RESUMEN
Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.
Asunto(s)
Epítopos/química , Interleucina-15/química , Complejos Multiproteicos/química , Receptores de Interleucina-15/química , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Humanos , Subunidad beta del Receptor de Interleucina-2/química , Modelos Moleculares , Transducción de SeñalRESUMEN
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-ß, 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.
Asunto(s)
Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Traslado Adoptivo/métodos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Receptores de Hialuranos/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rß/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15- and IL-2-dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2-dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.
Asunto(s)
Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-15/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad beta del Receptor de Interleucina-2/genética , Interleucina-2/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proliferación Celular , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucina-15/antagonistas & inhibidores , Interleucina-15/metabolismo , Subunidad alfa del Receptor de Interleucina-15/genética , Subunidad alfa del Receptor de Interleucina-15/inmunología , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad beta del Receptor de Interleucina-2/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Ratones , Unión Proteica , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.
Asunto(s)
Adenocarcinoma/terapia , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Carcinoma de Células Renales/terapia , Neoplasias del Colon/terapia , Inmunoterapia/métodos , Interleucina-15/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Adenocarcinoma/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Memoria Inmunológica/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacosRESUMEN
Interleukin (IL)-15 and its specific receptor chain, IL-15Rα, support the development of various effector cells, including NK and CD8 T cells via a mechanism called trans-presentation. Whereas the dynamic of trans-presentation has been shown to involve the recycling of IL-15Rα by presenting cells, the way responding cells integrate, or take advantage of this process has not been evaluated yet. To address this question, we set up a trans-presentation model using a membrane-bound IL-15.IL-15Rα fusion protein, and found that IL-15 is detectable within responding cells following IL-15 trans-presentation. The role of the proteolytic cleavage of IL-15Rα in this process was investigated by generating an uncleavable form of IL-15Rα. We showed that IL-15 entry into responding cells necessitates the cleavage of IL-15.IL-15Rα complex from the surface of IL-15 presenting cells, and observed that IL-15Rα cleavage is associated with a decrease of the duration of Stat5 signaling. Once separated from presenting cells, responding cells are able to recycle IL-15.IL-15Rα complexes via intracellular compartments, for residual proliferation in a time-limited manner. These studies define an unprecedented cytokine pathway in which the IL-15.IL-15Rα complex cleaved from presenting cells allows responding cells to internalize, store and use IL-15.IL-15Rα complex for their own proliferation and survival.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-15/inmunología , Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Presentación de Antígeno/inmunología , Comunicación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/inmunología , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Células HEK293 , Células HeLa , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Subunidad alfa del Receptor de Interleucina-15/genética , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Células Asesinas Naturales/metabolismo , Microscopía Fluorescente , Unión Proteica/inmunología , Transporte de Proteínas/inmunología , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismoRESUMEN
Cytokines are crucial molecules for maintaining the proper functioning of the immune system. Nevertheless, a dysregulation of cytokine expression could be involved in the pathogenesis of autoimmune diseases. Interleukin (IL)-15 is a key factor for natural killer cells (NK) and CD8 T cells homeostasis, necessary to fight cancer and infections but could also be considered as a pro-inflammatory cytokine involved in autoimmune inflammatory disease, including rheumatoid arthritis, psoriasis, along with tumor necrosis factor alpha (TNF-α), IL-6, and IL-1ß. The molecular mechanisms by which IL-15 exerts its inflammatory function in these diseases are still unclear. In this study, we generated an IL-15-derived molecule called NANTIL-15 (New ANTagonist of IL-15), designed to selectively inhibit the action of IL-15 through the high-affinity trimeric IL-15Rα/IL-2Rß/γc receptor while leaving IL-15 signaling through the dimeric IL-2Rß/γc receptor unaffected. Administrating of NANTIL-15 in healthy mice did not affect the IL-15-dependent cell populations such as NK and CD8 T cells. In contrast, we found that NANTIL-15 efficiently reduced signs of inflammation in a collagen-induced arthritis model. These observations demonstrate that the inflammatory properties of IL-15 are linked to its action through the trimeric IL-15Rα/IL-2Rß/γc receptor, highlighting the interest of selectively targeting this receptor.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-15 , Interleucina-15 , Animales , Citocinas , Inflamación/tratamiento farmacológico , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Células Asesinas Naturales , RatonesRESUMEN
INTRODUCTION: Novel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity. METHODS: BALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated. RESULTS: Adoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion. CONCLUSIONS: Anti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Vacunas contra el Cáncer/inmunología , Tolerancia Inmunológica , Neoplasias Ováricas/inmunología , Receptor ErbB-2/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Antiidiotipos/sangre , Células CHO , Línea Celular Tumoral , Supervivencia Celular/inmunología , Cricetinae , Femenino , Células HEK293 , Humanos , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Neoplasias Ováricas/terapia , Células TH1/inmunología , Células Th2/inmunología , TrastuzumabRESUMEN
PURPOSE: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. EXPERIMENTAL DESIGN: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. RESULTS: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. CONCLUSIONS: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Carcinoma/inmunología , Sinergismo Farmacológico , Receptores ErbB/inmunología , Neoplasias Pancreáticas/inmunología , Receptor ErbB-2/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Línea Celular Tumoral , Receptores ErbB/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Receptor ErbB-2/química , TrastuzumabRESUMEN
Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.