Optimierte biologische Aktivität allo- und tumorreaktiver CD8-+-T-Lymphozyten im humanisierten Mausmodell durch IL-15-Superagonisten

Interleukin 15 (IL-15) gilt als eines der vielversprechendsten zukünftigen Medikamente für die Krebstherapie. Es fördert die Proliferation, Persistenz und Funktion von CD8+ T-Zellen und vermittelt zahlreiche Effekte, die es als überlegene Alternative für das derzeit in der Klinik verwendete IL-2 erscheinen lassen. Für den Einsatz von IL-15 in der vorliegenden Arbeit wurde zunächst ein Protokoll zur Herstellung von rekombinantem IL-15 in E. coli etabliert. Das hergestellte Protein hatte eine zu kommerziellen Produkten vergleichbare Bioaktivität und begünstigte die Persistenz und Aktivität antigenspezifischer, humaner CD8+ T Zellen nach adoptivem Transfer in NSG-Mäuse, wobei unter anderem ein verstärkter Effekt auf T Zellen mit TSCM-Phänotyp beobachtet wurde. Um die Bioaktivität von IL-15 zu steigern, wurden super-agonistische IL-15-Fusions¬proteine entworfen und im Expi293-System hergestellt. Dabei wurde IL 15 kovalent mit der Sushi-Domäne, der IL-15Rα-Kette und einer IgG1-Fc-Domäne verbunden, was zu einer gesteigerten Affinität der IL 15-Superagonisten zum physiologischen, niederaffinen IL 15Rβγ und zu einer stark erhöhten Halbwertszeit in Mausserum führte. Die gesteigerte Affinität der IL-15-Super¬agonisten wurde durch die IL 15Rα-Sushi-Domäne vermittelt. Eine um 13 Amino¬säuren verlängerte Sushi-Domäne zeigte im Vergleich zur normalen Form eine nochmals ge¬steigerte Affinität. Die längere Halbwertszeit wurde von der Sushi- und der IgG1-Fc-Domäne vermittelt. Die IgG1-Fc-Domäne verstärkte die Wirkung der Fusionsproteine zusätzlich über einen Mechanismus, der wahrscheinlich mit der Transpräsentation durch Fc Re¬ze¬ptoren zusammen–hängt. Die gesteigerte Bioaktivität der IL-15-Superagonisten wurde im Tiermodell mit humanen und murinen T-Zellen bestätigt und ILR13+-Fc wurde als das Fusionsprotein mit der höchsten Bioaktivität identifiziert. Im Vergleich zu anderen IL-15-Superagonisten vereint es alle derzeit bekannten Eigenschaften zur Bioaktivitätssteigerung in einem einzigen Protein. In therapeutischen Versuchen mit adoptivem Transfer tumorreaktiver T-Zellen konnte der Antitumoreffekt durch ILR13+-Fc maßgeblich verstärkt werden. Als Modellsysteme wurden NSG-Mäuse, die mit humanen AML-Blasten oder einem soliden Ovarialkarzinom engraftet wurden, verwendet. Dabei wurden sowohl antigenspezifische als auch unspezifische Effekte beobachtet. Die unspezifischen Effekte wurden wahrscheinlich durch eine ILR13+-Fc-vermittelte Überexpression von NKG2D, einem Rezeptor der angeborenen Immunantwort, auf den adoptiv transferierten T Zellen vermittelt. Die Ergebnisse dieser Arbeit zeigen, dass IL-15 und die IL-15-Superagonisten die Proliferation und Reaktivität von CD8+ T-Zellen im Rahmen der Immuntherapie fördern können. Aufgrund der hohen Bioaktivität und potenzierten Wirksamkeit, könnten vor allem die IL 15-Superagonisten in Zukunft bei der Entwicklung effizienter Therapiemethoden eingesetzt werden und dadurch einen wichtigen Beitrag zu Behandlung von Krebs leisten. rnrn

Hit parade for adoptive cell transfer therapy: the best T cells for superior clinical responses.

Adoptive cell transfer (ACT) of T cells has great clinical potential, but the numerous variables of this therapy make choices difficult. A new study takes advantage of a novel technology for characterizing the T-cell responses of patients. If applied systematically, this approach may identify biomedical correlates of protection, thereby supporting treatment optimization.

Decreased tumor surveillance after adoptive T-cell therapy

The effect of cancer immunotherapy on the endogenous immune response against tumors is largely unknown. Therefore, we studied immune responses against murine tumors expressing the glycoprotein (GP) and/or nucleoprotein of lymphocytic choriomeningitis virus (LCMV) with or without adoptive T-cell therapy. In nontreated animals, CTLs specific for different epitopes as well as LCMV-GP-specific antibodies contributed to tumor surveillance. Adoptive immunotherapy with monoclonal CTLs specific for LCMV-gp33 impaired the endogenous tumor-specific antibody and CTL response by targeting antigen cross-presenting cells. As a consequence and in contrast to expectations, immunotherapy enhanced tumor growth. Thus, for certain immunogenic tumors, a reduction of tumor-specific B- and T-cell responses and enhanced tumor growth may be an unwanted consequence of adoptive immunotherapy.

MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7S15; CD45RA⁺/S15;) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1S15;CD160S15;TIM3S15;LAG3S15;2B4⁺/S15;). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Pharmacological inhibition of interleukin-15 prevents colitis and associated bone loss in IL-10 knockout mice

Bone loss secondary to inflammatory bowel diseases (IBD) is largely explained by activated T cells producing cytokines that trigger osteoclastogenesis and accelerate bone resorptionwhile inhibiting bone formation. In IBD, elevated expression of interleukin (IL)-15, a T cell growth factor, plays a central role in T cell activation, pro-inflammatory cytokine production and the development of colitis. We previously reported that IL-15 enhances RANKL-induced osteoclastogenesis and that an IL-15 antagonist, CRB-15, prevents weight and bone loss in a mousemodel of dextran sulfate sodium-induced colitis.We hypothesized that inhibition of IL-15 signalingmight prevent bone loss in IL-10 deficient (IL10−/−) mice, that develop spontaneous bowel inflammation associatedwith osteopeniawhen they are no longer raised under germ-free conditions.Mice received anIL-15 antagonist (CRB-15, 5 μg/day, n=5) or IgG2a (5 μg/day, n=4) fromweek 10 to 14 of age. The severity of colitis was assessed by histology and bowel cytokine gene expression by real time PCR. Bone mass and architecturewere evaluated by ex vivo DXA on femur and micro-computed tomography on femur and vertebra. Bodyweight gainwas similar in the two groups. After 4 weeks, colonwas 29% shorter in CRB-15 treatedmice (p<0.006), a sign of reduced inflammation. Histological analysis indicated a transmural infiltration of inflammatory cells, lymphoepithelial lesions and increased size of villi (histological score=4/6) in IgG2a treated mice, whereas colon from CRB-15 treated mice exhibited mild infiltration of inflammatory cells of the lamina propria, no mucosal damages and a minimal increased size of villi (histological score=1.6/6). Levels of TNFα, IL-17 and IL-6 mRNA in the colon were significantly reduced in CRB-15 treated mice (p<0.04 vs IgG2), indicating a decrease in colon inflammation. CRB-15 improved femur BMD (+10.6% vs IgG2a, p<0.002), vertebral trabecular bone volume fraction (BV/TV, +19.7% vs IgG2a, p<0.05) and thickness (+11.6% vs IgG2a, p<0.02). A modest but not significant increase in trabecular BV/TV was observed at the distal femur. Cortical thicknesswas also higher at themidshaft femur in CRB-15 treatedmice (+8.3% vs IgG2a, p<0.02). In conclusion, we confirm and extend our results about the effects of CRB-15 in colitis. Antagonizing IL-15 may exert favorable effects on intestinal inflammation and prevent bone loss and microarchitecture alterations induced by colitis. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: B. Brounais-Le Royer Grant / Research Support from Novartis Consumer Health Foundation, S. Ferrari-Lacraz: none declared, D. Velin: none declared, X. Zheng: none declared, S. Ferrari: none declared, D. Pierroz: none declared.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

Potential approaches for more successful dendritic cell-based immunotherapy.

INTRODUCTION: Dendritic cells (DCs) are the most important antigen-presenting cell population for activating antitumor T-cell responses; therefore, they offer a unique opportunity for specific targeting of tumors. AREAS COVERED: We will discuss the critical factors for the enhancement of DC vaccine efficacy: different DC subsets, types of in vitro DC manufacturing protocol, types of tumor antigen to be loaded and finally different adjuvants for activating them. We will cover potential combinatorial strategies with immunomodulatory therapies: depleting T-regulatory (Treg) cells, blocking VEGF and blocking inhibitory signals. Furthermore, recommendations to incorporate these criteria into DC-based tumor immunotherapy will be suggested. EXPERT OPINION: Monocyte-derived DCs are the most widely used DC subset in the clinic, whereas Langerhans cells and plasmacytoid DCs are two emerging DC subsets that are highly effective in eliciting cytotoxic T lymphocyte responses. Depending on the type of tumor antigens selected for loading DCs, it is important to optimize a protocol that will generate highly potent DCs. The future aim of DC-based immunotherapy is to combine it with one or more immunomodulatory therapies, for example, Treg cell depletion, VEGF blockage and T-cell checkpoint blockage, to elicit the most optimal antitumor immunity to induce long-term remission or even cure cancer patients.

Interleukin-15 as a potential regulator of the innate immune response

Interleukin-15 (IL-15) is a newly-discovered cytokine that is produced by activated monocytes early in the course of the innate immune response. IL-15 is able to bind to components of the interleukin-2 receptor (IL-2R) despite the fact that it has no sequence homology with IL-2. IL-15 stimulates human natural killer cell proliferation, cytotoxicity, and cytokine production and can substitute for IL-2 under most conditions. In vitro studies indicate that monocyte-derived IL-15 may be an important determinant of IFN-gamma production by NK cells. In addition, IL-15 is able to promote the survival of natural killer cells under serum-free conditions. The IL-15 receptor is a heterotrimeric complex which is composed of the IL-2Rß and T FACE="Symbol">gt> chains in combination with a unique alpha chain (IL-15T FACE="Symbol">at>). The IL-15RT FACE="Symbol">at> chain has strong sequence homology to the IL-2RT FACE="Symbol">at> chain and confers high affinity binding to the IL-15R. In contrast to IL-2, transcript for IL-15 and IL-15T FACE="Symbol">at> is expressed in a number of tissues and indicates that IL-15 may be an important ligand for cells that express components of the IL-2R

Interleukin-15 in the pathogenesis of multiple sclerosis and its animal models

L'interleukine-15 (IL-15) contribue au développement et à l’activation des lymphocytes T CD8, des cellules immunes qui ont été impliquées dans plusieurs maladies auto-immunes telle la sclérose en plaques. Des niveaux élevés de l'IL-15 ont été trouvés chez les patients atteints de cette maladie comparativement aux témoins, mais aucune étude n'a examiné les effets de tels niveaux élevés sur les lymphocytes T CD8. Les objectifs de notre étude étaient 1- de caractériser l’expression de l'IL-15 par des lymphocytes B humains et de déterminer ses effets sur les fonctions des lymphocytes T CD8, et 2- d’évaluer l'expression in vivo de l'IL-15 dans des modèles murins de la sclérose en plaques. Nous avons établi que les cellules B humaines augmentaient leur expression de l'IL-15 suite à une stimulation via le CD40. De plus, les fonctions effectrices des lymphocytes T CD8 ont été significativement augmentées lors des co-cultures avec des cellules B alloréactives exprimant l'IL-15. Dans les modèles murins de la sclérose en plaques, nous avons détecté au sein du système nerveux central des cellules immunes exprimant l’IL-15 ainsi que des cellules T CD8 exprimant le récepteur pour cette cytokine à différents stades de la maladie. Nous avons démontré que les cellules B modulent des réponses des lymphocytes T CD8 via l’IL-15, ce qui suggère un rôle pour les cellules B dans la pathogenèse de la sclérose en plaques. Nous avons aussi mis en évidence la présence de cellules exprimant l’IL-15 dans le système nerveux central dans des modèles murins de cette maladie.

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a pro-inflammatory cytokine produced by a wide range of different cell types, especially monocytes and macrophages, in response to infective agents, playing a crucial and modulatory role in innate and adaptive immune response. Infections by intracellular microorganisms such as some bacteria, protozoa and fungi point out the role of IL-15 in the activation of monocytes/macrophages and neutrophils, a process that represents an important defense mechanism in early periods of infection during the development of innate immune response. The aims of the present study were to evaluate the effects of IL-15 on human neutrophil fungicidal activity against a high virulent Paracoccidioides brasiliensis strain ( Pb18) and to verify whether this activity was mediated by oxidative metabolism such as the production of superoxide anion and H2O2 and if it was associated with an alteration of cytokine ( IL-8 and TNF-alpha) levels. Neutrophils from peripheral blood of healthy individuals were incubated in the presence and absence of IL-15 ( 12.5 - 250ng/ml) for 18h, at 37 degrees C, under tension of 5% CO2, then infected with Pb18 for 4h and evaluated for fungicidal activity, production of superoxide anion and H2O2, and quantification of cytokines IL-8 and TNF-a in the supernatant. Preincubation of neutrophils with IL-15 induced a significant increase in the fungicidal activity of such cells in a dose-dependent manner. After activation, there was an increase in the production of superoxide anion and H2O2 by these cells, suggesting participation of such metabolites in fungicidal activity. Catalase inhibits fungicidal activity, confirming the role of H2O2 in fungus killing. However, the levels of TNF-alpha and IL-8 were not modified after incubation with IL-15, which suggests that its role is not mediated by those cytokines. Taken together, results showed that IL-15 had a modulatory effect on human neutrophils infected in vitro with a high virulent strain of P. brasiliensis, which was characterized by an increased fungicidal activity mediated by a dependent mechanism of oxidative metabolism.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for IS h increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism. (c) 2007 Elsevier Ltd. All rights reserved.

Interleukin-15: Its role in microbial infections

Interleukin-15 (IL-15) is a pleiotropic cytokine which regulates the proliferation, survival and the secretory activities of many distinct cell types in the body. This cytokine is produced by macrophages and many other cell types in response to infectious agents; it controls growth and differentiation of T and B lymphocytes, activation of Natural Killer (NK) and phagocytic cells, and contributes to the homeostasis of the immune system. The present review focuses on the biological and modulatory effects of IL-15 in microbial infections and shows that this cytokine may play a role in the host defense against infections by inducing activation of effector cells from both innate and adaptive immune system.

Selezione e processazione di cellule Natural Killer da donatore aploidentico "KIR-ligand" incompatibile per l'immunoterapia adottiva di pazienti con Leucemia Acuta Mieloblastica ad alto rischio

The effector function of natural killer (NK) cells is regulated by activating and inhibitory receptors, termed killer immunoglobulin-like receptors (KIRs). In haploidentical T-cell depleted transplantation the donor/recipient KIR mismatch significantly impacts on NK-mediated tumor cell killing, particularly in acute myeloid leukaemia (AML). Thirty-four high risk AML patients entered a phase I-II study of adoptive NK-cell based immunotherapy and were screened for the availability of one haploidentical KIR ligand mismatched donor. Thirteen of them resulted as having one suitable donor. NK cells were enriched from steady-state leukaphereses by using a double-step immunomagnetic separation system, consisting in depletion of CD3+ T cells followed by positive selection of CD56+ NK cells. CD56+ cells were enriched from 7,70% (1,26-11,70) to 93,50% (66,41-99,20) (median recovery 53,05% (30,97-72,85), median T-depletion 3,03 log (2,15-4,52) viability >92%) and their citotoxic activity was inalterate. All patients (4 progressions, 1 partial remission and 8 complete remissions) received NK cell infusion which was preceeded by immunosuppressive chemotherapy (fludarabine and cyclophosphamide) and followed by interleukin 2 injections. The median number of reinfused NK cells was 2,74x10(e)6/kg(1,11-5,00) and contamining CD3+ T cells were always less than 1x10(e)5/kg. The procedure was well-tolerated and no significant toxicity, including GvHD, related to NK cell infusion was observed. The donor NK cells were demonstrated in 5/10 patients. Among the 8 patients in complete remission 5 patients are stable after 18, 15, 4, 2 months of follow-up. Three other patients relapsed after 2 and 7 months. The patient in partial remission obtained a complete remission, which lasted for 6 months. The 4 patients with active/progressive disease showed the persistence of disease. This clinical observation may be correlated with in vitro studies, indicating that AML cells are capable to induce NK cell apoptosis in a dose-depend manner. In summery, a two-step enrichment of CD56+ NK cells allows the collection of a suitable number of target cells to be used as adoptive immunotherapy in AML patients. Infusion of NK cells is feasible and safe and adoptively transferred NK cells can be detected after infusion.