Intestinal epithelial cell-derived IL-15 determines local maintenance and maturation of intra-epithelial lymphocytes in the intestine.

Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intra-epithelial lymphocytes (IELs), especially CD8αα + IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells was deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased, whereas Fas expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. Forced expression of Bcl-2 by a Bcl-2 transgene partially restored CD8αα IELs in Vil-Cre IL-15cKO mice, suggesting that some IL-15 signal other than Bcl-2 is required for maintenance of CD8αα IELs. Furthermore, granzyme B production was reduced, whereas PD-1 expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. These results collectively suggested that IEC-derived IL-15 is essential for homeostasis of IELs by promoting their survival and functional maturation.

Attenuation of Allergen-, IL-13-, and TGF-α-induced Lung Fibrosis after the Treatment of rIL-15 in Mice.

Endogenous IL-15 deficiency promotes lung fibrosis; therefore, we examined the effect of induced IL-15 in restricting the progression of lung fibrosis. Our objective in this work was to establish a novel therapeutic molecule for pulmonary fibrosis. Western blot, qPCR, and ELISA were performed on the lung tissues of IL-15-deficient mice, and recombinant IL-15 (rIL-15)-treated CC10-IL-13 and CC10-TGF-α mice, and allergen-challenged CC10-IL-15 mice were examined to establish the antifibrotic effect of IL-15 in lung fibrosis. We show that endogenous IL-15 deficiency induces baseline profibrotic cytokine and collagen accumulation in the lung, and pharmacological delivery of rIL-15 downregulates Aspergillus antigen-induced lung collagen, the profibrotic cytokines IL-13 and TGF-ß1, and α-SMA+ and FSP1+ cells in mice. To confirm that overexpression of IL-15 diminishes pulmonary fibrosis, we generated CC10-rtTA-tetO7-IL-15 transgenic mice and challenged them with Aspergillus antigen. Aspergillus antigen-challenged, doxycycline (DOX)-treated CC10-IL-15 transgenic mice exhibited decreased collagen accumulation, profibrotic cytokine (IL-13 and TGF-ß1) expression, and α-SMA+ and FSP1+ cells compared with IL-15-overexpressing mice not treated with DOX. Additionally, to establish that the antifibrotic effect of IL-15 is not limited to allergen-induced fibrosis, we showed that rIL-15 or IL-15 agonist treatment restricted pulmonary fibrosis even in CC10-IL-13 and CC10-TGF-α mice. Mechanistically, we show that T-helper cell type 17 suppressor IL-15-responsive RORγ+ T regulatory cells are induced in DOX-treated, allergen-challenged IL-15-overexpressing mice, which may be a novel pathway for restricting progression of pulmonary fibrosis. Taken together, our data establishes antifibrotic activity of IL-15 that might be a novel therapeutic molecule to combat the development of pulmonary fibrosis.

Natural killer-cell deficiency alters placental development in rats.

Natural killer (NK) cells are the most prevalent leukocyte population in the uterus during early pregnancy. Natural killer cells contribute to uterine vascular (spiral artery) remodeling in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification is directed by invasive trophoblast cells. The significance of the initial wave of NK-cell-mediated vascular remodeling in species exhibiting deep trophoblast invasion such as humans and rats is not known. The purpose of this study was to generate a genetic model of NK-cell deficiency in rats, and determine the consequences of NK-cell deficiency on spiral artery remodeling and reproductive outcomes. To accomplish this task, we utilized zinc finger nuclease-mediated genome editing of the rat interleukin-15 (Il15) gene. Il15 encodes a cytokine required for NK-cell lineage development. Using this strategy, a founder rat was generated containing a frameshift deletion in Il15. Uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable NK cells. NK-cell deficiency did not impact fetal growth or viability. However, NK-cell deficiency caused major structural changes to the placenta, including expansion of the junctional zone and robust, early-onset activation of invasive trophoblast-guided spiral artery remodeling. In summary, we successfully generated an NK-cell-deficient rat and showed, using this model, that NK cells dampen the extent of trophoblast invasion and delay trophoblast-directed spiral artery remodeling. This study furthers our understanding of the role of NK cells on uterine vascular remodeling, trophoblast invasion, and placental development.

IL-15-deficient mice develop enhanced allergic responses to airway allergen exposure.

BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.

Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes.

Interleukin (IL)-15 has multiple roles in innate and adaptive immunity, especially regarding CD8+ T cells and natural killer cells. However, the role of IL-15 in regulating differentiation of T helper cell subsets and mononuclear phagocytes (MPs) in different tissues in vivo is unknown. Here we report that IL-15 indirectly regulates Th17 but not other Th subsets in the intestinal lamina propria (LP), apparently through effects on MPs. Th17 cells in the LP were more prevalent in IL-15 KO mice than their wild-type counterparts, and less prevalent in IL-15 transgenic mice than their wild-type littermates, even co-caged. MPs from the LP of these mice were sufficient to mimic the in vivo finding in vitro by skewing of cocultured wild type OVA-specific CD4+ T cells. However, production of IL-15 or lack thereof by these MPs was not sufficient to explain the skewing, as addition or blockade of IL-15 in the cultures had no effect. Rather, a skewing of the relative proportion of CD11b+, CD103+ and double positive LP MP subsets in transgenic and KO could explain the differences in Th17 cells. Thus, IL-15 may influence MP subsets in the gut in a novel way that alters the frequency of LP Th17 cells.

Differential effects of IL-15 on the generation, maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination.

IL-15 is an important cytokine for the regulation of lymphocyte homeostasis. However, the role of IL-15 in the generation, maintenance and cytotoxic potential of antigen specific T cells is not fully understood. Because the route of antigenic delivery and the vaccine modality could influence the IL-15 requirement for mounting and preserving cytotoxic T cell responses, we have investigated the immunogenicity of DNA-based vaccines in IL-15 KO mice. DNA vaccination with SIV Gag induced antigen-specific CD4(+) and CD8(+) T cells in the absence of IL-15. However, the absolute number of antigen-specific CD8(+) T cells was decreased in IL-15 KO mice compared to WT animals, suggesting that IL-15 is important for the generation of maximal number of antigen-specific CD8(+) T cells. Interestingly, antigen-specific memory CD8 cells could be efficiently boosted 8 months after the final vaccination in both WT and KO strains of mice, suggesting that the maintenance of antigen-specific long-term memory T cells induced by DNA vaccination is comparable in the absence and presence of IL-15. Importantly, boosting by DNA 8-months after vaccination revealed severely reduced granzyme B content in CD8(+) T cells of IL-15 KO mice compared to WT mice. This suggests that the cytotoxic potential of the long-term memory CD8(+) T cells is impaired. These results suggest that IL-15 is not essential for the generation and maintenance of adaptive cellular responses upon DNA vaccination, but it is critical for the preservation of maximal numbers and for the activity of cytotoxic CD8(+) T cells.

The interleukin-15 system suppresses T cell-mediated autoimmunity by regulating negative selection and nT(H)17 cell homeostasis in the thymus.

The interleukin-15 (IL-15) system is important for regulating both innate and adaptive immune responses, however, its role in autoimmune disease remained unclear. Here we found that Il15(-/-) and Il15ra(-/-) mice spontaneously developed late-onset autoimmune phenotypes. CD4(+) T cells of the knockout mice showed elevated autoreactivity as demonstrated by the induction of lymphocyte infiltration in the lacrimal and salivary glands when transferred into nude mice. The antigen-presenting cells in the thymic medullary regions expressed IL-15 and IL-15Rα, whose deficiency resulted in insufficient negative selection and elevated number of natural IL-17A-producing CD4(+) thymocytes. These findings reveal previously unknown functions of the IL-15 system in thymocyte development, and thus a new layer of regulation in T cell-mediated autoimmunity.

Interleukin-15 modulates adipose tissue by altering mitochondrial mass and activity.

Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15-/-), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15-/- mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.

Development of innate CD4+ and CD8+ T cells in Itk-deficient mice is regulated by distinct pathways.

T cell development in the thymus produces multiple lineages of cells, including innate T cells such as γδ TCR(+) cells, invariant NKT cells, mucosal-associated invariant T cells, and H2-M3-specific cells. Although innate cells are generally a minor subset of thymocytes, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8(+) T cells develop as innate cells with characteristics of memory T cells. Thus, in Itk-deficient mice, mature CD4(-)CD8(+) (CD8 single-positive [SP]) thymocytes express high levels of the transcription factor eomesodermin (Eomes) and are dependent on IL-4 being produced in the thymic environment by a poorly characterized subset of CD4(+) thymocytes expressing the transcriptional regulator promyelocytic leukemia zinc finger. In this study, we show that a sizeable proportion of mature CD4(+)CD8(-) (CD4SP) thymocytes in itk(-/-) mice also develop as innate Eomes-expressing T cells. These cells are dependent on MHC class II and IL-4 signaling for their development, indicating that they are conventional CD4(+) T cells that have been converted to an innate phenotype. Surprisingly, neither CD4SP nor CD8SP innate Eomes(+) thymocytes in itk(-/-) or SLP-76(Y145F) mice are dependent on γδ T cells for their development. Instead, we find that the predominant population of Eomes(+) innate itk(-/-) CD4SP thymocytes is largely absent in mice lacking CD1d-specific invariant NKT cells, with no effect on innate itk(-/-) CD8SP thymocytes. In contrast, both subsets of innate Eomes(+)itk(-/-) T cells require the presence of a novel promyelocytic leukemia zinc finger-expressing, SLAM family receptor adapter protein-dependent thymocyte population that is essential for the conversion of conventional CD4(+) and CD8(+) T cells into innate T cells with a memory phenotype.

Cutting edge: resident memory CD8 T cells occupy frontline niches in secondary lymphoid organs.

Resident memory CD8 T cells (TRM) are a nonrecirculating subset positioned in nonlymphoid tissues to provide early responses to reinfection. Although TRM are associated with nonlymphoid tissues, we asked whether they populated secondary lymphoid organs (SLO). We show that a subset of virus-specific memory CD8 T cells in SLO exhibit phenotypic signatures associated with TRM, including CD69 expression. Parabiosis revealed that SLO CD69(+) memory CD8 T cells do not circulate, defining them as TRM. SLO TRM were overrepresented in IL-15-deficient mice, suggesting independent regulation compared with central memory CD8 T cells and effector memory CD8 T cells. These cells were positioned at SLO entry points for peripheral Ags: the splenic marginal zone, red pulp, and lymph node sinuses. Consistent with a potential role in guarding SLO pathogen entry points, SLO TRM did not vacate their position in response to peripheral alarm signals. These data extend the range of tissue resident memory to SLO.

IL-15 deficient tax mice reveal a role for IL-1α in tumor immunity.

IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL) and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/-) TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra) were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.

The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria.

Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity among residents of malaria endemic areas indicate that memory responses to Plasmodium Ags are not adequately developed or maintained, as people who survive episodes of childhood malaria are still vulnerable to either persistent or intermittent malaria infections. In contrast, multiple exposures to radiation-attenuated Plasmodium berghei sporozoites (Pb γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge. We previously demonstrated that sterile protection induced by Pb γ-spz is MHC class I-dependent and CD8 T cells are the key effectors. IFN-γ(+) CD8 T cells that arise in Pb γ-spz-immunized B6 mice are found predominantly in the liver and are sensitive to levels of liver-stage Ag depot and they express CD44(hi)CD62L(lo) markers indicative of effector/effector memory phenotype. The developmentally related central memory CD8 T (TCM) cells express elevated levels of CD122 (IL-15Rß), which suggests that CD8 TCM cells depend on IL-15 for maintenance. Using IL-15-deficient mice, we demonstrate in this study that although protective immunity is inducible in these mice, protection is short-lived, mainly owing to the inability of CD8 TCM cells to survive in the IL-15-deficient milieu. We present a hypothesis consistent with a model whereby intrahepatic CD8 TCM cells, being maintained by IL-15-mediated survival and basal proliferation, are conscripted into the CD8 effector/effector memory T cell pool during subsequent infections.

Deficiency of interleukin-15 enhances susceptibility to acetaminophen-induced liver injury in mice.

Hepatocytes have a direct necrotic role in acetaminophen (APAP)-induced liver injury (AILI), prolonged secondary inflammatory response through innate immune cells and cytokines also significantly contributes to APAP hepatotoxicity. Interleukin 15 (IL-15), a multifunction cytokine, regulates the adaptive immune system and influences development and function of innate immune cells. To better understand the role of IL-15 in liver injury, we treated wild-type (WT) and IL-15-knockout (Il15⁻/⁻) mice with a hepatotoxic dose of APAP to induce AILI and evaluated animal survival, liver damage, APAP metabolism in livers and the inflammatory response. Production of pro-inflammatory cytokines/chemokines was greater in Il15⁻/⁻ than WT mice. Subanalysis of hepatic infiltrated monocytes revealed greater neutrophil influx, along with greater hepatic induction of inducible nitric oxide synthase (iNOS), in Il15⁻/⁻ than WT mice. In addition, the level of hepatic hemeoxygenase 1 (HO-1) was partially suppressed in Il15⁻/⁻ mice, but not in WT mice. Interestingly, elimination of Kupffer cells and neutrophils did not alter the vulnerability to excess APAP in Il15⁻/⁻ mice. However, injection of galactosamine, a hepatic transcription inhibitor, significantly reduced the increased APAP sensitivity in Il15⁻/⁻ mice but had minor effect on WT mice. We demonstrated that deficiency of IL-15 increased mouse susceptibility to AILI. Moreover, Kupffer cell might affect APAP hepatotoxicity through IL-15.

Subcapsular sinus macrophages promote NK cell accumulation and activation in response to lymph-borne viral particles.

Natural killer (NK) cells become activated during viral infection in response to cytokines or to engagement of NK cell activating receptors. However, the identity of cells sensing viral particles and mediating NK cell activation has not been defined. Here, we show that local administration of a modified vaccinia virus Ankara vaccine in mice results in the accumulation of NK cells in the subcapsular area of the draining lymph node and their activation, a process that is strictly dependent on type I IFN signaling. NK cells located in the subcapsular area exhibited reduced motility and were found associated with CD169(+)-positive subcapsular sinus (SCS) macrophages and collagen fibers. Moreover, depletion of SCS macrophages using clodronate liposomes abolished NK cell accumulation and activation. Our results identify SCS macrophages as primary mediators of NK cell activation in response to lymph-borne viral particles suggesting that they act as early sensors of local infection or delivery of viral-based vaccines.

The role of IL-15 in activating STAT5 and fine-tuning IL-17A production in CD4 T lymphocytes.

IL-15 is an important IL-2-related cytokine whose role in Th17 cell biology has not been fully elucidated. In this study, we show that exogenous IL-15 decreased IL-17A production in Th17 cultures. Neutralization of IL-15 using an Ab led to increases in IL-17A production in Th17 cultures. Both Il15(-/-) and Il15r(-/-) T cell cultures displayed higher frequency of IL-17A producers and higher amounts of IL-17A in the supernatants compared with those of wild-type (WT) cells in vitro. IL-15 down-modulated IL-17A production independently of retinoic acid-related orphan receptor-γt, Foxp3, and IFN-γ expression. Both Th17 cells and APCs produced IL-15, which induced binding of STAT5, an apparent repressor to the Il17 locus in CD4 T cells. Also, in a model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE), Il15(-/-) mice displayed exacerbated inflammation-correlating with increased IL-17A production by their CD4(+) T cells-compared with WT controls. Exogenous IL-15 administration and IL-17A neutralization reduced the severity of EAE in Il15(-/-) mice. Taken together, these data indicate that IL-15 has a negative regulatory role in fine-tuning of IL-17A production and Th17-mediated inflammation.

Interleukin 15 mediates joint destruction in Staphylococcus aureus arthritis.

BACKGROUND: Staphylococcus aureus arthritis causes severe and rapid joint damage despite antibiotics. Thus, there is a need to identify new treatment targets in addition to antibiotics. Lately, interleukin 15 (IL-15) has been implicated both in osteoclastogenesis and in bacterial clearance-2 important issues in S. aureus-induced joint destruction. This has prompted us to investigate the importance of IL-15 in S. aureus-induced arthritis. METHODS: Toxic shock syndrome toxin-1 producing S. aureus was intravenously inoculated in IL-15 knockout and wildtype mice and in wildtype mice treated with anti-IL-15 antibodies (aIL-15ab) or isotype control antibody. RESULTS: Absence of IL-15, either in knockout mice or after treatment with aIL-15ab, significantly reduced weight loss compared with controls during the infection. The severity of synovitis and joint destruction was significantly decreased in IL-15 knockout and aIL-15ab treated mice compared with controls. In IL-15 knockout mice there was a reduced number of osteoclasts in the joints. The host's ability to clear bacteria was not influenced in the IL-15 knockout mice, but significantly increased after treatment with aIL-15ab. CONCLUSIONS: IL-15 is a mediator of joint destruction in S. aureus-induced arthritis and contributes to general morbidity, which makes this cytokine an interesting treatment target in addition to conventional antibiotics.

Regulating mammalian target of rapamycin to tune vaccination-induced CD8(+) T cell responses for tumor immunity.

Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

IL-15 regulates homeostasis and terminal maturation of NKT cells.

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

IL-15-high-responder developing NK cells bearing Ly49 receptors in IL-15-/- mice.

In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-15(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D(+) NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions.