Interleukin-15 Trans-presentation Research Articles

Abstract CT258: A phase 1/2 study of JK08, an IL-15 antibody fusion protein targeting CTLA-4, in patients with advanced solid tumors

Abstract Background: CTLA-4 is a transmembrane receptor that functions as an immune checkpoint and fundamental negative regulator of T cell activation. CTLA-4 is highly expressed on most regulatory T cells (Tregs) but only upregulated in effector T cells following activation. Emerging evidence suggests anti-tumor activity of antibodies targeting CTLA-4 may be modulated by the local presence of NK cells. Interleukin 15 (IL-15) is a pleiotropic cytokine important in both innate and adaptive immunity. The IL-15/IL-15Rα complex can stimulate adjacent cells through the IL-2Rβ/γ complex. This receptor complex is present on many hematopoietic cells; however, IL-2Rβ expression is highest on NK cells and CD8+ T cells. Trans-presentation of IL-15 is believed to be the optimal mode of action for IL-15-induced proliferation and activation of NK cells and CD8+ T cells, two essential mediators of anti-tumor responses. JK08 is a recombinant fusion protein consisting of two functional elements - a fully human monoclonal antibody directed against CTLA-4 and a protein complex consisting of human IL-15 and the Sushi domain of human IL-15Rα. JK08 is intended to widen the therapeutic window for IL-15 cytokine-mediated cancer therapy and CTLA-4-targeted antibody-mediated cancer therapy. This widening of the therapeutic window can be achieved by local activation and expansion of NK cells at sites of Tregs, and by IL-15 enhancement of the activity and potency of the proximal CTLA-4 antibody, mirroring the endogenous trans-presentation orientation. Preclinical studies demonstrate JK08 can elicit ADCC-mediated killing of CTLA-4-expressing cells and interact with IL-2Rβ to promote robust T cell proliferation independent of IL-15Rα expression, suggesting that JK08 could effectively activate IL-2Rβ/γC-expressing cells preferentially at sites of Tregs, and achieve enhanced anti-tumor responses including through ADCC-mediated depletion of T regulatory cells. In vivo studies show JK08 induces robust NK and CD8+ T cell expansion in cynomolgus monkeys and anti-tumor activity in syngeneic murine models. In summary, JK08 treatment may lead to clinical activity in multiple cancer indications by counteracting the contribution of Tregs to cancer progression. Methods: The Phase 1/2 study of JK08 will enroll patients with advanced relapsed/refractory solid tumors. The study will employ an accelerated 3+3 escalation design to explore the safety, PK, immuno-regulatory activity, and preliminary anti-tumor activity of JK08. Patients will receive treatment with JK08 subcutaneously once weekly until confirmed disease progression or intolerable toxicity. Four tumor specific expansion cohorts will be initiated once dose and schedule are established from dose escalation and include melanoma, breast cancer, colorectal cancer, and a basket of advanced solid tumors. Response will be assessed every 9 weeks per RECIST v1.1. Citation Format: Nuria Kotecki, Sylvie Rottey, Elena Garralda, Maria de de Miguel, Omar Saavedra, Valentina Boni, Judit W. Johnson, Jonathan P. McNally, Mohit Mathew, Adam Hughes, Sam Murphy, Naimish Pandya. A phase 1/2 study of JK08, an IL-15 antibody fusion protein targeting CTLA-4, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT258.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Trans-presentation of interleukin-15 by interleukin-15 receptor alpha is dispensable for the pathogenesis of autoimmune type 1 diabetes.

Interleukin-15 (IL-15) is a pro-inflammatory cytokine that is required for the survival and activation of memory CD8+T cells, natural killer (NK) cells, innate lymphoid cells, macrophages and dendritic cells. IL-15 is implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and autoimmune type 1 diabetes (T1D). IL-15 receptor (IL-15R) consists of a specific α chain, the β chain that is shared with IL-2R and the common γ chain. IL-15 is unique in the manner in which it binds and signals through its receptor subunits. IL-15 that is complexed with IL-15Rα binds to the βγ receptor complex present on the responding cell to mediate its biological effects through a process referred to as trans-presentation. The trans-presented IL-15 is essential to mediate the biological effects on T lymphocytes and NK cells. Here we show that IL-15, but not IL-15Rα, is required for the development of spontaneous and virus-induced T1D, viral clearance and for antigen cross-presentation to CD8+ T lymphocytes. Our findings provide insight into the complexities of IL-15 signalling in the initiation and maintenance of CD8+ T cell-mediated immune responses.

Transpresentation of interleukin-15 by IL-15/IL-15Rα mRNA-engineered human dendritic cells boosts antitumoral natural killer cell activity.

In cancer immunotherapy, the use of dendritic cell (DC)-based vaccination strategies can improve overall survival, but until now durable clinical responses remain scarce. To date, DC vaccines are designed primarily to induce effective T-cell responses, ignoring the antitumor activity potential of natural killer (NK) cells. Aiming to further improve current DC vaccination outcome, we engineered monocyte-derived DC to produce interleukin (IL)-15 and/or IL-15 receptor alpha (IL-15Rα) using mRNA electroporation. The addition of IL-15Rα to the protocol, enabling IL-15 transpresentation to neighboring NK cells, resulted in significantly better NK-cell activation compared to IL-15 alone. Next to upregulation of NK-cell membrane activation markers, IL-15 transpresentation resulted in increased NK-cell secretion of IFN-γ, granzyme B and perforin. Moreover, IL-15-transpresenting DC/NK cell cocultures from both healthy donors and acute myeloid leukemia (AML) patients in remission showed markedly enhanced cytotoxic activity against NK cell sensitive and resistant tumor cells. Blocking IL-15 transpresentation abrogated NK cell-mediated cytotoxicity against tumor cells, pointing to a pivotal role of IL-15 transpresentation by IL-15Rα to exert its NK cell-activating effects. In conclusion, we report an attractive approach to improve antitumoral NK-cell activity in DC-based vaccine strategies through the use of IL-15/IL-15Rα mRNA-engineered designer DC.

Rituximab‐induced interleukin‐15 reduction associated with clinical improvement in rheumatoid arthritis

Rituximab therapy alters all aspects of B-cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B-cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin-15 (IL-15) along with the frequency of IL-15-related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin-15 trans-presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin-15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL-15 (138±21pg/ml at baseline, 48±18pg/ml after third cycle, P=0·03) along with IL-17 (1197±203pg/ml at baseline, 623±213pg/ml after third cycle, P=0·03) and tended to increase the frequency of circulating regulatory T cells (3·1±1cells/μl at baseline, 7·7±2cells/μl after third cycle). Rituximab also significantly decreased IL-15 trans-presentation on surface monocytes of patients negative for IL-15 serum (mean fluorescence intensity: 4·82±1·30 at baseline, 1·42±0·69 after third cycle P=0·05). Reduction of serum IL-15 was associated with decrease in CD8(+) CD45RO(+) /RA(+) ratio (1·17±0·21 at baseline, 0·36± 0·06 at third cycle, P=0·02). DAS28, erythrocyte sedimentation rate and C-reactive protein correlated significantly with CD8(+) CD45RO(+) /RA(+) ratio (R=0·323, R=0·357, R=0·369 respectively, P<0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL-15/memory T-cell-related mechanisms beyond circulating B cells.

IL-15 trans-presentation regulates homeostasis of CD4+ T lymphocytes

Interleukin-15 (IL-15) is essential for the survival of memory CD8(+) and CD4(+) T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4(+) T cells. Adoptive transfer of splenocytes from non-obese diabetic (NOD) mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.Il15(-/-) mice when compared to NOD.scid recipients. The increased accumulation of CD4(+) T cells is also observed in NOD.Il15(-/-) mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the IL-15Rα chain, but not those lacking the common gamma chain, also show increased accumulation of CD4(+) T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4(+) T cells and requires trans-presentation of IL-15. CD4(+) T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4(+) T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-γ production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4(+) T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.

Interleukin-15 in Gene Therapy of Cancer

Interleukin-15 (IL-15) exerts powerful stimulatory effects on lymphocyte subsets that result in antiviral and antitumoral activities. The functions of this cytokine are mainly mediated in a cell-to-cell contact fashion termed IL-15 trans-presentation. This function is mediated by a cell which tethers IL-15 to its plasmatic membrane complexed to IL-15 receptor alpha (IL-15Rα). Such surface complexes interact with interleukin-2 receptor beta and gamma on the adjacent cell to elicit signaling. Unlike interleukin-2, IL-15 protects from activation-induced cell death and does not promote regulatory cells. These features underlie its activity against transplanted tumors and its adjuvanticity in tumor and viral vaccines. The GMP-manufactured recombinant protein is undergoing clinical trials but its rapid renal clearance calls for biotechnological strategies to increase molecular weight and ensure IL-15Rα. trans-presentation. Since early efforts with stable transfected tumor cells, IL-15 has been tested in a variety gene therapy approaches. Those mainly include transfer of expression cassettes to tumor cells, T cells, dendritic cells, vaccination sites and the liver as a biofactory organ. Detailed mechanistic knowledge of IL-15 biology is envisaged to make the most of a powerful immunotherapeutic tool ranked as one of the most promising for cancer immunotherapy.

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

Identification of a potent anti‐IL‐15 antibody with opposing mechanisms of action in vitro and in vivo

Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15. DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized. DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor β chain (IL-15Rβ) and common gamma chain (γ(c) ). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rβ/γ(c) subunits. Human IL-15 injected into mice caused an increase in NK1.1(+) and CD3(+) cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα. The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.

Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation

AbstractInvariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα−/− mice, CD44HighNK1.1+ iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-γ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation.

Macrophage- and Dendritic-Cell-Derived Interleukin-15 Receptor Alpha Supports Homeostasis of Distinct CD8+ T Cell Subsets

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.

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Elucidation of the Interleukin-15 Binding Site on Its Alpha Receptor by NMR

The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Trans-presentation of donor-derived interleukin 15 is necessary for the rapid onset of acute graft-versus-host disease but not for graft-versus-tumor activity

AbstractThe “holy grail” of allogeneic stem cell transplantation is to preserve the graft-versus-tumor (GVT) effect while eliminating graft-versus-host disease (GVHD). Endogenous donor-derived interleukin 15 (IL-15) has been implicated in the pathogenesis of acute GVHD, yet the mechanism by which it impacts this lethal process remains unclear. Using the well-described and clinically relevant C57BL/6 → B6D2F1 murine model of acute GVHD, we demonstrate that in trans presentation of IL-15 by donor bone marrow–derived cells is required for the rapid onset of acute GVHD. Recipients of IL-15–/– C57BL/6 bone marrow cells show diminished type 1 polarization of T cells, yet there is no decrease in donor T-cell reconstitution. A molecular basis for these findings is provided with the observation that expression of T-bet, the master control gene for type 1 T-cell functions, is necessary for IL-15–mediated acute GVHD lethality. Finally, we demonstrate that in the absence of donor-derived IL-15, the GVT effect is maintained. These findings thus establish a mechanism by which endogenous donor-derived IL-15 impacts the pathobiology of acute GVHD and GVT activity.

The interleukin‐15/interleukin‐15 receptor system as a model for juxtacrine and reverse signaling

Interleukin-15 (IL-15) is a pleiotropic cytokine of the 4 alpha-helix bundle family, which binds to a receptor complex that displays common elements with the IL-2 receptor and a unique high-affinity alpha chain. This review focuses on juxtacrine and reverse signaling levels in the IL-15/IL-15R system. Specifically, we discuss how agonistic stimulation of membrane-bound IL-15 induces phosphorylation of members of the MAP kinase family and of focal adhesion kinase (FAK), thereby upregulating processes including cytokine secretion, cell adhesion and migration. In addition, we explore IL-15 trans-presentation and intracellular signaling, and define promising molecular targets for future pharmacological intervention in infectious diseases and immunological disorders. These frontiers in IL-15/IL-15Ralpha research serve as highly instructive examples for key concepts, unsolved problems and therapeutic opportunities in juxtacrine and reverse signaling in general.

Soluble Interleukin-15 Receptor α (IL-15Rα)-sushi as a Selective and Potent Agonist of IL-15 Action through IL-15Rβ/γ: HYPERAGONIST 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-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha 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-15R alpha, 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-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma 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-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, 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.

Membrane-bound and soluble IL-15/IL-15Rα complexes display differential signaling and functions on human hematopoietic progenitors

AbstractMembrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor α (Rα) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Rα complex with the IL-15Rβγc chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1β cells). This behavior is likely due to the IL-15Rα chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Rα complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rβγc). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Rα/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Rα.