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In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed. © 2011 Published by Elsevier Ltd.

Allogeneic cord blood transplantation (CBT) is increasingly used in children with beta-thalassemia major. Donor/recipient mixed chimerism (MC) is frequently observed in patients with beta-thalassemia major given bone marrow transplantation (BMT) in the early post-transplant period, due to the persistence of haematopoietic and immune cells of host origin. Previous clinical data reported on these patients demonstrated that the majority of them exhibiting MC convert to full donor chimerism (FDC), some maintain MC stable over time, while a non negligible number of patients develop graft failure, in particular when increasing proportions of cells of host origin are observed in serial monitoring. Aims of the present study were to retrospectively investigate the proportion of children with beta-thalassemia major experiencing either transient, or long-lasting persistence of hematopoietic cells of host origin after CBT, as compared to those given BMT, and to analyze the relationship existing between the evolution of MC over time and clinical outcome. One hundred and six consecutive patients, 27 given CBT from an HLA-identical related donor (RD) and 79 given BMT (42 from a RD and 37 from an HLA-compatible unrelated volunteer, UD), were enrolled in the study. Donor/recipient chimerism status was evaluated by PCR-based assay, analyzing selected polymorphic short tandem repeat (STR) loci. CBT and BMT recipients received similar preparative regimens. Anti-thymocyte globulin (ATG 10 mg/Kg on days −4, −3 and −2; Fresenius, Graefelfing, Germany) was used in all patients given an UD-BMT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (Cs-A) alone in CBT recipients; either CsA alone (12 RD-BMT recipients) or CsA in combination with methotrexate (MTX) was given to BMT patients. All patients given CBT engrafted and displayed MC early after transplantation, 13/27 converted to FDC (6 within 1 month and 7 within 1 year after CBT), 14/27 maintained stable MC, and all of them are alive, transfusionin-dependent and free of any immune-suppressive drug, the follow-up ranging between 15 and 89 months (median value 42 months). In the group of children given BMT, 24/79 (12 transplanted from UD and 12 from RD) exhibited FDC at all time points examined, 4/79 (1 transplanted from UD and 3 from RD) did not engraft and 51/79 (23 from UD and 28 from RD) displayed MC at time of haematological reconstitution. Forty out of these 51 BMT recipients with MC status converted to FDC (17 from UD and 23 from RD; 29 within 1 month and 11 within 1 year after BMT), 3/51 maintained stable MC (1 undergoing UD- and 2 RD-BMT), while 8/51 (5 from UD and 3 from RD) progressively lost the graft, returning to be transfusion-dependent. The median follow-up time was 51 months (range: 15–110 months) and 48 months (range: 16–81 months) in the RD-BMT and UD-BMT group, respectively. None of the patients given CBT experienced either grade II-IV acute GVHD, which was, by contrast, diagnosed in 3 and 7 patients given RD- and UD-BMT, respectively. Chronic GVHD was diagnosed in 8 children (2 from RD- and 6 from UDBMT). Five out of 106 pediatric patients enrolled in the study, 2 included in the RD-BMT and 3 in the UD-BMT group, died of transplantation-related complications. These results indicate that MC is frequently observed in children with beta-thalassemia after RD-CBT, without, however, predicting an increased risk of treatment failure. CBT from an HLA-identical related donor is a successful option in beta-thalassemia major, possibly also due to the establishment of an effective state of immune tolerance between host and donor that decreases the risk of both graft rejection and GVHD.