Publications by authors named "Martino Introna"

88 Publications

Third-party bone marrow-derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial.

Am J Transplant 2020 Dec 28. Epub 2020 Dec 28.

Aldo & Cele Daccò Clinical Research Center for Rare Diseases, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy.

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56 NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.
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http://dx.doi.org/10.1111/ajt.16468DOI Listing
December 2020

Tolerance to Bone Marrow Transplantation: Do Mesenchymal Stromal Cells Still Have a Future for Acute or Chronic GvHD?

Front Immunol 2020 11;11:609063. Epub 2020 Dec 11.

Center of Cellular Therapy "G. Lanzani", Division of Haematology, Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Mesenchymal Stromal Cells (MSCs) are fibroblast-like cells of mesodermal origin present in many tissues and which have the potential to differentiate to osteoblasts, adipocytes and chondroblasts. They also have a clear immunosuppressive and tissue regeneration potential. Indeed, the initial classification of MSCs as pluripotent stem cells, has turned into their identification as stromal progenitors. Due to the relatively simple procedures available to expand large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential . One pathological condition where MSCs have been quite extensively tested is steroid resistant (SR) graft versus host disease (GvHD), a devastating condition that may occur in acute or chronic form following allogeneic hematopoietic stem cell transplantation. The clinical and experimental results obtained have outlined a possible efficacy of MSCs, but unfortunately statistical significance in clinical studies has only rarely been reached and effects have been relatively limited in most cases. Nonetheless, the extremely complex pathogenetic mechanisms at the basis of GvHD, the fact that studies have been conducted often in patients who had been previously treated with multiple lines of therapy, the variable MSC doses and schedules administered in different trials, the lack of validated potency assays and clear biomarkers, the difference in MSC sources and production methods may have been major factors for this lack of clear efficacy . The heterogeneity of MSCs and their different stromal differentiation potential and biological activity may be better understood through more refined single cell sequencing and proteomic studies, where either an "anti-inflammatory" or a more "immunosuppressive" profile can be identified. We summarize the pathogenic mechanisms of acute and chronic GvHD and the role for MSCs. We suggest that systematic controlled clinical trials still need to be conducted in the most promising clinical settings, using better characterized cells and measuring efficacy with specific biomarkers, before strong conclusions can be drawn about the therapeutic potential of these cells in this context. The same analysis should be applied to other inflammatory, immune or degenerative diseases where MSCs may have a therapeutic potential.
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http://dx.doi.org/10.3389/fimmu.2020.609063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759493PMC
December 2020

Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities.

J Clin Invest 2020 11;130(11):6021-6033

Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, Monza, Italy.

BACKGROUNDChimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability.METHODSWe report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells.RESULTSThe cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD-). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion.CONCLUSIONSB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.TRIAL REGISTRATIONClinicalTrials.gov NCT03389035.FUNDINGThis study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).
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http://dx.doi.org/10.1172/JCI138473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598053PMC
November 2020

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

Mol Ther 2020 09 30;28(9):1974-1986. Epub 2020 May 30.

Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, 20900 Monza, Italy.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.
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http://dx.doi.org/10.1016/j.ymthe.2020.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474266PMC
September 2020

Protective Effects of Human Nonrenal and Renal Stromal Cells and Their Conditioned Media in a Rat Model of Chronic Kidney Disease.

Cell Transplant 2020 Jan-Dec;29:963689720965467

Institute of Biomedical Ethics and History of Medicine, University of Zurich, Zurich, Switzerland.

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.
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http://dx.doi.org/10.1177/0963689720965467DOI Listing
March 2021

Kidney transplant tolerance associated with remote autologous mesenchymal stromal cell administration.

Stem Cells Transl Med 2020 04 24;9(4):427-432. Epub 2019 Dec 24.

Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy.

Here we report the case of successful immune tolerance induction in a living-donor kidney transplant recipient remotely treated with autologous bone marrow-derived mesenchymal stromal cells (MSC). This case report, which to the best of our knowledge is the first in the world in this setting, provides evidence that the modulation of the host immune system with MSC can enable the safe withdrawal of maintenance immunosuppressive drugs while preserving optimal long-term kidney allograft function.
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http://dx.doi.org/10.1002/sctm.19-0185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103624PMC
April 2020

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin.

J Vis Exp 2019 04 30(146). Epub 2019 Apr 30.

Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche;

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 10) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.
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http://dx.doi.org/10.3791/58922DOI Listing
April 2019

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation.

Blood 2019 03 17;133(13):1395-1405. Epub 2019 Jan 17.

Center of Cellular Therapy "G. Lanzani," Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab') antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.
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http://dx.doi.org/10.1182/blood-2018-07-864538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484458PMC
March 2019

Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease.

Stem Cell Res Ther 2018 08 14;9(1):220. Epub 2018 Aug 14.

Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Via Stezzano 87, 24126, Bergamo, Italy.

Background: Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties.

Methods: The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 10 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigen cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h.

Results: Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system.

Conclusions: Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients.
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http://dx.doi.org/10.1186/s13287-018-0960-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092807PMC
August 2018

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19 tumors.

Cytotherapy 2018 08 7;20(8):1077-1088. Epub 2018 Aug 7.

Center of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy. Electronic address:

Background: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19 malignancies.

Methods: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs.

Results: CB-CIKs, like PB-CIKs, were mostly CD3 T cells with mean 45% CD3CD56 and expressing mostly TCR(T cell receptor)αβ with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4 cells, mostly CD56, as well as a greater proportion of naïve CCR7CD45RA and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8 cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19 tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph CD19 acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 10 CIK/kg.

Discussion: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
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http://dx.doi.org/10.1016/j.jcyt.2018.06.003DOI Listing
August 2018

Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation.

Biol Blood Marrow Transplant 2018 11 20;24(11):2365-2370. Epub 2018 Jul 20.

School of Cancer & Pharmaceutical Sciences, King's College London, London, United Kingdom. Electronic address:

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.
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http://dx.doi.org/10.1016/j.bbmt.2018.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299357PMC
November 2018

Long-Term Clinical and Immunological Profile of Kidney Transplant Patients Given Mesenchymal Stromal Cell Immunotherapy.

Front Immunol 2018 14;9:1359. Epub 2018 Jun 14.

IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy.

We report here the long-term clinical and immunological results of four living-donor kidney transplant patients given autologous bone marrow-derived mesenchymal stromal cells (MSCs) as part of a phase 1 study focused on the safety and feasibility of this cell therapy. According to study protocols implemented over time, based on initial early safety findings, the patients were given MSC at day 7 posttransplant ( = 2) or at day -1 pretransplant ( = 2) and received induction therapy with basiliximab and low-dose rabbit anti-thymocyte globulin (RATG) or RATG alone, and were maintained on low-dose ciclosporin (CsA)/mycophenolate mofetil (MMF). All MSC-treated patients had stable graft function during the 5- to 7-year follow-up, without increased susceptibility to infections or neoplasm. In three MSC recipients, but not historical control patients, circulating memory CD8 T cell percentages remained lower than basal, coupled with persistent reduction of donor-specific cytotoxicity. Two patients showed a long-lasting increase in the regulatory T cell/memory CD8 T cell ratio, paralleled by high circulating levels of naïve and transitional B cells. In one of these two patients, CsA was successfully discontinued, and currently the low-dose MMF monotherapy is on the tapering phase. The study shows that MSC therapy is safe in the long term and could promote a pro-tolerogenic environment in selected patients. Extensive immunomonitoring of MSC-treated kidney transplant recipients could help selection of patients for safe withdrawal of maintenance immunosuppressive drugs (NCT00752479 and NCT02012153).
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http://dx.doi.org/10.3389/fimmu.2018.01359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014158PMC
June 2018

Mesenchymal stromal cells from human umbilical cord prevent the development of lung fibrosis in immunocompetent mice.

PLoS One 2018 1;13(6):e0196048. Epub 2018 Jun 1.

Dipartimento di Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Ancona, Italy.

Lung fibrosis is a severe condition resulting from several interstial lung diseases (ILD) with different etiologies. Current therapy of ILD, especially those associated with connective tissue diseases, is rather limited and new anti-fibrotic strategies are needed. In this study, we investigated the anti-fibrotic activity in vivo of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC). Adult immunocompetent C57BL/6 mice (n. = 8 for each experimental condition) were injected intravenously with hUC-MSC (n. = 2.5 × 105) twice, 24 hours and 7 days after endotracheal injection of bleomycin. Upon sacrifice at days 8, 14, 21, collagen content, inflammatory cytokine profile, and hUC-MSC presence in explanted lung tissue were analyzed. Systemic administration of a double dose of hUC-MSC significantly reduced bleomycin-induced lung injury (inflammation and fibrosis) in mice through a selective inhibition of the IL6-IL10-TGFβ axis involving lung M2 macrophages. Only few hUC-MSC were detected from explanted lungs, suggesting a "hit and run" mechanism of action of this cellular therapy. Our data indicate that hUC-MSC possess strong in vivo anti-fibrotic activity in a mouse model resembling an immunocompetent human subject affected by inflammatory ILD, providing proof of concept for ad-hoc clinical trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196048PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983506PMC
November 2018

Innovative Clinical Perspectives for CIK Cells in Cancer Patients.

Int J Mol Sci 2018 Jan 25;19(2). Epub 2018 Jan 25.

USS Center of Cell Therapy "G. Lanzani", USC Ematologia, ASST Papa Giovanni XXIII Bergamo, 24124 Bergamo, Italy.

Cytokine-induced killer (CIK) cells are T lymphocytes that have acquired, in vitro, following extensive manipulation by Interferon gamma (IFN-γ), OKT3 and Interleukin 2 (IL-2) addition, the expression of several Natural Killer (NK) cell-surface markers. CIK cells have a dual "nature", due to the presence of functional TCR as well as NK molecules, even if the antitumoral activity can be traced back only to the NK-like structures (DNAM-1, NKG2D, NKp30 and CD56). In addition to antineoplastic activity in vitro and in several in-vivo models, CIK cells show very limited, if any, GvHD toxicity as well as a strong intratumoral homing. For all such reasons, CIK cells have been proposed and tested in many clinical trials in cancer patients both in autologous and allogeneic combinations, up to haploidentical mismatching. Indeed, genetic modification of CIK cells as well as the possibility of combining them with specific monoclonal antibodies will further expand the possibility of their clinical utilization.
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http://dx.doi.org/10.3390/ijms19020358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855580PMC
January 2018

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance.

Cytotherapy 2018 02 12;20(2):262-270. Epub 2017 Dec 12.

Centre of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Background: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs).

Methods: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms.

Results: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium.

Discussion: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.
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http://dx.doi.org/10.1016/j.jcyt.2017.11.009DOI Listing
February 2018

Multiple intracerebroventricular injections of human umbilical cord mesenchymal stem cells delay motor neurons loss but not disease progression of SOD1G93A mice.

Stem Cell Res 2017 12 10;25:166-178. Epub 2017 Nov 10.

Department of Neuroscience, IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy. Electronic address:

Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1β) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression.
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http://dx.doi.org/10.1016/j.scr.2017.11.005DOI Listing
December 2017

Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.

Nat Commun 2017 10 17;8(1):983. Epub 2017 Oct 17.

IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, 24126, Bergamo, Italy.

Mesenchymal stromal cells (MSCs) are renoprotective and drive regeneration following injury, although cellular targets of such an effect are still ill-defined. Here, we show that human umbilical cord (UC)-MSCs transplanted into mice stimulate tubular cells to regain mitochondrial mass and function, associated with enhanced microtubule-rich projections that appear to mediate mitochondrial trafficking to create a reparative dialogue among adjacent tubular cells. Treatment with UC-MSCs in mice with cisplatin-induced acute kidney injury (AKI) regulates mitochondrial biogenesis in proximal tubuli by enhancing PGC1α expression, NAD biosynthesis and Sirtuin 3 (SIRT3) activity, thus fostering antioxidant defenses and ATP production. The functional role of SIRT3 in tubular recovery is highlighted by data that in SIRT3-deficient mice with AKI, UC-MSC treatment fails to induce renoprotection. These data document a previously unrecognized mechanism through which UC-MSCs facilitate renal repair, so as to induce global metabolic reprogramming of damaged tubular cells to sustain energy supply.Mesenchymal stromal cells drive renal regeneration following injury. Here, the authors show that human mesenchymal stromal cells, when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.
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http://dx.doi.org/10.1038/s41467-017-00937-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754365PMC
October 2017

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2017 Dec 13;23(12):2070-2078. Epub 2017 Jul 13.

USC Hematology and Bone Marrow Transplant Unit ASST Papa Giovanni XXIII Bergamo, Bergamo, Italy; Università degli Studi di Milano, Milan Italy.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.005DOI Listing
December 2017

The specific Bruton tyrosine kinase inhibitor acalabrutinib (ACP-196) shows favorable activity against chronic lymphocytic leukemia B cells with CD20 antibodies.

Haematologica 2017 10 22;102(10):e400-e403. Epub 2017 Jun 22.

Center of Cellular Therapy "G. Lanzani, USC Hematology, and Fondazione per la Ricerca Ospedale Maggiore, ASST Papa Giovanni XXIII, Bergamo, Italy.

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http://dx.doi.org/10.3324/haematol.2017.169334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622871PMC
October 2017

Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies.

Blood 2017 05 13;129(19):2636-2644. Epub 2017 Mar 13.

Center of Cellular Therapy G. Lanzani, Division of Hematology, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo.
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http://dx.doi.org/10.1182/blood-2016-08-735605DOI Listing
May 2017

Phenotypical and Functional Characteristics of In Vitro-Expanded Adipose-Derived Mesenchymal Stromal Cells From Patients With Systematic Sclerosis.

Cell Transplant 2017 05 31;26(5):841-854. Epub 2017 Jan 31.

Mesenchymal stromal cells (MSCs) have received attention as an ideal source of regenerative cells because of their multipotent differentiation potential. Adipose tissue is an attractive source of MSCs. Recent studies have shown that autologous fat grafting may be effective in the treatment of systemic sclerosis (SSc), but no specific study exists that aimed at investigating whether adipose tissue-derived stromal cells (ADSCs) from SSc patients maintain normal phenotypic and functional characteristics. The purpose of the current study was to investigate whether ADSCs from patients with SSc (SSc-ADSCs) are phenotypically and functionally identical to those from healthy controls (HC-ADSCs). Adipose tissue samples were obtained from 10 patients with SSc and from 8 HCs. Both MSC populations were evaluated for their capacity to (a) express specific MSC surface antigens by flow cytometry analysis, (b) proliferate, (c) differentiate along the adipogenic and osteogenic lineages, (d) suppress in vitro lymphocyte proliferation induced by a mitogenic stimulus, and (e) support endothelial cell (EC) tube formation. ADSCs from SSc patients and HCs showed similar surface phenotype and multilineage differentiation capabilities. In PBMC proliferation inhibition assays, no significant differences were observed between SSc- and HC-ADSCs. Using ADSC/EC cocultures, both SSc- and HC-ADSCs improved tube formation by both HC- and SSc-ECs. This effect was enhanced under hypoxic conditions in all of the cocultures. SSc-ADSCs exhibited the same phenotypic pattern, proliferation and differentiation potentials, and immunosuppressive properties as those from HCs. The proangiogenic activity shown by SSc-ADSCs, namely, under hypoxic conditions, suggests that autologous ADSC grafting may represent a possible therapeutic option for SSc.
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http://dx.doi.org/10.3727/096368917X694822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657721PMC
May 2017

Thrombospondin-1 promotes mesenchymal stromal cell functions via TGFβ and in cooperation with PDGF.

Matrix Biol 2016 09 16;55:106-116. Epub 2016 Mar 16.

Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy. Electronic address:

Mesenchymal stromal cells (MSC) are characterized by unique tropism for wounded tissues, high differentiating capacity, ability to induce tissue repair, and anti-inflammatory and immunoregulatory activities. This has generated interest in their therapeutic use in severe human conditions as well as in regenerative medicine and tissue engineering. Identification of factors involved in the regulation of MSC proliferation, migration and differentiation could provide insights into the pathophysiological regulation of MSC and be exploited to optimize clinical grade expansion protocols for therapeutic use. Here we identify thrombospondin-1 (TSP-1) as a major regulator of MSC. TSP-1 induced MSC proliferation. This effect was mediated by TSP-1-induced activation of endogenous TGFβ, as shown by the inhibitory effects of anti-TGFβ antibodies and by the lack of activity of TSP-2 - that does not activate TGFβ. Moreover, TSP-1 strongly potentiated the proliferative and migratory activity of PDGF on MSC. TSP-1 directly bound to PDGF, through a site located within the TSP-1 type III repeats, and protected the growth factor from degradation by MSC-derived proteases, hence increasing its stability and bioavailability. The studies presented here identify a more comprehensive picture of the pleiotropic effect of TSP-1 on MSC behavior, setting the basis for further studies aimed at investigating the possible use of PDGF and TSP-1 in the in vitro expansion of MSC for therapeutic applications.
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http://dx.doi.org/10.1016/j.matbio.2016.03.003DOI Listing
September 2016

Development of advanced therapies in Italy: Management models and sustainability in six Italian cell factories.

Cytotherapy 2016 Apr;18(4):481-6

Laboratory of Cell and Gene Therapy Stefano Verri, San Gerardo Hospital, Monza, Italy; Tettamanti Research Center, Pediatric Clinic Monza Brianza per il Bambino e la sua Mamma (MBBM) Foundation, Monza, Italy.

On November 10, 2014, the representatives of all six certified Good Manufacturing Practices (GMP) cell factories operating in the Lombardy Region of Italy convened a 1-day workshop in Milan titled "Management Models for the Development And Sustainability of Cell Factories: Public-Private Partnership?" The speakers and panelists addressed not only the many scientific, technological and cultural challenges faced by Lombardy Cell Factories, but also the potential impact of advanced therapy medicinal products (ATMPs) on public health and the role played by translational research in this process. Future perspectives for research and development (R&D) and manufacturing processes in the field of regenerative medicine were discussed as well. This report summarizes the most important issues raised by the workshop participants with particular emphasis on strengths and limitations of the R&D and manufacturing processes for innovative therapeutics in Lombardy and what can be improved in this context while maintaining GMP standards. The participants highlighted several strategies to translate patient-specific advanced therapeutics into scaled manufacturing products for clinical application. These included (i) the development of a synergistic interaction between public and private institutions, (ii) better integration with Italian regulatory agencies and (iii) the creation of a network among Lombardy cell factories and other Italian and European institutions.
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http://dx.doi.org/10.1016/j.jcyt.2016.01.002DOI Listing
April 2016

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis.

Stem Cell Res 2015 Jul 27;15(1):243-53. Epub 2015 Jun 27.

IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.
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http://dx.doi.org/10.1016/j.scr.2015.06.010DOI Listing
July 2015

Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

Stem Cell Reports 2015 Apr 5;4(4):685-98. Epub 2015 Mar 5.

IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri," Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy.

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.
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http://dx.doi.org/10.1016/j.stemcr.2015.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400646PMC
April 2015

Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration.

Stem Cells Dev 2015 Jul 18;24(13):1570-81. Epub 2015 Mar 18.

1 Department of Experimental Medicine (DIMES), University of Genoa , Genoa, Italy .

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms.
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http://dx.doi.org/10.1089/scd.2014.0490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499786PMC
July 2015

Mesenchymal stromal cells for prevention and treatment of graft-versus-host disease: successes and hurdles.

Curr Opin Organ Transplant 2015 Feb;20(1):72-8

USC Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy.

Purpose Of Review: The aim of the present review was to give a critical opinion on the use of mesenchymal stromal cells (MSCs) to treat or to prevent graft-versus-host disease (GVHD).

Recent Findings: The first part includes a summary of the many clinical trials published so far either to prevent or to treat GVHD in recipients of haematopoietic stem cell transplantation. We discuss in more detail a comparison in a subgroup of studies, including our own clinical work, which have in common the use of the platelet lysate to expand the MSCs from bone marrow origin.In the second part, we describe a few crucial elements of the biology of the GVHD and the biology of the MSCs themselves, showing their possible role in the immune modulation and in the inflammation in several in-vivo experimental models.

Summary: The complexity of the clinical condition that is the object of the trials and the paucity of information on the mechanisms of action in vivo of MSCs at different anatomical sites and in different times of the development of the disease preclude at the moment the identification of a strong rationale for MSC therapeutic schedule. Moreover, the typical development of GVHD requires different time points of clinical evaluation than those previously generally utilized in studies conducted on MSCs.
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http://dx.doi.org/10.1097/MOT.0000000000000158DOI Listing
February 2015

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy.

Haematologica 2015 Jan 24;100(1):77-86. Epub 2014 Oct 24.

Center of Cellular Therapy "G. Lanzani", Division of Hematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.
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http://dx.doi.org/10.3324/haematol.2014.107011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281316PMC
January 2015

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

J Immunol 2014 Nov 29;193(9):4739-47. Epub 2014 Sep 29.

Centro Trapianto Midollo Osseo, USC Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, 24127 Bergamo, Italy;

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1401550DOI Listing
November 2014