycle (●), one cycle followed by a 3-wk resting period (▴), or two cycles separated by a 3-wk resting period (●) performed with indicated molecules. Dotted lines represent the numbers of NK and CD44+ CD8 T cells in mice injected with PBS. (Statistical analyses are comparing one cycle and two cycles of stimulation). (E) Flow cytometric analysis of CD3NK1.1+ NK cells in blood, bone marrow, lungs, and liver of C57BL/6 mice after one cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period. The untreated (UT) group was injected with PBS. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Next, we wondered whether this decreased NK cell response was dependent on the strength of the first stimulation. The expansion of NK cells was examined in the spleen of C57BL/6 mice after one cycle, one cycle followed by 3 wk of rest (one cycle plus 20 d), or two cycles of stimulation separated by a 20-d period (two cycles), with either IL-15 or the two IL-15/IL-15Rα complexes, IL-15clpx and Fc-15clpx (Fig. 1C). Throughout this study, CD44+ CD8 T cell expansion was analyzed in parallel to that of NK cells, with both cell types responding to IL-15 (5). As expected, the numbers of both splenic NK and CD44+ CD8 T cells were increased in mice treated with one cycle of stimulation with either IL-15, IL-15clpx, or Fc-15clpx. As compared with the untreated mice (Fig. 1D, dotted line), the respective strengths of stimulation induced by IL-15, IL-15clpx, and Fc-15clpx were ∼1.3-fold, 4.3-fold, and 12.3-fold increases in number (Fig. 1D). NK and CD44+ CD8 T cell numbers then returned to the baseline 3 wk later (Fig. 1D). Surprisingly, upon mice rechallenge (two-cycle schedule), NK cell expansion was significantly impaired regardless of the strength of the first stimulation (Fig. 1D). In contrast, the numbers of splenic CD44+ CD8 T cells at rechallenge were comparable to those at the first stimulation (Fig. 1D). Thus, it appears that NK cells, unlike CD44+ CD8 T cells, are refractory to a second cycle of IL-15 or IL-15 agonist stimulation. As similar results were obtained with IL-15clpx or Fc-15clpx, one or the other agonist was used throughout this study.

Next, we wondered whether increasing doses of IL-15clpx at the second cycle could restore the original response. IL-15clpx was injected at the second cycle at escalating doses, that is, 1-, 2-, or 5-fold the one injected at the first cycle. NK cell expansion was tested after two cycles, separated by 20 d of rest. The results showed that the NK cell numbers, upon rechallenge with higher IL-15clpx doses, remained lower than after a single cycle of stimulation (Supplemental Fig. 1A), indicating that the NK refractory state to restimulation was not influenced by the strength of this restimulation.

To evaluate the dissemination of NK cell hyporesponsiveness, NK cell expansion was measured in other lymphoid or nonlymphoid organs. A significant increase in the percentages of NK cells was observed in blood, bone marrow, lungs, and liver after one cycle of IL-15clpx stimulation (Fig. 1E), and then returned to baseline 20 d later. Nevertheless, when two cycles were performed, the levels of NK cells in these organs increased but never reached those obtained after only one cycle. This suggests that the NK cell defect observed was systemic and not restricted to the lymphoid organs, ruling out the hypothesis that NK cells could have escaped from the spleen by trafficking through organs.

To further investigate whether endogenous increase of IL-15 into mice was deleterious for NK cell re-expansion, we adapted our protocol and replaced the first cycle of IL-15clpx by an injection of LPS, known to indirectly trigger IL-15 production and lead to the expansion of NK and CD44+ CD8 T cells (26) (Fig. 2A). This alternative two-cycle protocol was compared with a single IL-15clpx stimulation cycle. Despite the return to baseline of splenic NK cells after one cycle with LPS followed by 3 wk of rest, splenic NK cell expansion within the alternative two-cycle protocol was lower compared with the one obtained after one cycle of stimulation with IL-15clpx, and not significantly different from the untreated control mice. In contrast, CD44+ CD8 T cell expansion remained significantly higher than for the untreated control mice (Fig. 2B). Moreover, when two injections of LPS were performed separated by a 3-wk resting period, NK cell expansion was also impaired, as the number of splenic NK cells was equivalent to that of untreated mice (Supplemental Fig. 1B). Thus, the hyporesponsive state of NK cells after two cycles was not only observed when IL-15 or IL-15 agonists were directly administered to mice but also occurred when an indirect, endogenous production of IL-15 was first triggered in vivo. Moreover, this phenomenon appears NK cell specific because CD44+ CD8 T cells kept responding to two cycles of stimulation. Additionally, within the alternative two cycles, a lower expansion of NK cells, as compared with one cycle of IL-15clpx, was also observed in bone marrow, lungs and liver, establishing that this phenomenon was indeed systemic (Fig. 2C).

FIGURE 2.

Indirect exposure of IL-15 during the first cycle induces NK cell hyporesponsiveness at a second IL-15 stimulation cycle. (A) Experimental design. C57BL/6 mice were injected with LPS or PBS at the first cycle and 3 wk later received a cycle of stimulation with IL-15clpx or PBS. (B) Graphs represent the numbers of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells of mice treated as indicated. (C) Flow cytometric analysis of the percentages of CD3NK1.1+ NK cells in bone marrow, lungs, and liver of mice treated as indicated. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

FIGURE 2.

Indirect exposure of IL-15 during the first cycle induces NK cell hyporesponsiveness at a second IL-15 stimulation cycle. (A) Experimental design. C57BL/6 mice were injected with LPS or PBS at the first cycle and 3 wk later received a cycle of stimulation with IL-15clpx or PBS. (B) Graphs represent the numbers of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells of mice treated as indicated. (C) Flow cytometric analysis of the percentages of CD3NK1.1+ NK cells in bone marrow, lungs, and liver of mice treated as indicated. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

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To further investigate the phenotype of NK cell hyporesponsiveness, NK cell maturation was analyzed in the spleen of C57BL/6 mice after one cycle, one cycle followed by 3 wk of rest, or two cycles of stimulation performed with IL-15clpx. NK cell maturation was studied using the expression of the two surface markers CD11b and CD27, allowing study of the four subpopulations of NK cells (27, 28). The effector CD11b+CD27+ NK cell population was significantly increased in response to one cycle with IL-15clpx and then returned to the baseline 3 wk later. In contrast, the percentage of this population after two cycles was similar to that of untreated mice (Fig. 3A). Thus, NK cell maturation was impaired after two cycles, with an absence of expansion of the CD11b+CD27+ effector subset. A similar result was observed using KLRG1 maturation marker (29), confirming that two cycles of IL-15 stimulation impaired NK cell maturation (Supplemental Fig. 1C).

FIGURE 3.

Impaired NK cell maturation, intracellular signaling, and activation after two IL-15 stimulation cycles. (A) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated (UT) group was injected with PBS. Mice were sacrificed the day following the last injection or after the resting period for analysis. Left panel, Representative flow cytometric analysis of the maturation state of splenic CD3NK1.1+ NK cells. Numbers indicate the percentage of cells in the indicated gate. Right panel, Respective percentages of CD11b+CD27+ cells among splenic NK cells of mice treated as indicated. (B) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed 2 h following the last injection or after the resting period for analysis. Graphs represent the percentages of p-Stat5 among splenic (upper panel) CD3NK1.1+ NK cells and (lower panel) CD44+ CD8 T cells for mice treated as indicated. (C) Representative flow cytometric analysis of (left panel) CD122 and (right panel) CD132 expression at the surface of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells in untreated C57BL/6 mice (gray-filled histograms) or 20 d after a first cycle of stimulation with IL-15clpx (black histograms). Isotype control is shown in gray histograms. (DF) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed 2 h following the last injection for (E) and 24 h after the last injection for (D) and (F) or after the resting period for analysis. Graphs represent the percentages of (C) Ki67 and (D) CD69 among splenic (upper panel) CD3NK1.1+ NK cells and (lower panel) CD44+ CD8 T cells for mice treated as indicated. (F) Graphs represent the percentages of Ly49D among splenic CD3NK1.1+ NK cells and NKG2D among splenic CD44+ CD8 T cells for mice treated as indicated. All data are a pool of at least two independent experiments, including at least three mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01. UT, untreated.

FIGURE 3.

Impaired NK cell maturation, intracellular signaling, and activation after two IL-15 stimulation cycles. (A) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated (UT) group was injected with PBS. Mice were sacrificed the day following the last injection or after the resting period for analysis. Left panel, Representative flow cytometric analysis of the maturation state of splenic CD3NK1.1+ NK cells. Numbers indicate the percentage of cells in the indicated gate. Right panel, Respective percentages of CD11b+CD27+ cells among splenic NK cells of mice treated as indicated. (B) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed 2 h following the last injection or after the resting period for analysis. Graphs represent the percentages of p-Stat5 among splenic (upper panel) CD3NK1.1+ NK cells and (lower panel) CD44+ CD8 T cells for mice treated as indicated. (C) Representative flow cytometric analysis of (left panel) CD122 and (right panel) CD132 expression at the surface of splenic CD3NK1.1+ NK cells and CD44+ CD8 T cells in untreated C57BL/6 mice (gray-filled histograms) or 20 d after a first cycle of stimulation with IL-15clpx (black histograms). Isotype control is shown in gray histograms. (DF) One cycle, one cycle followed by a 3-wk resting period, or two cycles of IL-15clpx stimulation separated by a 3-wk resting period were administered to C57BL/6 mice. The untreated group was injected with PBS. Mice were sacrificed 2 h following the last injection for (E) and 24 h after the last injection for (D) and (F) or after the resting period for analysis. Graphs represent the percentages of (C) Ki67 and (D) CD69 among splenic (upper panel) CD3NK1.1+ NK cells and (lower panel) CD44+ CD8 T cells for mice treated as indicated. (F) Graphs represent the percentages of Ly49D among splenic CD3NK1.1+ NK cells and NKG2D among splenic CD44+ CD8 T cells for mice treated as indicated. All data are a pool of at least two independent experiments, including at least three mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01. UT, untreated.

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As IL-15 binding to the CD122/CD132 receptor induces activation of the JAK/STAT pathway, leading to the recruitment and activation of Stat5, intracellular signaling was then studied by monitoring Stat5 phosphorylation. An increased level of Stat5 phosphorylation within splenic NK cells was observed after one cycle of stimulation with IL-15clpx, followed by a return to the baseline 3 wk later. Stat5 phosphorylation within splenic NK cells was increased after two IL-15clpx cycles, but it was lower as compared with a single cycle of stimulation (Fig. 3B, upper panel). Regarding CD44+ CD8 T cells, the phosphorylation rate of Stat5 was equivalent after one or two cycles of stimulation (Fig. 3B, lower panel). As Stat5 phosphorylation is related to the activation of the heterodimeric receptor CD122/CD132, we wondered whether the differential behaviors between NK and CD44+ CD8 T cells could be due to defects in IL-2/15 receptor chain expression. In fact, the expressions of either CD122 or CD132 at the surface of splenic NK and CD44+ CD8 T cells remained equivalent before the first and the second cycle of stimulation (Fig. 3C). NK cell hyporesponsiveness seems therefore to be correlated to a perturbation of intracellular signaling rather than to a receptor chain expression defect. Accordingly, when a first cycle was performed with Fc-15clpx, followed 3 wk later by another cycle using either IL-15 or IL-2, NK cells again failed to expand whereas the numbers of CD44+ CD8 T cells kept increasing (Supplemental Fig. 1D, 1E). This indicates that one cytokine could not compensate the other despite their redundant property.

To further characterize NK cell hyporesponsiveness, NK cell proliferation and activation were studied. First, the levels of splenic NK and CD44+ CD8 T cell proliferation were determined by the analysis of Ki67 expression. Pursuant to NK and CD44+ CD8 T cell expansions, one cycle of stimulation with IL-15clpx led to a full upregulation of Ki67 within these cells (93 ± 0.8 and 83 ± 12%, respectively) followed by a return to the baseline 3 wk later. Ki67 expression among splenic CD44+ CD8 T cells increased after two IL-15clpx cycles at a level (94 ± 3%) equivalent to the one obtain after a single cycle. In contrast, Ki67 expression among splenic NK cells after two cycles of stimulation was reduced (50 ± 10%) (Fig. 3D).

We also analyzed the CD69 activation marker that has also been shown to be upregulated by IL-15 during the activation of NK and CD8 T cells (30, 31). Its expression on splenic NK and CD44+ CD8 T cell was assessed after one or two cycles of IL-15clpx. One cycle of IL-15clpx stimulation increased the expression of CD69 at the NK and CD44+ CD8 T cell surfaces, followed by a return to the baseline 3 wk later. Interestingly, CD69 expression level on splenic NK cells was impaired in response to two cycles of IL-15clpx stimulation as compared with that obtained after a single stimulation cycle, whereas its upregulation was not affected on splenic CD44+ CD8 T cells (Fig. 3E). Moreover, after two cycles with IL-15clpx, the expression of the Ly49D activating receptor at the surface of NK cells was lower compared with it expression after one cycle of IL-15, supporting a reduced NK cell activity after two stimulation cycles (Fig. 3F, upper panel). In contrast, NKG2D expression, an activating receptor of CD8 T cells, was upregulated as much after one cycle as two cycles of IL-15clpx stimulation (Fig. 3F, lower panel).

Upon stimulation, NK cells rapidly produce cytokines, including IFN-γ, which possesses antitumor properties (32) and the production of which has been shown to be upregulated with IL-15 (31). We wondered whether splenic NK cells extracted from mice treated with one or two cycles of IL-15clpx in vivo would produce the same amount of IFN-γ after ex vivo NKp46 stimulation. When splenic NK cells were extracted from mice that had received one cycle of stimulation with IL-15clpx just before the ex vivo activation test (at day 22 and day 24), the production of IFN-γ within splenic NK cells was increased compared with splenic NK cells extracted from untreated mice. Interestingly, when splenic NK cells were extracted from mice that had received one cycle with IL-15clpx 3 wk before the ex vivo activation test (at day 1 and day 3), the percentage of IFN-γ was also increased compared with splenic NK cells extracted from untreated mice. However, when splenic NK cells were extracted from mice that had received two cycles of stimulation, the percentage of IFN-γ was lower and equivalent to that of splenic NK cells extracted from untreated mice (Fig. 4A, 4B).

FIGURE 4.

NK cell hyporesponsiveness prevents the antitumor efficacy of IL-15 agonists in a disseminated tumor model. (AC) (A) Experimental design. PBS or IL-15clpx was injected to C57BL/6 mice at the first cycle, followed 3 wk later by a second cycle of stimulation with PBS or IL-15clpx. Mice were sacrificed the day following the last injection or after the resting period for ex vivo activation tests. (B) Splenocytes were incubated for 4 h at 37°C on a NKp46-coated plate. The graph represents the percentages of IFN-γ among splenic CD3NK1.1+ NK cells extracted from mice treated in vivo as indicated. (C) Splenocytes were incubated for 18 h at 37°C with CFSE-labeled B16F10 tumor cells. The graph represents the percentage of dead cells among CFSE+ tumor cells. (D and E) (D) Experimental design. B16F10 tumor cells (1 × 105) were injected i.v. to C57BL/6 mice before the second cycle of stimulation. Cycles were performed using PBS or IL-15clpx. Mice were sacrificed 21 d following the i.v. graft and lungs were harvested and weighed. (E) Lung weights from mice treated as indicated. Dotted line represents the lung weight of healthy, untreated mice. Data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

FIGURE 4.

NK cell hyporesponsiveness prevents the antitumor efficacy of IL-15 agonists in a disseminated tumor model. (AC) (A) Experimental design. PBS or IL-15clpx was injected to C57BL/6 mice at the first cycle, followed 3 wk later by a second cycle of stimulation with PBS or IL-15clpx. Mice were sacrificed the day following the last injection or after the resting period for ex vivo activation tests. (B) Splenocytes were incubated for 4 h at 37°C on a NKp46-coated plate. The graph represents the percentages of IFN-γ among splenic CD3NK1.1+ NK cells extracted from mice treated in vivo as indicated. (C) Splenocytes were incubated for 18 h at 37°C with CFSE-labeled B16F10 tumor cells. The graph represents the percentage of dead cells among CFSE+ tumor cells. (D and E) (D) Experimental design. B16F10 tumor cells (1 × 105) were injected i.v. to C57BL/6 mice before the second cycle of stimulation. Cycles were performed using PBS or IL-15clpx. Mice were sacrificed 21 d following the i.v. graft and lungs were harvested and weighed. (E) Lung weights from mice treated as indicated. Dotted line represents the lung weight of healthy, untreated mice. Data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

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In the light of this result, we wondered whether NK cell antitumor efficacy would be affected. To test this hypothesis, we took advantage of the B16F10 mouse metastatic model, in which IL-15clpx has proved its efficacy to reduce the number of metastases in an NK-dependent manner (3335). First, we tested ex vivo the ability of splenocytes extracted from mice that had received one or two cycles of stimulation with IL-15clpx to induce B16F10 tumor cell death. As expected, when splenocytes were extracted from mice that had received one cycle of stimulation with IL-15clpx just before the ex vivo cytotoxic test (at day 22 and day 24), the percentage of tumor dead cells was significantly increased compared with that induced by splenocytes extracted from untreated mice. In a similar manner to the IFN-γ activation test, when splenocytes were extracted from mice that had received one cycle with IL-15clpx 3 wk before the ex vivo cytotoxic test, the percentage of dead cells was also increased as compared with that induced by splenocytes extracted from untreated mice. However, when splenocytes were extracted from mice that had received two cycles of stimulation, the percentage of dead cells was equivalent to that induced by splenocytes extracted from untreated mice (Fig. 4A, 4C). To further confirm our results, we set up a mouse metastatic model with B16F10 tumor cells. For that purpose, B16F10 melanoma cells were injected i.v. to C57BL/6 mice, 3 wk after they had received the first stimulation cycle of IL-15clpx or its vehicle. A second cycle of stimulation with IL-15clpx or PBS was performed the day after the tumor graft (Fig. 4D). As expected, and compared with the PBS-treated control mice, a reduction in lungs metastases was observed when C57BL/6 mice received only one cycle with IL-15clpx. In accordance with the ex vivo results, the therapeutic effect obtained after only one cycle of IL-15clpx was similar whether IL-15clpx was administered 20 d before or 1 d after the i.v. graft. However, when mice were pretreated with IL-15clpx, i.v. injected with B16F10 melanoma cells, and retreated with another cycle of IL-15clpx, a loss in therapeutic efficacy was observed, illustrated by the lung’s weight being comparable to that of PBS-treated control mice (Fig. 4E). Thus, the impairment of NK cell activity, as a consequence of two IL-15 cycles, is also critical for their therapeutic efficacy.

To evaluate whether this hyporesponsiveness was inherent to NK cells or could be environment-dependent, an ex vivo NK cell proliferation assay was performed with IL-15clpx, using splenocytes isolated from untreated mice or from mice having received one cycle of stimulation with IL-15clpx in vivo. Interestingly, NK cell proliferation profiles, after ex vivo IL-15clpx stimulation, were similar when mice received or did not receive one cycle of stimulation (Fig. 5A). This ability of NK cells to proliferate ex vivo after an in vivo pretreatment suggested that NK cell hyporesponsiveness was related to the in vivo environment. Then, to further characterize the origin of this detrimental environment for NK cell expansion, splenocytes were harvested from mice that had received one cycle of IL-15clpx, labeled with the VPD-450 tracker, and adoptively transferred into recipient mice, just before their second cycle of injection. First and second cycles of stimulation were performed either with PBS or IL-15clpx and control mice received PBS injection at both cycles. Three days after the last injection, recipient mice were sacrificed and the number of splenic VPD-450+ cells was analyzed (Fig. 5B, left). In accordance with our previous observations, the number of exogenous VPD-450+ NK cells was increased compared with that of control mice when the first stimulation cycle was performed with PBS and the second cycle with IL-15clpx. However, when two cycles of IL-15clpx stimulation were administered to mice, the number of exogenous labeled NK cells was equivalent to that of control mice. In contrast, the numbers of exogenous labeled CD44+ CD8 T cells were equivalent in recipient mice whether mice received one or two cycles of stimulation with IL-15clpx (Fig. 5B, right). Furthermore, similar results were observed when the splenocytes used for the adoptive transfer were extracted from untreated mice (Supplemental Fig. 2A), indicating that this phenomenon was not inherent to NK cells but rather due to the environment surrounding NK cells at the time of the second cycle of stimulation.

FIGURE 5.

NK cell hyporesponsiveness is an environment-dependent phenomenon. (A) Left panel, Experimental design. One cycle of stimulation with IL-15clpx or PBS was administered to C57BL/6 mice. At day 10, mice were sacrificed; splenocytes were harvested, stained with VPD-450, and cultured in the presence or absence of 1 nM IL-15clpx for 5 d. Proliferation was estimated by VPD-450 dilution by flow cytometry. Right panel, Representative histograms of VPD-450 dilution within CD3NK1.1+ cells treated as indicated. (B) Left panel, Experimental design. Two cycles of stimulation with PBS or IL-15clpx, separated by 10 d, were administered to recipient C57BL/6 mice. In parallel, a cycle of IL-15clpx stimulation was administrered to donor C57BL/6 mice. At day 9, splenocytes from donor mice were harvested, labeled with the VPD-450 tracker, and adoptively transferred to recipient mice prior to the second IL-15clpx stimulation cycle. C57BL/6 mice were sacrificed on day 15 for analysis. Right panel, Flow cytometric analysis of the numbers of exogenous VPD-450+CD3NK1.1+ NK cells and VPD-450+CD44+ CD8 T cells. (C) Left panel, Experimental design. PBS or Fc-15clpx was injected to C57BL/6 mice at the first cycle and the second cycle 5 wk later. Mice were depleted for CD25+ cells or neutralized for IL-10R or TGF-β during the second cycle of stimulation. Right panel, Graph represents the flow cytometric analysis of the numbers of splenic CD3NK1.1+ NK cells of mice treated as indicated. All data are a pool of at least two independent experiments, including at least three mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

FIGURE 5.

NK cell hyporesponsiveness is an environment-dependent phenomenon. (A) Left panel, Experimental design. One cycle of stimulation with IL-15clpx or PBS was administered to C57BL/6 mice. At day 10, mice were sacrificed; splenocytes were harvested, stained with VPD-450, and cultured in the presence or absence of 1 nM IL-15clpx for 5 d. Proliferation was estimated by VPD-450 dilution by flow cytometry. Right panel, Representative histograms of VPD-450 dilution within CD3NK1.1+ cells treated as indicated. (B) Left panel, Experimental design. Two cycles of stimulation with PBS or IL-15clpx, separated by 10 d, were administered to recipient C57BL/6 mice. In parallel, a cycle of IL-15clpx stimulation was administrered to donor C57BL/6 mice. At day 9, splenocytes from donor mice were harvested, labeled with the VPD-450 tracker, and adoptively transferred to recipient mice prior to the second IL-15clpx stimulation cycle. C57BL/6 mice were sacrificed on day 15 for analysis. Right panel, Flow cytometric analysis of the numbers of exogenous VPD-450+CD3NK1.1+ NK cells and VPD-450+CD44+ CD8 T cells. (C) Left panel, Experimental design. PBS or Fc-15clpx was injected to C57BL/6 mice at the first cycle and the second cycle 5 wk later. Mice were depleted for CD25+ cells or neutralized for IL-10R or TGF-β during the second cycle of stimulation. Right panel, Graph represents the flow cytometric analysis of the numbers of splenic CD3NK1.1+ NK cells of mice treated as indicated. All data are a pool of at least two independent experiments, including at least three mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

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In cancer and leukemia contexts, alteration of the environment leads to immunosuppression that could impair NK cell functions (3640). To investigate whether such immunosuppressive mechanisms could be involved, the impacts of depleting immune regulatory cells or neutralizing immunosuppressive cytokines were analyzed in immunocompetent C57BL/6 mice. CD4 regulatory T cells, which have been shown to inhibit NK cell cytotoxic activity (36), express the CD25 receptor chain at their surface (41). Depleting CD25+ cells at the second cycle of Fc-15clpx stimulation (Supplemental Fig. 2B) was ineffective in restoring NK cell expansion, as the numbers of splenic NK cells after two cycles of stimulation with Fc-15clpx with or without CD25+ depleting conditions were equivalent (Fig. 5C). Then, as TGF-β and IL-10 have been described as immunosuppressive cytokines (37, 4244), treatments with anti–TGF-β or anti–IL-10R blocking Abs were performed at the second cycle of Fc-15clpx stimulation. These neutralizations also failed to induce the expansion of NK cells after two Fc-15clpx stimulation cycles (Fig. 5C). However, IL-10 has been shown to play an important role during a first cycle of stimulation. Indeed, IL-10 expression during a first antigenic priming was deleterious to a secondary enhancement of CD8 T cells responses (45). To evaluate the presence of this phenomenon in our study, IL-10R was blocked during the first stimulation cycle and splenic expansion was assessed after the second cycle of stimulation. Even in those conditions, NK cell expansion was not restored, as the number of splenic NK cells after two cycles of IL-15clpx was equivalent to that of untreated mice (Supplemental Fig. 2C). Collectively, our results indicate that NK cell hyporesponsiveness does not seem to be related to the immunosuppressive properties driven by either CD25+ regulatory cells or IL-10 and TGF-β.

To further investigate the environment responsible for NK cell hyporesponsiveness, we took advantage of NMRI-nude immunodeficient mice, which lack T cells. As expected, the number of splenic NK cells from NMRI-nude mice was increased by 17-fold after Fc-15clpx stimulation, compared with untreated mice, and then returned to the baseline 5 wk later (Fig. 6A, left). Surprisingly, a similar increase was obtained after two cycles of stimulation with Fc-15clpx, showing that, in mice lacking T cells, NK cells were actually responsive to the second cycle of stimulation (Fig. 6A, left). Moreover, the NK cell maturation profile in NMRI-nude mice (percentage of mature CD11b+CD27+ NK cells) was found equivalent whether mice received one or two cycles of Fc-15clpx stimulation (Fig. 6A, right). As controls, NK cells from NMRI-wt mice failed to re-expand after two Fc-15clpx stimulation cycles (Fig. 6B, left), and the percentage of mature CD11b+CD27+ NK cells was equivalent to that of untreated mice (Fig. 6B, right), in agreement with our previous results observed in C57BL/6 mice. This confirms that the defect in NK cell expansion is linked to the environment, and it suggests that T cells play a role in that phenomenon.

FIGURE 6.

Role of T cells in NK cell hyporesponsiveness. (A and B) One cycle, one cycle followed by 5 wk of rest, or two cycles of stimulation with Fc-15clpx separated by a 5-wk resting period were administered to NMRI-nude and NMRI-wt mice. The untreated (UT) group was injected with PBS. Mice were sacrificed the day following the last injection or after the resting period for analysis. Graphs represent the flow cytometric analysis of the numbers of splenic CD3NKp46+ NK cells and the percentages of CD11b+CD27+ cells among splenic NK cells in (A) NMRI-nude mice and (B) NMRI-wt mice. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

FIGURE 6.

Role of T cells in NK cell hyporesponsiveness. (A and B) One cycle, one cycle followed by 5 wk of rest, or two cycles of stimulation with Fc-15clpx separated by a 5-wk resting period were administered to NMRI-nude and NMRI-wt mice. The untreated (UT) group was injected with PBS. Mice were sacrificed the day following the last injection or after the resting period for analysis. Graphs represent the flow cytometric analysis of the numbers of splenic CD3NKp46+ NK cells and the percentages of CD11b+CD27+ cells among splenic NK cells in (A) NMRI-nude mice and (B) NMRI-wt mice. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01.

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We therefore tested directly whether T cells could impact the NK cell response. For this purpose, T lymphocytes were extracted from NMRI-wt mice 30 d after one cycle of PBS or Fc-15clpx stimulation and were adoptively transferred into NMRI-nude mice prior to the second Fc-15clpx stimulation cycle (Supplemental Fig. 2D). The adoptive transfer of T cells extracted from prestimulated NMRI-wt donor mice diminished NK cell expansion in NMRI-nude mice following the second Fc-15clpx stimulation (Supplemental Fig. 2E), whereas adoptive transfer of T cells extracted from untreated NMRI-wt mice did not affect NK cell number (Supplemental Fig. 2E) or NK cell maturation (Supplemental Fig. 2F) in NMRI-nude mice following the second Fc-15clpx stimulation. Besides, maturation of these NK cells (percentage of CD11b+ CD27+) was also decreased (Supplemental Fig. 2F). Thus, these results further argue in favor of a role of prestimulated T cells in the regulation of NK cell expansion. Moreover, when T lymphocytes, extracted from untreated or Fc-15clpx prestimulated NMRI-wt mice, were adoptively transferred before the first cycle of Fc-15clpx, the expansion and maturation of splenic NK cells from NMRI-nude mice were not affected, indicating that there was no competition between NK cells and adoptively transferred T cells for using IL-15 as a stimulating cytokine (Supplemental Fig. 2G–I). Moreover, this result also showed that prestimulated T cells are efficient in limiting NK cell expansion only at the time of the second stimulation. This indicates that the first IL-15 exposure is needed to create a detrimental environment for NK cell response at the second cycle.

We finally wondered which types of T cell subsets could be implicated in NK cell hyporesponsiveness. For this purpose, CD4+ or CD8+ cells were depleted in C57BL/6 mice during the second cycle of Fc-15clpx stimulation (Fig. 7A, Supplemental Fig. 2B). CD4+ cell depletion failed in restoring NK cell expansion because equivalent numbers of splenic NK cells and mature CD11b+CD27+ NK cells were obtained after two cycles with or without depletion (Fig. 7B, Supplemental Fig. 3A). In contrast, CD8+ cell depletion allowed a significant increase in the number of splenic NK cells and mature CD11b+CD27+ NK cells (Fig. 7B, Supplemental Fig. 3A). This result suggests that among T cells, CD8 T cells play a role in NK cell hyporesponsiveness.

FIGURE 7.

CD44+ CD8 T cells are involved in NK cells hyporesponsiveness at the time of the second cycle of stimulation. (A and B) (A) Experimental design. PBS or Fc-15clpx was injected to C57BL/6 mice at the first cycle and the second cycle 5 wk later. Mice were depleted for CD4+ or CD8+ cells during the second cycle of stimulation. (B) Graph represents the flow cytometric analysis of the numbers of splenic CD3NK1.1+ NK cells of mice treated as indicated. (CF) (C) Experimental design. Two cycles of stimulation with PBS or IL-15clpx, separated by 3 wk, were administered to C57BL/6 mice. Mice were depleted for CD8+ during the second cycle of stimulation. Graphs represent the flow cytometric analysis of (D) the numbers of splenic CD3NK1.1+ NK cells and the percentages of (E) CD11b+CD27+ and (F) Ki67+ cells among splenic NK cells for mice treated as indicated. (GJ) (G) Experimental design. Two cycles of stimulation with PBS or Fc-15clpx, separated by a 3 wk resting period, were administered to NMRI-nude recipient mice. In parallel, a cycle of Fc-15clpx stimulation was administered to NMRI-wt mice. At day 21, splenic CD44CD8+ T or CD44+CD8+ T cells were purified and adoptively transferred (AT) to NMRI-nude mice prior to the second cycle. NMRI-nude mice were sacrificed the following day for analysis. Graphs represent the flow cytometric analysis of the number of (H) splenic NKp46+ NK cells and the percentages of (I) CD11b+ CD27+ and (J) Ki67+ cells among splenic NK cells for mice treated as indicated. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7.

CD44+ CD8 T cells are involved in NK cells hyporesponsiveness at the time of the second cycle of stimulation. (A and B) (A) Experimental design. PBS or Fc-15clpx was injected to C57BL/6 mice at the first cycle and the second cycle 5 wk later. Mice were depleted for CD4+ or CD8+ cells during the second cycle of stimulation. (B) Graph represents the flow cytometric analysis of the numbers of splenic CD3NK1.1+ NK cells of mice treated as indicated. (CF) (C) Experimental design. Two cycles of stimulation with PBS or IL-15clpx, separated by 3 wk, were administered to C57BL/6 mice. Mice were depleted for CD8+ during the second cycle of stimulation. Graphs represent the flow cytometric analysis of (D) the numbers of splenic CD3NK1.1+ NK cells and the percentages of (E) CD11b+CD27+ and (F) Ki67+ cells among splenic NK cells for mice treated as indicated. (GJ) (G) Experimental design. Two cycles of stimulation with PBS or Fc-15clpx, separated by a 3 wk resting period, were administered to NMRI-nude recipient mice. In parallel, a cycle of Fc-15clpx stimulation was administered to NMRI-wt mice. At day 21, splenic CD44CD8+ T or CD44+CD8+ T cells were purified and adoptively transferred (AT) to NMRI-nude mice prior to the second cycle. NMRI-nude mice were sacrificed the following day for analysis. Graphs represent the flow cytometric analysis of the number of (H) splenic NKp46+ NK cells and the percentages of (I) CD11b+ CD27+ and (J) Ki67+ cells among splenic NK cells for mice treated as indicated. All data are a pool of at least two independent experiments, including at least four mice per condition, and are represented as mean ± SEM. Statistical analyses were performed using a Kruskal–Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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To further evaluate this phenomenon, CD8+ cells were depleted in C57BL/6 mice before the second cycle of IL-15clpx stimulation (Fig. 7C). First, when CD8+ depletion occurred before a primary cycle of stimulation with IL-15clpx, the number of NK cells (6 ± 1.6%, Fig. 7D) was similar to that obtained without depletion (5.4 ± 0.9%, Fig. 1D), indicating that NK cell hyporeponsiveness was not related to an IL-15 competition between these two populations. However, when CD8+ depletion occurred before the second cycle of stimulation with IL-15clpx, the number of splenic NK cells was significantly increased compared with the control mice, at a level equivalent to that obtained after only one cycle of stimulation (Fig. 7D). Similar results were obtained for the percentage of CD11b+CD27+ cells among splenic NK cells (Fig. 7E), as well as the proliferation level of NK cells (Fig. 7F), confirming that CD8+ depletion, during the second cycle, not only restores NK cell expansion but also their proliferation and maturation in response to IL-15clpx. Additionally, when CD8+ cells were depleted during the second cycle of stimulation, NK cell expansion was also restored in other organs than the spleen, such as bone marrow and blood, indicating a systemic effect of restoration of NK cell responsiveness (Supplemental Fig. 3B). Thus, NK cell hyporesponsiveness appears to be linked to the presence of CD8+ cells whose depletion during the second cycle of stimulation rescues NK cell responsiveness. Besides, when CD8+ cells were depleted before the first cycle of Fc-15clpx stimulation or in the middle of the resting period (D16), NK cell responsiveness was not restored at the second cycle of stimulation, showing that this phenomenon was not dependent on a specific CD8+ cell population emerging over time (Supplemental Fig. 3C). Thus, in agreement with previous studies showing a greater expansion of NK cells in the absence of CD8 T cells (46, 47), we have shown in the present study that precise timing of CD8+ cell depletion at the second cycle of stimulation is decisive for NK cell expansion. Finally, such punctual CD8+ cell depletion offers a new opportunity in restoring NK cell responsiveness.

Then, we wondered whether CD44+ CD8 T cells could be involved in this NK cell hyporesponsiveness. For this purpose, CD44CD8+ or CD44+CD8+ cells were extracted from NMRI-wt mice 30 d after one cycle of Fc-15clpx stimulation and were adoptively transferred into NMRI-nude mice prior to the second Fc-15clpx stimulation cycle (Fig. 7G). Interestingly, the adoptive transfer of CD44+ CD8+ decreased the expansion of NK cells after two cycles with Fc-15clpx, whereas adoptive of CD44CD8+ cells had no impact (Fig. 7H). The adoptive transfer of CD44+CD8+ cells also led to a lower percentage of mature NK cells and decreased proliferation compared with two cycles without adoptive transfer (Fig. 7I, 7J). Moreover, when CD44CD8+ or CD44+CD8+ cells, extracted from Fc-15clpx–prestimulated mice were adoptively transferred before a first cycle of Fc-15clpx, no decreases in NK cells expansion, maturation, and proliferation were observed, indicating again that there was no IL-15 competition between NK cells and adoptively transferred T cells (Supplemental Fig. 3D). Moreover, this result indicates that the first IL-15 exposure, in recipient mice, is needed to create a detrimental environment for NK cell response at the second IL-15 cycle. In these conditions, the adoptive transfer of prestimulated CD44+CD8+ cells could modulate NK cell expansion. Taken together, our data indicate that upon rechallenge, NK cells become hyporesponsive and this hyporesponsiveness is tightly linked to the presence of CD44+CD8+ cells. Finally, punctual depletion of these cells at the time of the second cycle of stimulation could be a way to restore NK cell responsiveness.

The present study has demonstrated that two cycles of injection of IL-15 or IL-15 agonists resulted in a weak or even no expansion of NK cells in vivo in immunocompetent mice, whereas a single cycle increased NK cell percentages both in lymphoid and nonlymphoid organs. This NK cell hyporesponsiveness was independent of the length of the resting period between two cycles of injection (from 10 to 50 d). Taking into consideration that the NK cell half-life has been estimated at ∼17 d (48), our results suggested that NK cells, even those newly generated during the 3-wk resting period, were unable to respond to another IL-15 cycle. Moreover, we have shown that NK cell hyporesponsiveness was independent of the strength of the first stimulation, as similar results were observed with IL-15 and its agonists, IL-15clpx or Fc-15clpx. Our results also indicated that NK cell hyporesponsiveness was systemic, as both lymphoid and nonlymphoid organs were affected. Accordingly, Tamaldge et al. (49) have shown that some members of the family of biological response modifiers (BRMs), including type 1 IFN and IL-2, also induced NK cell hyporesponsiveness after repeated treatment, and this hyporesponsiveness was indeed systemic for cytokine BRMs or specific to lymphoid organs for noncytokine BRMs.

TLR agonists such as LPS or polyinosinic-polycytidylic acid induce the production of IL-15 in vitro and in vivo (26, 50). Interestingly, we found that this NK cell hyporesponsiveness to IL-15 also occurred following a first exposure to LPS. Thus, NK cell hyporesponsiveness arose irrespective of whether the exposure to IL-15 during the first cycle was exogenous or induced endogenously by LPS. Furthermore, NK cell expansion was not even triggered after two cycles with LPS. TLR engagement has been shown to induce the production of type 1 IFN (51), which in turn stimulates the expression of IL-15 (26, 52). This suggests that NK cell hyporesponsiveness, after two LPS cycles, could be linked to type 1 IFN induction. Additionally, in the 1980s it was revealed that multiple treatments with type 1 IFN also resulted in NK cell hyporesponsiveness in mice (49, 53). Moreover, the first IFN-α clinical trials in humans have pointed out that repeated injections of IFN-α in patients failed to repeatedly sustain NK cell activity (54), suggesting that this phenomenon also exists in humans. In this regard, one might wonder about the existence of human and macaque NK cell hyporesponsiveness triggered by IL-15 repeated injections. Recent studies actually support this hypothesis. A recent study has shown that one IL-15 infusion led to an increase in NK cell expansion and proliferation in the blood of patients, whereas a second infusion of IL-15 resulted in a decrease in NK cell responses (55), in agreement with our results observed in mice. Furthermore, cycles of injection with an IL-15 agonist have also been shown to be detrimental for NK cell expansion in SIV+ macaque blood (56).

Regarding the T cell compartment dependent on IL-15 signaling, we found that CD44+ CD8 T cells were still responsive after a second cycle of stimulation with IL-15 or its agonists. This difference between NK cells and CD44+ CD8 T cells was not linked to a reduced expression of the IL-15 heterodimeric CD122/CD132 receptor at the cell surface. However, decreases in Stat5 phosphorylation and Ki67 expression in NK cells were observed in response to a second IL-15 cycle, whereas in CD44+ CD8 T cells, Stat5 phosphorylation and Ki67 expression were equivalent after one and two cycles of stimulation. This decreased Stat5 phosphorylation within NK cells is in agreement with previous studies showing that the suppression of the Stat5 pathway plays a major role in NK cell activity defects (12, 57, 58). Accordingly, it has been shown that CD122 and CD132 chain expression was not a limiting factor to explain T cell anergy (59, 60). We have also pointed out impaired NK cell activation after two IL-15 cycles through the decreased expression of the early activation marker CD69 or the activating receptor Ly49D. On the contrary, equivalent increases of the activation markers (CD69 and NKG2D) for CD44+ CD8 T cells were found after one or two cycles of stimulation.

Our results have shown that two cycles of stimulation with IL-15clpx led to decreased IFN-γ production within splenic NK cells. Consequently, this NK cell hyporesponsiveness after multiple IL-15 exposures led to a reduced therapeutic efficacy in a disseminated tumor model. Taken together, these data could suggest that a first endogenous IL-15 increase triggered by LPS or a primary infection could impair an IL-15–based metastatic cancer treatment aimed at stimulating NK cells. Interestingly, our results have pointed out that one cycle of IL-15 agonist was effective in decreasing tumor progression independently of whether this cycle occurred 3 wk before or just after the tumor cell injection. This result was intriguing and supports that priming mice with IL-15 could improve the therapeutic efficacy of the immune cells. However, performing several cycles with IL-15 would impede the antitumor efficacy instead of improving it.

Additionally, we have shown that naive or in vivo–prestimulated NK cells possessed the same ability to proliferate ex vivo. This result is in accordance with a recent study were authors have shown that both continuous or intermittent treatment with IL-15 in vitro result in increased proliferation of human NK cells. However, in this study, the authors also showed that this proliferation did not reflect the viability of the cells. Indeed, in contrast to intermittent treatment, continuous treatment with IL-15 decreased NK cell viability (61). Their study is in line with our result because the proliferation potential did not reflect the ex vivo activation ability of NK cells extracted from mice that have received two IL-15clpx cycles.

Furthermore, we have considered a role of the environment in preventing NK cell expansion during the second cycle of stimulation in immunocompetent mice. In diverse pathologies, loss of NK cell functionality is related to the presence of regulatory cells or immunosuppressive cytokines. For example, the decline of NK cell cytotoxic functions in patients suffering from acute myeloid leukemia is correlated with IL-10 production by malignant cells (3739). Moreover, CD4+CD25+ regulatory T cells are well described for their immunosuppressive functions and have been shown to inhibit NK cell effector functions through TGF-β (62, 63). Intriguingly, in our model, neither depletion of CD25+ cells nor blocking of IL-10R or TGF-β restored NK cell responsiveness, ruling out the implication of these immunosuppressive factors in the induction of NK cell hyporesponsiveness.

Interestingly, NK cells were able to expand after two cycles of IL-15 agonist in immunodeficient mice, suggesting the implication of T cells in NK cell hyporesponsiveness. Furthermore, adoptive transfer of prestimulated T cells to immunodeficient mice during the second cycle of stimulation impaired NK cell expansion. Conversely, and to confirm the involvement of T cells in this phenomenon, we showed that depletion of CD8+ cells but not CD4+ cells in immunocompetent mice was able to restore NK cell expansion in response to the second IL-15 cycle, therefore designating CD8 T cells as impairing NK cell responsiveness. More precisely, the CD44+CD8+ cell subtype was shown to be involved. Indeed, the adoptive transfer of this population in immunodeficient mice before the second cycle of stimulation reduced NK cell expansion, maturation, and proliferation. Furthermore, we demonstrated that this population of CD8 T cells played its deleterious effect on NK cells only at the time of the second stimulation in immunocompetent mice. Accordingly, in immunodeficient mice, only the adoptive transfer of CD44+CD8+ cells at the time of the second stimulation was detrimental for NK cell expansion. Interestingly, Salem and Hossain (46) have highlighted the importance of the depletion timing of CD8 T cells for expansion of NK cells in a murine CMV infection model. The authors have demonstrated that depletion of CD8 T cells before infection allowed the expansion of NK cells, in contrast to the depletions occurring immediately postinfection or chronically. The reason for the importance of this timing remains unclear. In this line, we wondered whether the timing for the other depletion or blocking conditions we used in that study was adequately chosen. Indeed, early depletion of CD25+ cells, or blocking TGF-β or IL-10R cytokines, could perhaps restore NK cell expansion. As IL-10 expression during a first antigenic priming has been shown to be deleterious to a secondary enhancement of CD8 T cells (45), we have tested blocking IL-10R during the period of the first IL-15 cycle. However, even in these conditions, NK cell expansion at the second cycle was not restored.

In summary, we have provided evidence that repeated treatments with IL-15 agonists would not only fail to be optimal but would actually be counterproductive, which is illustrated by a profound defect of NK cell activation as compared with a single cycle of treatment. Moreover, we found that this phenomenon was related to the environment surrounding NK cells, generated by the first exposure to IL-15. Unexpectedly, whereas CD4 T regulatory cells were not involved in the NK cell hyporesponsiveness, CD44+ CD8 T cells play an important role in this phenomenon. Collectively, these observations have to be taken into consideration regarding the IL-15–based treatment schedules for cancer therapy.

We thank Prof. Averil Ma for critically reading this manuscript and the Unité Thérapeutique Expérimentale and CytoCell facilities for technical assistance. This work was included in the LabEx IGO, Immunotherapy, Graft, Oncology program.

This work was supported by CNRS, INSERM, the University of Nantes, Cytune Pharma, Agence Nationale de la Recherche Grant ANR-15-CE-17-0023-02, and by the Agence Nationale de la Recherche et de la Technologie.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • BRM

    biological response modifier

  •  
  • Fc-15clpx

    fusion protein linking IL-15clpx to the COOH terminus of an Fc fragment from a human IgG1

  •  
  • IL-15clpx

    IL-15/IL-15Rα fusion protein

  •  
  • VPD-450

    violet proliferation dye 450

  •  
  • wt

    wild-type.

1
Fehniger
,
T. A.
,
M. H.
Shah
,
M. J.
Turner
,
J. B.
VanDeusen
,
S. P.
Whitman
,
M. A.
Cooper
,
K.
Suzuki
,
M.
Wechser
,
F.
Goodsaid
,
M. A.
Caligiuri
.
1999
.
Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response.
J. Immunol.
162
:
4511
4520
.
2
Chiossone
,
L.
,
M.
Vienne
,
Y. M.
Kerdiles
,
E.
Vivier
.
2017
.
Natural killer cell immunotherapies against cancer: checkpoint inhibitors and more.
Semin. Immunol.
31
:
55
63
.
3
Grabstein
,
K. H.
,
J.
Eisenman
,
K.
Shanebeck
,
C.
Rauch
,
S.
Srinivasan
,
V.
Fung
,
C.
Beers
,
J.
Richardson
,
M. A.
Schoenborn
,
M.
Ahdieh
, et al
.
1994
.
Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor.
Science
264
:
965
968
.
4
Kennedy
,
M. K.
,
M.
Glaccum
,
S. N.
Brown
,
E. A.
Butz
,
J. L.
Viney
,
M.
Embers
,
N.
Matsuki
,
K.
Charrier
,
L.
Sedger
,
C. R.
Willis
, et al
.
2000
.
Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice.
J. Exp. Med.
191
:
771
780
.
5
Lodolce
,
J. P.
,
D. L.
Boone
,
S.
Chai
,
R. E.
Swain
,
T.
Dassopoulos
,
S.
Trettin
,
A.
Ma
.
1998
.
IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation.
Immunity
9
:
669
676
.
6
Suzuki
,
H.
,
G. S.
Duncan
,
H.
Takimoto
,
T. W.
Mak
.
1997
.
Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor β chain.
J. Exp. Med.
185
:
499
505
.
7
Intlekofer
,
A. M.
,
N.
Takemoto
,
E. J.
Wherry
,
S. A.
Longworth
,
J. T.
Northrup
,
V. R.
Palanivel
,
A. C.
Mullen
,
C. R.
Gasink
,
S. M.
Kaech
,
J. D.
Miller
, et al
.
2005
.
Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. [Published erratum appears in 2006 Nat. Immunol. 7: 113.]
Nat. Immunol.
6
:
1236
1244
.
8
Banerjee
,
A.
,
S. M.
Gordon
,
A. M.
Intlekofer
,
M. A.
Paley
,
E. C.
Mooney
,
T.
Lindsten
,
E. J.
Wherry
,
S. L.
Reiner
.
2010
.
Cutting edge: the transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche.
J. Immunol.
185
:
4988
4992
.
9
Li
,
G.
,
Q.
Yang
,
Y.
Zhu
,
H.-R.
Wang
,
X.
Chen
,
X.
Zhang
,
B.
Lu
.
2013
.
T-Bet and eomes regulate the balance between the effector/central memory T cells versus memory stem like T cells.
PLoS One
8
:
e67401
.
10
Ma
,
A.
,
R.
Koka
,
P.
Burkett
.
2006
.
Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis.
Annu. Rev. Immunol.
24
:
657
679
.
11
Rochman
,
Y.
,
R.
Spolski
,
W. J.
Leonard
.
2009
.
New insights into the regulation of T cells by γc family cytokines.
Nat. Rev. Immunol.
9
:
480
490
.
12
Imada
,
K.
,
E. T.
Bloom
,
H.
Nakajima
,
J. A.
Horvath-Arcidiacono
,
G. B.
Udy
,
H. W.
Davey
,
W. J.
Leonard
.
1998
.
Stat5b is essential for natural killer cell–mediated proliferation and cytolytic activity.
J. Exp. Med.
188
:
2067
2074
.
13
Cooper
,
M. A.
,
J. E.
Bush
,
T. A.
Fehniger
,
J. B.
VanDeusen
,
R. E.
Waite
,
Y.
Liu
,
H. L.
Aguila
,
M. A.
Caligiuri
.
2002
.
In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells.
Blood
100
:
3633
3638
.
14
Dubois
,
S.
,
J.
Mariner
,
T. A.
Waldmann
,
Y.
Tagaya
.
2002
.
IL-15Rα recycles and presents IL-15 in trans to neighboring cells.
Immunity
17
:
537
547
.
15
Mortier
,
E.
,
T.
Woo
,
R.
Advincula
,
S.
Gozalo
,
A.
Ma
.
2008
.
IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation.
J. Exp. Med.
205
:
1213
1225
.
16
Marks-Konczalik
,
J.
,
S.
Dubois
,
J. M.
Losi
,
H.
Sabzevari
,
N.
Yamada
,
L.
Feigenbaum
,
T. A.
Waldmann
,
Y.
Tagaya
.
2000
.
IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice.
Proc. Natl. Acad. Sci. USA
97
:
11445
11450
.
17
Waldmann
,
T. A.
2006
.
The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design.
Nat. Rev. Immunol.
6
:
595
601
.
18
Cheever
,
M. A.
2008
.
Twelve immunotherapy drugs that could cure cancers.
Immunol. Rev.
222
:
357
368
.
19
Conlon
,
K. C.
,
E.
Lugli
,
H. C.
Welles
,
S. A.
Rosenberg
,
A. T.
Fojo
,
J. C.
Morris
,
T. A.
Fleisher
,
S. P.
Dubois
,
L. P.
Perera
,
D. M.
Stewart
, et al
.
2015
.
Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer.
J. Clin. Oncol.
33
:
74
82
.
20
Han
,
K.-P.
,
X.
Zhu
,
B.
Liu
,
E.
Jeng
,
L.
Kong
,
J. L.
Yovandich
,
V. V.
Vyas
,
W. D.
Marcus
,
P.-A.
Chavaillaz
,
C. A.
Romero
, et al
.
2011
.
IL-15:IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization.
Cytokine
56
:
804
810
.
21
Stoklasek
,
T. A.
,
K. S.
Schluns
,
L.
Lefrançois
.
2006
.
Combined IL-15/IL-15Rα immunotherapy maximizes IL-15 activity in vivo.
J. Immunol.
177
:
6072
6080
.
22
Mortier
,
E.
,
A.
Quémener
,
P.
Vusio
,
I.
Lorenzen
,
Y.
Boublik
,
J.
Grötzinger
,
A.
Plet
,
Y.
Jacques
.
2006
.
Soluble interleukine-15 receptor α (IL-15Rα)-sushi as a selective and potent agonist of IL-15 action through IL-15Rβ/γ.
J. Biol. Chem.
281
:
1612
1619
.
23
Rosenberg
,
S. A.
2014
.
IL-2: the first effective immunotherapy for human cancer.
J. Immunol.
192
:
5451
5458
.
24
Farina
,
G. A.
,
M. R.
York
,
M.
Di Marzio
,
C. A.
Collins
,
S.
Meller
,
B.
Homey
,
I. R.
Rifkin
,
A.
Marshak-Rothstein
,
T. R. D. J.
Radstake
,
R.
Lafyatis
.
2010
.
Poly(I:C) drives type I IFN- and TGFβ-mediated inflammation and dermal fibrosis simulating altered gene expression in systemic sclerosis.
J. Invest. Dermatol.
130
:
2583
2593
.
25
Barthelemy
,
A.
,
S.
Ivanov
,
J.
Fontaine
,
D.
Soulard
,
H.
Bouabe
,
C.
Paget
,
C.
Faveeuw
,
F.
Trottein
.
2017
.
Influenza A virus-induced release of interleukin-10 inhibits the anti-microbial activities of invariant natural killer T cells during invasive pneumococcal superinfection.
Mucosal Immunol.
10
:
460
469
.
26
Mattei
,
F.
,
G.
Schiavoni
,
F.
Belardelli
,
D. F.
Tough
.
2001
.
IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation.
J. Immunol.
167
:
1179
1187
.
27
Hayakawa
,
Y.
,
M. J.
Smyth
.
2006
.
CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity.
J. Immunol.
176
:
1517
1524
.
28
Chiossone
,
L.
,
J.
Chaix
,
N.
Fuseri
,
C.
Roth
,
E.
Vivier
,
T.
Walzer
.
2009
.
Maturation of mouse NK cells is a 4-stage developmental program.
Blood
113
:
5488
5496
.
29
Huntington
,
N. D.
,
H.
Tabarias
,
K.
Fairfax
,
J.
Brady
,
Y.
Hayakawa
,
M. A.
Degli-Esposti
,
M. J.
Smyth
,
D. M.
Tarlinton
,
S. L.
Nutt
.
2007
.
NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation.
J. Immunol.
178
:
4764
4770
.
30
Lanier
,
L. L.
,
D. W.
Buck
,
L.
Rhodes
,
A.
Ding
,
E.
Evans
,
C.
Barney
,
J. H.
Phillips
.
1988
.
Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen.
J. Exp. Med.
167
:
1572
1585
.
31
Huntington
,
N. D.
,
N.
Legrand
,
N. L.
Alves
,
B.
Jaron
,
K.
Weijer
,
A.
Plet
,
E.
Corcuff
,
E.
Mortier
,
Y.
Jacques
,
H.
Spits
,
J. P.
Di Santo
.
2009
.
IL-15 trans-presentation promotes human NK cell development and differentiation in vivo.
J. Exp. Med.
206
:
25
34
.
32
Schroder
,
K.
,
P. J.
Hertzog
,
T.
Ravasi
,
D. A.
Hume
.
2004
.
Interferon-γ: an overview of signals, mechanisms and functions.
J. Leukoc. Biol.
75
:
163
189
.
33
Dubois
,
S.
,
H. J.
Patel
,
M.
Zhang
,
T. A.
Waldmann
,
J. R.
Müller
.
2008
.
Preassociation of IL-15 with IL-15Rα-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44high T cells and its antitumor action.
J. Immunol.
180
:
2099
2106
.
34
Bessard
,
A.
,
V.
Solé
,
G.
Bouchaud
,
A.
Quéméner
,
Y.
Jacques
.
2009
.
High antitumor activity of RLI, an interleukin-15 (IL-15)–IL-15 receptor α fusion protein, in metastatic melanoma and colorectal cancer.
Mol. Cancer Ther.
8
:
2736
2745
.
35
Eckelhart
,
E.
,
W.
Warsch
,
E.
Zebedin
,
O.
Simma
,
D.
Stoiber
,
T.
Kolbe
,
T.
Rülicke
,
M.
Mueller
,
E.
Casanova
,
V.
Sexl
.
2011
.
A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development.
Blood
117
:
1565
1573
.
36
Smyth
,
M. J.
,
M. W. L.
Teng
,
J.
Swann
,
K.
Kyparissoudis
,
D. I.
Godfrey
,
Y.
Hayakawa
.
2006
.
CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer.
J. Immunol.
176
:
1582
1587
.
37
Costello
,
R. T.
,
S.
Sivori
,
E.
Marcenaro
,
M.
Lafage-Pochitaloff
,
M.-J.
Mozziconacci
,
D.
Reviron
,
J.-A.
Gastaut
,
D.
Pende
,
D.
Olive
,
A.
Moretta
.
2002
.
Defective expression and function of natural killer cell-triggering receptors in patients with acute myeloid leukemia.
Blood
99
:
3661
3667
.
38
Stringaris
,
K.
,
T.
Sekine
,
A.
Khoder
,
A.
Alsuliman
,
B.
Razzaghi
,
R.
Sargeant
,
J.
Pavlu
,
G.
Brisley
,
H.
de Lavallade
,
A.
Sarvaria
, et al
.
2014
.
Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia.
Haematologica
99
:
836
847
.
39
Khaznadar
,
Z.
,
N.
Boissel
,
S.
Agaugué
,
G.
Henry
,
M.
Cheok
,
M.
Vignon
,
D.
Geromin
,
J.-M.
Cayuela
,
S.
Castaigne
,
C.
Pautas
, et al
.
2015
.
Defective NK cells in acute myeloid leukemia patients at diagnosis are associated with blast transcriptional signatures of immune evasion.
J. Immunol.
195
:
2580
2590
.
40
Spallanzani
,
R. G.
,
N. I.
Torres
,
D. E.
Avila
,
A.
Ziblat
,
X. L. R.
Iraolagoitia
,
L. E.
Rossi
,
C. I.
Domaica
,
M. B.
Fuertes
,
G. A.
Rabinovich
,
N. W.
Zwirner
.
2015
.
Regulatory dendritic cells restrain NK cell IFN-γ production through mechanisms involving NKp46, IL-10, and MHC class I–specific inhibitory receptors.
J. Immunol.
195
:
2141
2148
.
41
Jonuleit
,
H.
,
E.
Schmitt
.
2003
.
The regulatory T cell family: distinct subsets and their interrelations.
J. Immunol.
171
:
6323
6327
.
42
Stassi
,
G.
,
M.
Todaro
,
M.
Zerilli
,
L.
Ricci-Vitiani
,
D.
Di Liberto
,
M.
Patti
,
A.
Florena
,
F.
Di Gaudio
,
G.
Di Gesù
,
R.
De Maria
.
2003
.
Thyroid cancer resistance to chemotherapeutic drugs via autocrine production of interleukin-4 and interleukin-10.
Cancer Res.
63
:
6784
6790
.
43
Todaro
,
M.
,
M.
Zerilli
,
L.
Ricci-Vitiani
,
M.
Bini
,
M.
Perez Alea
,
A.
Maria Florena
,
L.
Miceli
,
G.
Condorelli
,
S.
Bonventre
,
G.
Di Gesù
, et al
.
2006
.
Autocrine production of interleukin-4 and interleukin-10 is required for survival and growth of thyroid cancer cells. [Published erratum appears in 2016 Cancer Res. 76: 7292.]
Cancer Res.
66
:
1491
1499
.
44
Li
,
M. O.
,
Y. Y.
Wan
,
S.
Sanjabi
,
A.-K. L.
Robertson
,
R. A.
Flavell
.
2006
.
Transforming growth factor-β regulation of immune responses.
Annu. Rev. Immunol.
24
:
99
146
.
45
Kang
,
S. S.
,
P. M.
Allen
.
2005
.
Priming in the presence of IL-10 results in direct enhancement of CD8+ T cell primary responses and inhibition of secondary responses.
J. Immunol.
174
:
5382
5389
.
46
Salem
,
M. L.
,
M. S.
Hossain
.
2000
.
In vivo acute depletion of CD8+ T cells before murine cytomegalovirus infection upregulated innate antiviral activity of natural killer cells.
Int. J. Immunopharmacol.
22
:
707
718
.
47
Alvarez
,
M.
,
M. N.
Bouchlaka
,
G. D.
Sckisel
,
C. M.
Sungur
,
M.
Chen
,
W. J.
Murphy
.
2014
.
Increased antitumor effects using IL-2 with anti–TGF-β reveals competition between mouse NK and CD8 T cells.
J. Immunol.
193
:
1709
1716
.
48
Jamieson
,
A. M.
,
P.
Isnard
,
J. R.
Dorfman
,
M. C.
Coles
,
D. H.
Raulet
.
2004
.
Turnover and proliferation of NK cells in steady state and lymphopenic conditions.
J. Immunol.
172
:
864
870
.
49
Talmadge
,
J. E.
,
R. B.
Herberman
,
M. A.
Chirigos
,
A. E.
Maluish
,
M. A.
Schneider
,
J. S.
Adams
,
H.
Philips
,
G. B.
Thurman
,
L.
Varesio
,
C.
Long
, et al
.
1985
.
Hyporesponsiveness to augmentation of murine natural killer cell activity in different anatomical compartments by multiple injections of various immunomodulators including recombinant interferons and interleukin 2.
J. Immunol.
135
:
2483
2491
.
50
Lucas
,
M.
,
W.
Schachterle
,
K.
Oberle
,
P.
Aichele
,
A.
Diefenbach
.
2007
.
Dendritic cells prime natural killer cells by trans-presenting interleukin 15.
Immunity
26
:
503
517
.
51
Colonna
,
M.
2007
.
TLR pathways and IFN-regulatory factors: to each its own.
Eur. J. Immunol.
37
:
306
309
.
52
Colpitts
,
S. L.
,
T. A.
Stoklasek
,
C. R.
Plumlee
,
J. J.
Obar
,
C.
Guo
,
L.
Lefrançois
.
2012
.
Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo.
J. Immunol.
188
:
2483
2487
.
53
Saito
,
T.
,
R.
Ruffman
,
R. D.
Welker
,
R. B.
Herberman
,
M. A.
Chirigos
.
1985
.
Development of hyporesponsiveness of natural killer cells to augmentation of activity after multiple treatments with biological response modifiers.
Cancer Immunol. Immunother.
19
:
130
135
.
54
Maluish
,
A. E.
,
J. R.
Ortaldo
,
J. C.
Conlon
,
S. A.
Sherwin
,
R.
Leavitt
,
D. M.
Strong
,
P.
Weirnik
,
R. K.
Oldham
,
R. B.
Herberman
.
1983
.
Depression of natural killer cytotoxicity after in vivo administration of recombinant leukocyte interferon.
J. Immunol.
131
:
503
507
.
55
Miller
,
J. S.
,
C.
Morishima
,
D. G.
McNeel
,
M. R.
Patel
,
H. E. K.
Kohrt
,
J. A.
Thompson
,
P. M.
Sondel
,
H. A.
Wakelee
,
M. L.
Disis
,
J. C.
Kaiser
, et al
.
2018
.
A first-in-human phase I study of subcutaneous outpatient recombinant human IL15 (rhIL15) in adults with advanced solid tumors.
Clin. Cancer Res.
24
:
1525
1535
.
56
Ellis-Connell
,
A. L.
,
A. J.
Balgeman
,
K. R.
Zarbock
,
G.
Barry
,
A.
Weiler
,
J. O.
Egan
,
E. K.
Jeng
,
T.
Friedrich
,
J. S.
Miller
,
A. T.
Haase
, et al
.
2018
.
ALT-803 transiently reduces SIV replication in the absence of antiretroviral treatment.
J. Virol.
92
:
e01748-17
.
57
Bernasconi
,
A.
,
R.
Marino
,
A.
Ribas
,
J.
Rossi
,
M.
Ciaccio
,
M.
Oleastro
,
A.
Ornani
,
R.
Paz
,
M. A.
Rivarola
,
M.
Zelazko
,
A.
Belgorosky
.
2006
.
Characterization of immunodeficiency in a patient with growth hormone insensitivity secondary to a novel STAT5b gene mutation.
Pediatrics
118
:
e1584
e1592
.
58
Tabellini
,
G.
,
D.
Vairo
,
O.
Scomodon
,
N.
Tamassia
,
R. M.
Ferraro
,
O.
Patrizi
,
S.
Gasperini
,
A.
Soresina
,
G.
Giardino
,
C.
Pignata
, et al
.
2017
.
Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.
J. Allergy Clin. Immunol.
140
:
553
564.e4
.
59
Mortarini
,
R.
,
C.
Vegetti
,
A.
Molla
,
F.
Arienti
,
F.
Ravagnani
,
A.
Maurichi
,
R.
Patuzzo
,
M.
Santinami
,
A.
Anichini
.
2009
.
Impaired STAT phosphorylation in T cells from melanoma patients in response to IL-2: association with clinical stage.
Clin. Cancer Res.
15
:
4085
4094
.
60
Grundström
,
S.
,
M.
Dohlsten
,
A.
Sundstedt
.
2000
.
IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common γ-chain of the IL-2 receptor.
J. Immunol.
164
:
1175
1184
.
61
Felices
,
M.
,
A. J.
Lenvik
,
R.
McElmurry
,
S.
Chu
,
P.
Hinderlie
,
L.
Bendzick
,
M. A.
Geller
,
J.
Tolar
,
B. R.
Blazar
,
J. S.
Miller
.
2018
.
Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect.
JCI Insight
3
.
62
Ghiringhelli
,
F.
,
C.
Ménard
,
M.
Terme
,
C.
Flament
,
J.
Taieb
,
N.
Chaput
,
P. E.
Puig
,
S.
Novault
,
B.
Escudier
,
E.
Vivier
, et al
.
2005
.
CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner.
J. Exp. Med.
202
:
1075
1085
.
63
Zou
,
W.
2006
.
Regulatory T cells, tumour immunity and immunotherapy.
Nat. Rev. Immunol.
6
:
295
307
.

M.F. received income from Cytune Pharma. E.M. received grant support from Cytune Pharma. E.M., A.Q., and Y.J. have a patent related to the reagents used. The other authors have no financial conflicts of interest.

Supplementary data