Centro Interdipartimentale di Biotecnologie Molecolari "Guido Tarone"
Benedetto Bruno - PI
Benedetto Bruno - Professor of Hematology, Department of Molecular Biotechnology and Health Sciences, School of Medicine, University of Torino, Torino, Italy
- Alessia Melis, Student,
- Alessia Cargnino, Student,
- Giuseppe Lia, Research Associate
- Luisa Giaccone, MD, PhD, Associate Professor
Research activity
In addition to a robust clinical activity in the field of hematological malignancies, the group has focused on translational projects with particular emphasis on the role of the thymus in post-transplant immune-reconstitution (Figure 1), on the role of biomarkers such as extracellular vesicles (EVs) in the pathogenesis and early diagnosis of acute and chronic “graft versus host disease” (Figure 2), and, more recently, on the role of the endothelial damage in the complications following the infusion of chimeric antigen receptor T (CAR T) cells (Figure 3).
Briefly, thymus-dependent recovery of peripheral T cells ensures long term T cell-mediated protection and tolerance. We measured the TCR gene rearrangements in T cells.
This technique measures the extrachromosomal DNA excision circles that are generated in the thymus only in naïve T cells following TCR gene rearrangement [i.e. joined T cell receptor excision circles (sjTRECs)].
These stable DNA circles do not replicate during mitosis, but they are diluted within each cellular division and can persist in mature T cells, thus providing an excellent measure of thymic functions.
Our preliminary data demonstrated a progressive increase in absolute numbers of all T cell subsets and of sjTRECs from the 3rd month up to 2 years post-Haploidentical-HCT.
The increased sjTREC values provide evidence of an active thymic function despite age-dependent involution that substantially contributes to T cell IR after Haplo-HSCT. Moreover, we observed that both CD4+ and CD8+ T cell levels and sjTRECs levels are lower in recipients after 2 years from transplant compared to healthy donors. Of note, chronic GVHD and older age are significantly correlated with thymic activity.
EVs are natural carriers of several bioactive molecules, such as lipids, proteins, and nucleic acids (like DNA and miRNA), and their quantity relies on patient status. EVs are physiologically present in body fluids, including blood and urine from where they can be easily extracted without invasive procedures, making them very attractive targets for diagnostic applications.
The content inside each EV reflects the cellular origin and it can be quantified. EVs are shed by cells under both normal and pathological conditions. They carry nucleic acids and proteins from their host cells that are indicative of pathophysiological conditions, and, for these reasons, they are considered crucial for the understanding of pathophysiological processes.
Over the past few years, numerous studies have demonstrated that exosomes contain nucleic acids and proteins implicated in cancer as well as inflammatory conditions.
Figure 3.. Current chimeric antigen receptor (CAR) T-cell therapies have been a major breakthrough in immunotherapy. In particular, CD19-directed CAR T- cells have dramatically changed the treatment of relapse/refractory B-cell origin hematological malignancies. CAR T-cells are primarily derived from autologous T-cells, collected by leukopheresis, and genetically engineered to express a CAR, specific for a given antigen expressed on cancer cells, followed by ex vivo CAR T cell expansion and re-infusion into the patient. CARs are fusion proteins of a selected single-chain fragment variable from a specific monoclonal antibody and one or more T-cell receptor intracellular signaling domains. This genetic T-cell modification can be obtained by viral-based gene transfer or nonviral technologies such as DNA-based transposons, CRISPR/Cas9 technology or direct transfer of in vitro transcribed-mRNA by electroporation. In the clinical setting, treatment protocols (either for FDA/EMA approved products or for clinical trials) incorporate a short, non-intense lymphodepleting regimen prior to CAR T-cell infusion with the aim to increase efficacy. Overall, this “patient journey” from collection to infusion usually requires between two and four weeks.
We previously showed that the immune-profile of serum extra cellular vesicles, including their content, and the degree of endothelial dysfunction provide important insights in the pathophysiology and severity of post-transplant life-threatening complications (i.e. graft-vs.-host disease, venous-occlusive disease, transplant-associated microangiopathy). Novel cell therapies such as chimeric antigen receptor (CAR) – T cells and T cell receptor (TCR) T cells have recently changed the treatment landscape of hematological malignancies with high rates of remission, and potentially reaching a cure, in particular in relapsed/refractory lymphoproliferative diseases and multiple myeloma. However, these immunotherapies are associated with significant toxicities that not only limit their administration but have also become an area of intensive research with the goal of improving clinical outcomes by reducing untoward side effects. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are life-threatening complications that follow CAR T infusions. While CAR T cell expansion and their persistence after infusion appear associated with disease response, inflammation and a supraphysiological response appear pivotal processes in the development of both CRS and ICANS. Mechanisms of endothelial activation and dysfunction have also emerged as important factors associated with CAR T cell toxicity. A significant consequence of endothelial activation, reported in a number of inflammatory processes, is represented by a dysfunctional switch from an antithrombotic to a prothrombotic endothelial phenotype, sustained by the triggering of fibrin formation and platelet adhesion and aggregation. Overall our aim is to investigate the dynamic changes of endothelial activation in association with the immune-profile of EVs in peripheral blood from baseline to given time-points after the infusion of CAR T cells in patients with relapsed/refractory lymphoproliferative diseases. Importantly, laboratory findings will be correlated with clinical outcomes to design potential pre-emptive strategies to prevent CAR T cells toxicity and expand patient eligibility criteria.
We plan to compare the immune-reconstitution, and in particular the role of the thymus, in different settings of hematopoietic stem cell transplantation and novel cell therapies. We will continue to investigate the role of EVs as biomarkers of post-transplant complications of endothelial origin and infections. Some projects will be carried out with national and international Research Institutes.
- M.I.U.R. (Ministry of University and Scientific Research) Research Grants (2018-2023):
- Role of Plasmatic Extracellular vesicles as biomarkers of inflammatory and endothelial complications after hematopoietic stem cell transplantation
- Plasmatic Extracellular Vesicles in Cytomegalovirus reactivation and acute Graft Versus Host Disease after Haploidentical Stem Cell Transplantation
- Plasmatic Extracellular Vesicles as potential biomarkers of viral infection and endothelial complications after allogeneic stem cell transplantation
- Extracellular vesicles as potenzial biomarkers of chronic GVHD"
- Integrating Next Generation Sequencing in minimal residual disease and prognosis stratification of acute myeloid leukemia and high risk myelosplastic syndromes patients. (JANSSEN/CILAG)
- Efficacy of the biosimilar filgrastim Nivestim on the mobilization of hematopoietic CD34+ cells and on the kinetics of engraftment after high dose chemotherapy and mobilized peripheral hematopoietic cell support. (HOSPIRA ITALIA SRL)
- Bando PRIN 2022 PNRR Endothelial cell dysfunction and extracellular vesicles as biomarkers of clinical outcomes (toxicity and disease responses) after chimeric antigen receptor (CAR) T-cell therapy for lymph oproliferative diseases
Bruno B, Goerner MA, Nash RA, Storb R, Kiem HP, McSweeney PA. Purified canine CD34+Lin- marrow cells transduced with retroviral vectors give rise to long-term multi-lineage hematopoiesis. Biol Blood Marrow Tr. 2001;7:543-51. doi: 10.1016/s1083-8791(01)70020-1
Bruno B, Rotta M, Patriarca F, Mordini N, Allione B, Carnevale-Schianca F, Giaccone L, Sorasio R, Omedè P, Baldi I, Bringhen S, Massaia M, Aglietta M, Levis A, Gallamini A, Fanin R, Palumbo A, Storb R, Ciccone G, Boccadoro M. A comparison of allografting with autografting for newly diagnosed myeloma. New Engl J Med. 2007; 356:1110-20. doi: 10.1056/NEJMoa065464
Bruno B, Rotta M, Patriarca F, Mattei D, Allione B, Carnevale-Schianca F, Sorasio R, Rambaldi A, Casini M, Parma M, Bavaro P, Onida F, Busca A, Castagna L, Benedetti E, Iori AP, Giaccone L, Palumbo A, Corradini P, Fanin R, Maloney D, Storb R, Baldi I, Ricardi U, Boccadoro M. Non-myeloablative allografting for newly diagnosed multiple myeloma: the experience of the Gruppo Italiano Trapianti di Midollo. Blood. 2009;113:3375-82. doi: 10.1182/blood-2008-07-167379
Giaccone L, Storer B, Patriarca F, Rotta M, Sorasio R, Allione B, Carnevale-Schianca F, Festuccia M, Brunello L, Omedè P, Bringhen S, Aglietta M, Levis A, Mordini N, Gallamini A, Fanin R, Massaia M, Palumbo A, Ciccone G, Storb R, Gooley TA, Boccadoro M, Bruno B. Long-term follow-up of a comparison of nonmyeloablative allografting with autografting for newly diagnosed myeloma. Blood. 2011;117:6721-7. doi: 10.1182/blood-2011-03-339945
Lia G, Brunello L, Bruno S, Carpanetto A, Omedè P, Festuccia M, Tosti L, Maffini E, Giaccone L, Arpinati M, Ciccone G, Boccadoro M, Evangelista A, Camussi G, Bruno B. Extracellular vesicles as potential biomarkers of acute graft-vs-host disease. Leukemia. 2018;32:765-773. doi: 10.1038/leu.2017.277
Mariotti J, Raiola AM, Evangelista A, Carella AM, Martino M, Patriarca F, Risitano A, Bramanti S, Busca A, Giaccone L, Brunello L, Merla E, Savino L, Loteta B, Console G, Fanin R, Sperotto A, Marano L, Marotta S, Frieri C, Sica S, Chiusolo P, Harbi S, Furst S, Santoro A, Bacigalupo A, Blaise D, Angelucci E, Mavilio D, Castagna L, Bruno B. Impact of donor age and kinship on clinical outcomes after T-cell-replete haploidentical transplantation with PT-Cy. Blood Adv. 2020;4:3900-3912. doi: 10.1182/bloodadvances.2020001620
Bruno B, Wäsch W, Engelhardt M, Gay F, Giaccone L, D’Agostino M, Rodríguez-Lobato LG, Danhof S, Gagelmann N, Kröger N, Popat, van de Donk NWCJ, Terpos E, Dimopoulos MA, Sonneveld P, Einsele H, Boccadoro M. European Myeloma Network Perspective on CAR T-Cell Therapies for Multiple Myeloma. Haematologica, 2021;106:2054-2065. doi: 10.3324/haematol.2020.276402
Lia G, Di Vito C, Bruno S, Tapparo M, Brunello L, Santoro A, Mariotti J, Bramanti S, Zaghi E, Calvi M, Comba L, Fascì M, Giaccone L, Camussi G, Castagna L, Evangelista A, Mavilio D, Bruno B. Extracellular Vesicles as Biomarkers of acute Graft-vs.-host Disease after Haploidentical Stem Cell Transplantation and Post-transplant Cyclophosphamide. Front in Immunol ;12:816231. doi: 10.3389/fimmu.2021.816231
Boccalatte F, Mina R, Aroldi A, Leone S, Suryadevara C, Placantonakis D, Bruno B. Advances and hurdles of CAR T cell immune therapy in solid tumors. Cancers (Basel). 2022 Oct 18;14(20):5108. doi: 10.3390/cancers14205108
Rejeski K, Subklewe M, Aljurf M, Bachy E, Balduzzi AC, Barba P, Bruno B, Benjamin R, Carrabba MG, Chabannon C, Ciceri F, Corradini P, Delgado J, Di Blasi R, Greco R, Houot R, Iacoboni G, Jaeger U, Kersten MJ, Mielke S, Nagler A, Onida F, Peric Z, Roddie C, Ruggeri A, Sanchez-Guijo FM, Sánchez-Ortega I, Schneidawind D, Schubert ML, Snowden J, Thieblemont C, Topp MS, Zinzani PL, Gribben JG, Bonini C, Sureda Balari A, Yakoub-Agha I. Immune Effector Cell-Associated Hematotoxicity (ICAHT): EHA/EBMT Consensus Grading and Best Practice Recommendations. Blood. 2023. doi: 10.1182/blood.2023020578.
