The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.

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Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been IL-153 is a cytokine that was originally described, like IL-2, as a T cell growth factor (1). Both cytokines belong to the four ␣-helix bundle family, and their membrane receptors share two subunits (the IL-2R/ IL-15R ␤ and ␥ chains) responsible for signal transduction (2). The IL-2R␤/␥ complex is an intermediate affinity receptor for both cytokines that is expressed by most NK cells and can be activated in vitro by nanomolar concentrations of IL-2 or IL-15. The high affinity IL-2 and IL-15 receptors, such as those expressed on activated T cells, can be activated with picomolar concentrations of either cytokine, and additionally contain their own private ␣ chain (IL-2R␣ and IL-15R␣) that confers cytokine specificity and enhances the affinity of cytokine binding (3).Both cytokines play pivotal roles in innate and adaptative immunity. Whereas initial in vitro experiments have shown a large functional overlap in the effects of the two cytokines (induction of the proliferation and cytotoxicity of activated lymphocytes and NK cells, co-stimulation of B cell proliferation and immunoglobulin synthesis, and chemoattraction of T cells) (1, 4 -6), more recent experiments have indicated that they can exert complementary or even contrasting actions in vivo. Whereas IL-2 or IL-2R␣ knock-out in mice was associated with autoimmune phenotypes with increased populations of activated T and B cells, IL-15 and IL-15R␣ knock-out resulted in specific defects in NK, NK-T, intraepithelial lymphocytes, and memory CD8 T cells (7,8). Furthermore, IL-2 promotes peripheral tolerance by inducing activation-induced cell death, whereas IL-15 inhibits IL-2-mediated activation-induced cell death (9), and, unlike IL-2, IL-15 is a survival factor for CD8 memory T cells (10). In line with these observations, it has been suggested that the major role of IL-2 is to limit continuous expansion of activated T cells, whereas IL-15 is critical for initiation of T cell division and survival of memory T cells (11). A novel mechanism of IL-15 action described recently is that of transpresentation in which IL-15 and IL-15R␣ are coordinately expressed by antigen-presenting cells (monocytes and dendritic cells), and IL-15 bound to IL-15R␣ is presented in trans to neighboring NK or CD8 T cells expressing only the IL-15R␤/␥ receptor (12). As a co-stimulatory event occurring at the immunological synapse, IL-15 transpresentation now appears to be a dominant mechanism for IL-15 action in vivo (13, 14) and appears to play a major role in tumor immunosurveillance (15).The IL-15R␣ and IL-2R␣ subunits form a sub-family of cytokine receptors in that they comprise extracellular parts, so called "sushi" structural domains (one in IL-15R␣ and two in IL-2R␣), at their N terminus, which are also found in complement or adhesion molecules (16). In both cases, these ...

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor–associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain–containing adaptor inducing interferon (IFN) β–dependent nuclear factor κB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated.

Interleukin-15 receptor alpha (IL-15R alpha) is a pleiotropically expressed molecule that chaperones and trans-presents IL-15 to NK and T cells. To investigate whether IL-15R alpha presented by different cells perform distinct physiological functions, we have generated four lines of mice lacking IL-15R alpha in various cell types. We find that IL-15R alpha expression on macrophages but not dendritic cells (DCs) supports the early transition of antigen specific effector CD8(+) T cells to memory cells. After memory CD8(+) T cell differentiation, IL-15R alpha expression on DCs selectively supports central memory CD8(+) T cells, whereas IL-15R alpha expression on macrophages supports both central and effector memory CD8(+) T cells. By contrast, mice lacking IL-15R alpha on macrophages, DCs, or both, exhibit equivalent defects in NK cell homeostasis and activation. These studies define unique roles for macrophage expression of IL-15R alpha and show that NK cells rely upon distinct IL-15R alpha dependent IL-15 signals than memory CD8(+) T cells. Moreover, they demonstrate the diversity, specification, and geographic restriction of cytokine signals.

IL-15 and IL-2 are two structurally and functionally related cytokines whose high affinity receptors share the IL-2R β-chain and γ-chain in association with IL-15R α-chain (IL-15Rα) or IL-2R α-chain, respectively. Whereas IL-2 action seems restricted to the adaptative T cells, IL-15 appears to be crucial for the function of the innate immune responses, and the pleiotropic expression of IL-15 and IL-15Rα hints at a much broader role for the IL-15 system in multiple cell types and tissues. In this report, using a highly sensitive radioimmunoassay, we show the existence of a soluble form of human IL-15Rα (sIL-15Rα) that arises from proteolytic shedding of the membrane-anchored receptor. This soluble receptor is spontaneously released from IL-15Rα-expressing human cell lines as well as from IL-15Rα transfected COS-7 cells. This release is strongly induced by PMA and ionomycin, and to a lesser extent by IL-1β and TNF-α. The size of sIL-15Rα (42 kDa), together with the analysis of deletion mutants in the ectodomain of IL-15Rα, indicates the existence of cleavage sites that are proximal to the plasma membrane. Whereas shedding induced by PMA was abrogated by the synthetic matrix metalloproteinases inhibitor GM6001, the spontaneous shedding was not, indicating the occurrence of at least two distinct proteolytic mechanisms. The sIL-15Rα displayed high affinity for IL-15 and behaved as a potent and specific inhibitor of IL-15 binding to the membrane receptor, and of IL-15-induced cell proliferation (IC50 in the range from 3 to 20 pM). These results suggest that IL-15Rα shedding may play important immunoregulatory functions.

To identify the epitopes in human interleukin-15 (IL-15

IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPβ/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPβ/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.