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1.
Clin Cancer Res ; 25(17): 5231-5241, 2019 09 01.
Article in English | MEDLINE | ID: mdl-31171545

ABSTRACT

PURPOSE: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. PATIENTS AND METHODS: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. RESULTS: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. CONCLUSIONS: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.


Subject(s)
Dendritic Cells/transplantation , Immunotherapy, Adoptive/methods , Lymphoma, Follicular/therapy , Neoplasm Recurrence, Local/therapy , Rituximab/administration & dosage , Adult , Aged , Animals , Antineoplastic Agents, Immunological/administration & dosage , Combined Modality Therapy , Dendritic Cells/drug effects , Dendritic Cells/immunology , Drug Resistance, Neoplasm , Female , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Injections, Intralymphatic , Interferon-alpha/pharmacology , Lymphoma, Follicular/immunology , Lymphoma, Follicular/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/pathology , Remission Induction , Salvage Therapy , Xenograft Model Antitumor Assays
2.
Cancer Immunol Res ; 6(6): 658-670, 2018 06.
Article in English | MEDLINE | ID: mdl-29622580

ABSTRACT

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Interferon Type I/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Receptor, ErbB-2/metabolism , Signal Transduction , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Female , Gene Expression Profiling , Humans , Immunophenotyping , Mice, Knockout , Mice, Transgenic , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Receptor, ErbB-2/genetics , Tumor Stem Cell Assay
3.
Blood Transfus ; 13(3): 464-71, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25761309

ABSTRACT

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.


Subject(s)
Cell Culture Techniques/methods , Killer Cells, Natural/cytology , Cell Culture Techniques/instrumentation , Cryopreservation/methods , Female , Humans , Killer Cells, Natural/transplantation , Leukapheresis/methods , Male
4.
Vaccine ; 29(23): 3935-44, 2011 May 23.
Article in English | MEDLINE | ID: mdl-21481326

ABSTRACT

Human gammaherpesviruses such as Epstein-Barr virus (EBV) cause lifelong infections and associated diseases, by virtue of their ability to establish latent infection. Many studies performed in the past years in murine herpesvirus 68 (MHV-68) model of infection suggested that the limited immunity generated against isolated viral components by subunit vaccines cannot counteract the multiple immune evasion strategies operated by gammaherpesviruses. Indeed, a significant inhibition of long-term latency establishment could be observed in mice vaccinated with strains of genetically modified MHV-68 defective in reactivation or establishment of latency. In this study, we focused on the effects of interferon-α (IFN-α) on both the lytic and latent phase of MHV-68 infection, as exerted by the constitutive release of IFN-α1 by a clone of MHV-68 genetically modified to produce this cytokine (MHV-68mIFNα1). Although the MHV-68mIFNα1 recombinant virus exhibited in vitro replication features indistinguishable from those of the wild type MHV-68, its pathological properties were severely attenuated in vivo in immunocompetent mice and not in mice rendered genetically unresponsive to type I IFN, suggesting that a stronger immune response was primed in the presence of the cytokine. Notably, MHV-68mIFNα1 attenuation did not result in a reduced level of long-term spleen latency establishment. These results prompted us to evaluate the efficacy of MHV-68mIFNα1 in a prophylactic vaccination regimen aimed at inhibiting the symptoms of acute virus infection and the establishment of long-term latency after MHV-68 challenge. Our results show that mice vaccinated with MHV-68mIFNα1, administered as a live-attenuated or partially inactivated (by Psoralen and UV treatment) vaccine, were protected against the challenge with wt MHV-68 from all phases of infection. The ability of MHV-68mIFNα1 to produce IFN-α at the site of the infection, thus efficiently stimulating the immune system in case of virus reactivation from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the previously reported latency-deficient clones.


Subject(s)
Herpesviridae Infections/prevention & control , Interferon-alpha/metabolism , Organisms, Genetically Modified/physiology , Rhadinovirus/pathogenicity , Tumor Virus Infections/prevention & control , Vaccines, Attenuated/administration & dosage , Animals , Cell Line , Female , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Humans , Interferon-alpha/genetics , Mice , Mice, Inbred C57BL , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism , Recombination, Genetic , Rhadinovirus/genetics , Rhadinovirus/metabolism , Spleen , Tumor Virus Infections/immunology , Tumor Virus Infections/virology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Virus Replication
5.
J Immunol ; 170(10): 5195-202, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12734367

ABSTRACT

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14(+) monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-gamma with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8(+) T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8(+) T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8(+) T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8(+) T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Dendritic Cells/immunology , Epitopes, T-Lymphocyte/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Herpesvirus 4, Human/immunology , Interferon-alpha/pharmacology , Monocytes/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/transplantation , Cell Culture Techniques/methods , Cell Division/immunology , Cell Line, Transformed , Cytotoxicity, Immunologic , Dendritic Cells/cytology , Dendritic Cells/transplantation , Female , Humans , Immunologic Memory/immunology , Injections, Intraperitoneal , Lymphocyte Activation/immunology , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/prevention & control , Mice , Mice, SCID , Monocytes/cytology , Peptide Fragments/administration & dosage , Peptide Fragments/immunology , Peptide Fragments/pharmacology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology
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