Search PubMed⌕ Search

Biomedical subjects

Barbara Savoldo

Publications and source records attributed to Barbara Savoldo.

12 recordsLinked to original sources
PubMed2005

Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis.

Effector-memory T cells expressing Fas (Apo-1/CD95) are switched to an apoptotic program by cross-linking with Fas-ligand (FasL). Consequently, tumors that express FasL can induce apoptosis of infiltrating Fas-positive T lymphocytes and subdue any antitumor host immune response. Since Epstein-Barr virus (EBV)-associated tumors such as Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) express FasL, we determined whether EBV-specific cytotoxic T lymphocytes (EBV-CTLs) could be modified to resist this evasion strategy. We show that long-term down-modulation of Fas can be achieved in EBV-CTLs by transduction with small interfering RNA (siRNA) encoded in a retrovirus. Modified T cells resisted Fas/FasL-mediated apoptosis compared with control cells and showed minimal cleavage of the caspase3 substrate poly(ADP-ribose) polymerase (PARP) protein after Fas engagement. Prolonged Fas stimulation selected a uniformly Fas(low) and FasL resistant population. Removal of responsiveness to this single death signal had no other discernible effects on EBV-CTLs. In particular, it did not lead to their autonomous growth since the modified EBV-CTLs remained polyclonal, and their survival and proliferation retained dependence on antigen-specific stimulation and on the presence of other physiologic growth signals. EBV-CTLs with knocked down Fas should have a selective functional and survival advantage over unmodified EBV-CTLs in the presence of tumors expressing FasL and may be of value for adoptive cellular therapy.

Animals↗
PubMed2005

Cellular immunity to Epstein-Barr virus in liver transplant recipients treated with rituximab for post-transplant lymphoproliferative disease.

The evaluation of long-term cellular immunity to EBV in pediatric orthotopic liver transplant (OLT) recipients after treatment with the humanized anti-CD20 monoclonal antibody (Rituximab) has not yet been explored. At our institution, one child with EBV-related mononucleosis-like syndrome and five children with polymorphic-EBV-PTLD occurring 6-88 months after OLT were treated with Rituximab. Treatment was well tolerated. All children achieved complete remission. After Rituximab, B-lymphocytes were undetectable in the peripheral blood and EBV-load, monitored with real-time PCR, decreased to undetectable levels in all children from >4000 copies/microg DNA at diagnosis. Four to eight months after Rituximab, EBV-load increased (>4000 copies/microg DNA) in four children, and PTLD recurred in three. Their frequency of EBV-specific T-cell precursors, measured by Elispot analysis, remained lower than in healthy controls. Rituximab effectively induced regression of PTLD in OLT recipients. However, EBV-specific T-cell immunocompetence, which may be crucial for the long-term control of EBV-mediated proliferation, did not improve.

Antibodies, Monoclonal↗
PubMed2005

Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients.

Epstein-Barr virus (EBV) is a common viral infection in pediatric liver transplant patients and can lead to development of post-transplant lymphoproliferative disorder (PTLD). Differing studies have used immunosuppression reduction, antiviral medications or i.v. CMV-immunogloublin for EBV prevention and treatment. The purpose of this study was to determine whether implementation of a protocol for frequent EBV monitoring and EBV viral load-driven immunosuppression reduction could decrease the incidence of PTLD in our patient population. All data were prospectively collected between 2001 and 2004 at a single institution. Seventy-three patients were entered into the study. Patients were divided into a historical control group (pre-2001, 30 patients) and a treatment group (post-2001, 43 patients). Approximately 1271 blood samples of 73 patients were collected between 2001 and 2004. Eleven out of 43 patients received immunosuppression tapering due to high EBV viral loads (>4000 copies/microg DNA). One patient developed allograft rejection after immunosuppression modulation. Prior to 2001, the incidence of PTLD at our institution was 16%. After instituting a protocol for EBV monitoring, the incidence of PTLD decreased to 2% (p-value<0.05). These findings illustrate that frequent EBV viral load monitoring and preemptive immunosuppression modulation have an integral role in preventing PTLD in the pediatric liver transplant population.

Adolescent↗
PubMed2004

Targeted delivery of adenoviral vectors by cytotoxic T cells.

Effective targeting of vectors to tumor cells that have metastasized to multiple different tissue sites remains a major challenge for gene therapy. Tumor-specific cytotoxic T lymphocytes (CTLs) have been shown in animal models and in humans to be able to cross tissue barriers and traffic to tumor cells. However, their capacity to eliminate malignancy has been limited by tumor immune evasion strategies. We now use a model of Epstein-Barr virus-mediated malignancy to show that human CTLs themselves may be modified to release therapeutic vectors following engagement of their antigen-specific receptors and that these vectors will effectively transduce and destroy tumor targets. We generated EBV-specific CTLs that were transgenic for the adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand (CD40L) promoter. Following transduction with E1-deficient adenoviral vectors, these CTLs produced infectious virus when exposed to HLA-matched EBV-expressing targets, but not on exposure to major histocompatibility complex (MHC)-mismatched or otherwise irrelevant cells. This approach provides a means of delivering oncolytic/therapeutic vectors not only to locally accessible macroscopic tumors as is presently the case, but also to disseminated metastatic disease, while avoiding the risks associated with systemic administration of large doses of adenoviral vectors.

Adenoviridae↗
PubMed2004

Cellular therapy of Epstein-Barr-virus-associated post-transplant lymphoproliferative disease.

During the immunodeficiency that follows hemopoietic stem cell transplant or solid organ transplant, lymphoproliferation can develop due to uncontrolled expansion of Epstein-Barr-virus (EBV)-infected B cells that express the full spectrum of EBV latent antigens. As development of post-transplant lymphoproliferative disease (PTLD) in these patients is clearly associated with a deficient EBV-specific cellular immune response, immunotherapy strategies to restore the EBV-specific immune response have been evaluated. In hemopoietic stem cell transplant recipients, adoptively transferred donor-derived EBV-specific T cells have been able to restore immunity and eradicate overt lymphoproliferation. Autologous or closely matched allogeneic EBV-specific cytotoxic T lymphocytes have also shown promise in recipients of solid organ transplant. The use of genetically modified T cells or newer suicide genes may result in improved safety and efficacy. Current challenges are to define indications for immunotherapy or antibody therapy in patients with incipient or overt PTLD.

Adoptive Transfer↗
PubMed2003

Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications.

Adenovirus (Ad) infections are responsible for considerable morbidity and mortality, particularly in pediatric hematopoietic stem cell transplant (HSCT) recipients. To date there is no therapy. The present study was motivated by the potential for using adoptive immunotherapy as either prophylaxis or treatment for Ad infections and associated diseases. The authors have developed a protocol to reactivate Ad-specific memory T cells from peripheral blood mononuclear cells (PBMCs) using a clinical-grade adenoviral vector. Such lines contain a specific CD4 and CD8 T-cell component and are capable of recognizing and lysing target cells infected with wild-type Ad serotypes from different Ad groups. Furthermore, the frequency of Ad-specific precursors can be determined in PBMCs ex vivo and used as a means to assess changes in Ad-specific T-cell memory responses after infusion. This is the first report of a simple and reproducible method to activate and expand Ad-specific cytotoxic T lymphocytes (CTLs), which should be protective against the range of different Ad subtypes that affect transplant recipients.

Adenoviridae↗
PubMed2003

Adoptive T-cell therapy for EBV-associated post-transplant lymphoproliferative disease.

Increased understanding of the mechanisms by which T lymphocytes recognize virus and tumor-specific antigens has fueled the use of adoptive immunotherapy for viral and malignant diseases. An ideal candidate for such treatment is Epstein-Barr virus (EBV). EBV-associated post-transplant lymphoproliferative disorder (PTLD) is a serious complication post-solid organ transplant (SOT) or hematopoietic stem cell transplant (HSCT). The disease is essentially the result of suppression of cytotoxic T-cell function and despite various treatment strategies the course may still be fulminant and lethal. Therefore, an adoptive immunotherapeutic approach using ex vivo derived EBV-specific CTL offers a promising solution not only for the treatment but also as prophylaxis for PTLD. The infusion of EBV-CTL has been demonstrated to be safe and effective in allogeneic HSCT recipients and their use post-SOT is being evaluated.

Adoptive Transfer↗
PubMed2002

Autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for the treatment of persistent active EBV infection.

Chronic active Epstein-Barr virus (CAEBV) infection syndrome is a heterogeneous EBV-related disorder characterized by chronic fatigue, fever, lymphadenopathy, and/or hepatosplenomegaly, associated with abnormal patterns of antibody to EBV. CAEBV can range from disabling mild/moderate forms to rapidly lethal disorders. Even patients with mild/moderate disease frequently suffer adverse effects from long-term anti-inflammatory agents and have a quality of life that progressively deteriorates. It is still unknown why these individuals are unable to produce an effective immune response to control EBV, and no effective treatment is currently available. Since ex vivo-expanded EBV-specific cytotoxic T lymphocytes (EBV-CTLs) can safely restore EBV-specific cellular immune responses in immunodeficient patients, we assessed the possibility that adoptive immunotherapy might also effectively treat CAEBV infection. Following stimulation with irradiated EBV-transformed lymphoblastoid cell lines (LCLs), EBV-CTLs were successfully generated from 8 of 8 patients with the mild/moderate form of CAEBV infection. These CTLs were predominantly CD3(+) CD8(+) cells and produced specific killing of the autologous LCLs. There were 5 patients with 1- to 12-year histories of disease who were treated with 1 to 4 injections of EBV-CTLs. Following infusion, there was resolution of fatigue and malaise, disappearance of fever, and regression of lymphadenopathy and splenomegaly. The pattern and titers of anti-EBV antibodies also normalized. No toxicity was observed. There were 4 patients who did not show any relapse of disease within 6 to 36 months follow-up; one patient had recurrence of fatigue and myalgia one year after CTL infusion. We suggest that adoptive immunotherapy with autologous EBV-CTLs may represent a safe and feasible alternative treatment for patients affected with mild/moderate CAEBV infection and that this approach should be evaluated in the more severe forms of the disease.

Adolescent↗
PubMed2002

Transgenic expression of CD40 ligand produces an in vivo antitumor immune response against both CD40(+) and CD40(-) plasmacytoma cells.

Because tumor-specific antigens have been identified in multiple myeloma (MM), immunotherapy might provide an additional treatment modality for the disease. Expression of CD40 ligand (CD40L) proximate to the MM cells might serve this purpose, either by increasing their capacity to present self-antigens by activation through their CD40 receptor or by the recruitment of professional antigen-presenting cells (APCs) able to take up and present tumor-associated antigens. To distinguish between these possibilities and predict whether human CD40(-) myeloma might respond to this approach, we examined 3 murine plasmacytoma cell lines, 2 (MPC-11 and S107) expressing the CD40 molecule and 1 (X-24) lacking such expression. Syngeneic BALB/CBYJ mice were inoculated subcutaneously with tumor cells mixed with CL7.1 fibroblasts, retrovirally transduced to express either the mCD40L or the neo gene. For all 3 plasmacytoma cell lines, coinjection with CL7.1/mCD40L significantly reduced local tumor growth compared with controls. This effect was mediated by a systemic antitumor immune response, since mice immunized with tumor and CL7.1/mCD40L were resistant to subsequent challenge with tumor, and tumor growth inhibition was abolished when CD8(+) or CD4(+) lymphocytes were depleted. Because expression of CD40L gave equivalent protection from CD40(+) and CD40(-) tumors and transgenic-CD40L failed to up-regulate costimulatory molecules in either tumor, the protective effects of CD40L probably resulted from recruitment/activation of professional APCs rather than from CD40 activation of plasmacytoma cells. As further support of this concept, we found that mice were also well protected if CL7.1 and CD40L were injected together with apoptotic plasmacytoma cells from these tumors. Hence, transgenic CD40L expression may produce an antimyeloma immune response against either CD40(+) or CD40(-) tumors and may be of therapeutic value for both types of myeloma in humans.

Animals↗
PubMed2002

Generation of EBV-specific CD4+ cytotoxic T cells from virus naive individuals.

Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.

Adult↗
PubMed2002

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the prevention and treatment of EBV-associated post-transplant lymphomas.

The Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD) that occur in individuals immunosuppressed by solid organ transplant (SOT) or T cell-depleted stem cell transplantation (SCT) are unequivocally a result of T cell dysfunction. Reconstitution of "at-risk" patients with EBV-specific cytotoxic T lymphocyte (CTL) lines that have been reactivated and expanded in vitro, should prevent the development of post-transplant lymphoproliferative disease or treat pre-existing disease. We have provided over 125 infusions of donor-derived EBV-specific CTL to 60 recipients of T cell-depleted stem cells. As prophylaxis, infusions were safe and effective, as no patient developed EBV-LPD, in contrast to 11.5% of controls who did not receive CTL. The CTL-reconstituted cellular immune responses to EBV, persisted for up to 80 months following infusion and reduced the high virus load seen in about 12% of patients. CTL were also effective in two of three patients who received CTL as treatment for fulminant disease. SOT recipients are also good candidates for CTL therapy, but present problems not seen in bone marrow transplant recipients. First the CTL product must be autologous, since the majority of tumors are recipient-derived and allogeneic CTL are unlikely to survive in vivo. Second most patients continue to receive immunosuppressive drugs, which may compromise the function of infused CTL. Third, unlike SCT recipients SOT recipients do not have an empty niche for EBV-specific CTL. Finally, standard protocols are not effective in generating CTL from seronegative recipients of EBV-carrying organs, who are the patients most at risk for the development of EBV-LPD. For CTL to be an option for the management of EBV in these patients, a sensitive and specific assay for the prediction of high-risk patients is required as well as an effective method for the generation of EBV-specific CTL from seronegative recipients.

Cell Line, Transformed↗