Yannick Jacques

IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

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.

Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor

Consistent with their function in immune surveillance, natural killer (NK) cells are distributed throughout lymphoid and nonlymphoid tissues. However, the mechanisms governing the steady-state trafficking of NK cells remain unknown. The lysophospholipid sphingosine 1-phosphate (S1P), by binding to its receptor S1P1, regulates the recirculation of T and B lymphocytes. In contrast, S1P5 is detected in the brain and regulates oligodendrocyte migration and survival in vitro. Here we show that S1P5 was also expressed in NK cells in mice and humans and that S1P5-deficient mice had aberrant NK cell homing during steady-state conditions. In addition, we found that S1P5 was required for the mobilization of NK cells to inflamed organs. Our data emphasize distinct mechanisms regulating the circulation of various lymphocyte subsets and raise the possibility that NK cell trafficking may be manipulated by therapies specifically targeting S1P5.

Randomized Controlled Trial of a Monoclonal Antibody against the Interleukin-2 Receptor (33B3.1) as Compared with Rabbit Antithymocyte Globulin for Prophylaxis against Rejection of Renal Allografts

Interleukin-2 is a major growth factor for activated T lymphocytes, and antibodies reacting with the Tac-chain component of the interleukin-2 receptor can prevent allograft rejection in animals. Because Tac chains are expressed only on a small fraction of activated lymphocytes, monoclonal antibodies against the interleukin-2 receptor may offer a more specific means of immunosuppression than polyclonal antilymphocyte globulin in prophylaxis against graft rejection. Therefore, we compared the immunosuppressive effect of 33B3.1, a rat monoclonal antibody against the human Tac chain, with the effect of a rabbit polyclonal antithymocyte globulin in a randomized study of 100 recipients of first renal transplants. Injections of 33B3.1 (10 mg per day) were tolerated well, whereas major side effects in 15 of 47 patients (32 percent) receiving antithymocyte globulin required discontinuation of treatment before day 14. The incidence of rejection episodes was not statistically different in the two groups at days 14, 30, 60, and 90 after transplantation. Patient and graft survival was also equal in the two groups at one year (96 and 85 percent, respectively, in both groups), and graft function was similar. The total number of infectious episodes within the first three months was lower in the 33B3.1 group than in the antithymocyte group (47 vs. 72). The drop in peripheral-blood lymphocyte concentrations was significantly larger in the patients treated with antithymocyte globulin. The level of circulating Tac-chain-bearing lymphocytes remained below 1 percent during 33B3.1 treatment, as compared with 4 to 5 percent during antithymocyte-globulin treatment (P not significant). We conclude that 33B3.1 is as effective as antithymocyte globulin in the prevention of renal-transplant rejection, and its use results in fewer infections and side effects.

Soluble IL-15Rα sushi as a selective and potent agonist of IL-15 action through IL-15Rβ/γ: hyper-agonist IL-15-IL-15Rα fusion proteins

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 suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15Rα chain corresponding to the entire extracellular domain of IL-15Rα behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15Rα, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15Rβ/γ heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15Rα/β/γ membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15Rα-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15Rα-sushi. After binding to IL-15Rβ/γ, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

Natural, Proteolytic Release of a Soluble Form of Human IL-15 Receptor α-Chain That Behaves as a Specific, High Affinity IL-15 Antagonist

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.

PREVENTION OF REJECTION OF KIDNEY TRANSPLANTS BY MONOCLONAL ANTIBODY DIRECTED AGAINST INTERLEUKIN 2

The effect of 33B3.1, a rat IgG2a monoclonal antibody (MAb) directed against interleukin 2 receptors on activated T lymphocytes, was studied during the first two weeks after transplantation in an attempt to prevent rejection in primary, cadaveric, kidney transplant recipients. 9 patients received 5 mg 33B3.1 daily by intravenous infusion for 14 days (group A) and 18 received 10 mg daily (group B). Both groups also received prednisone and azathioprine. In threatened rejection, rescue treatment consisted of anti-thymocyte globulin (ATG). 33B3.1 was well tolerated, with mild fever during the first 2 days being the most common side-effect. 3 patients in group A had a reversible acute rejection episode before day 14, whereas only 1 patient in group B had reversible rejection. 20 of 30 historical control patients (prednisone/azathioprine) and 2 of 55 patients previously treated with ATG had a rejection episode in a similar period after transplantation. Trough levels of 33B3.1 in the blood ranged from undetectable to 1.6 micrograms/ml in group A and from 0.3 to 9 micrograms/ml in group B. Most patients had antibodies against 33B3.1 by the end of treatment.

NATURAL SPLICING OF EXON 2 OF HUMAN INTERLEUKIN-15 RECEPTOR ALPHA -CHAIN MRNA RESULTS IN A SHORTENED FORM WITH A DISTINCT PATTERN OF EXPRESSION

We report the existence of eight different interleukin-15 receptor α-chain (IL-15Rα) transcripts resulting from exon-splicing mechanisms within the IL-15Rα gene. Two main classes of transcripts can be distinguished that do or do not (Δ2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Rα bound IL-15 with high affinity, Δ2IL-15Rα was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Rα was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15·IL-15Rα complex at the nuclear level. In sharp contrast, Δ2IL-15Rα was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Rα is a natural process that might play regulatory roles at different levels.

High antitumor activity of RLI, an interleukin-15 (IL-15)–IL-15 receptor α fusion protein, in metastatic melanoma and colorectal cancer

Interleukin (IL)-15 has an important role in tumor immunosurveillance and has a contemplated use in tumor immunotherapy. We have previously engineered the fusion protein RLI, composed of the NH2-terminal (amino acids 1–77, sushi+) domain of IL-15 receptor α coupled via a linker to IL-15, and shown that it displayed far better efficacy than IL-15 in vitro. In this report, we investigated in vivo whether RLI would be a better alternative than IL-15 and IL-2 for cancer treatment using two distinct animal models. B16F10 mouse melanoma cells were injected in C57BL/6 mice either i.v. or intrasplenically for lung or liver metastasis, respectively. HCT-116 human colorectal cancer cells were injected in the cecum of nude mice. We show that RLI has a higher efficiency than IL-15 or IL-2 to reduce lung and liver metastasis and enhance survival in the mouse B16F10 melanoma model, a result that was associated with a higher half-life in vivo. We also found that the antitumoral effect of RLI was completely abolished by in vivo depletion of natural killer cells using anti-asialoGM1 antibody. Moreover, RLI was also efficient to reduce by 50% tumor growth and the progression of metastasis of human colon carcinoma cells in an orthotopic nude mouse model. The fusion protein RLI has revealed strong anticancer effect in two different cancer models overcoming the limited effect of IL-15 by increasing its bioavailability and efficiency. These findings hold significant importance for the use of RLI as a potential adjuvant/therapeutic. [Mol Cancer Ther 2009;8(9):2736–45]

The Soluble α Chain of Interleukin-15 Receptor: A Proinflammatory Molecule Associated with Tumor Progression in Head and Neck Cancer

Interleukin (IL)-15 is a proinflammatory cytokine, as it induces the production of inflammatory cytokines [IL-6, tumor necrosis factor alpha (TNFalpha), IL-17, etc.]. A correlation between high intratumoral IL-15 concentrations and poor clinical outcome in lung and head and neck cancer patients has been recently reported. The purpose of this study was to investigate the role of the soluble alpha chain of IL-15 receptor (sIL-15Ralpha), a natural regulator of IL-15, in head and neck cancer. Fifty-three newly diagnosed untreated head and neck cancer patients were included in this study. Quantification of sIL-15Ralpha was performed with a newly developed RIA. Increased serum sIL-15Ralpha concentrations were found in head and neck cancer patients and were closely correlated with poor clinical outcome both in terms of locoregional control and survival even on multivariate analysis. sIL-15Ralpha was mainly produced by tumor cells via proteolytic cleavage of IL-15Ralpha mediated by ADAM-17. A correlation was observed between ADAM-17 expression in tumor cells and serum sIL-15Ralpha concentrations. Surprisingly, sIL-15Ralpha did not act in vitro as an IL-15 antagonist but rather as an enhancer of IL-15-induced proinflammatory cytokines (IL-6, TNFalpha, and IL-17) that may promote tumor progression. This new tumor evasion mechanism based on amplification of the intratumoral inflammatory reaction is probably not restricted to head and neck cancer, as other tumors have been shown to release sIL-15Ralpha. Overall, these results support for the first time an original protumor role of sIL-15Ralpha in cancer.

Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Mediates Internalization and Degradation of Leukemia Inhibitory Factor but Not Signal Transduction

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886–20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF availablein vivo.