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Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro , with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo . Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Capelli, Cuofano, Pavoni, Frigerio, Lisini, Nava, Quaroni, Colombo, Galli, Bezukladova, Panina-Bordignon, Gaipa, Comoli, Cossu, Martino, Biondi, Introna and Golay.)

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 bright 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., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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 in vitro large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential in vivo . 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 in vivo . 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Introna and Golay.)

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).

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., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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., (© 2019 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

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 5 ) 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.

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') 2 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., (© 2019 by The American Society of Hematology.)

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 ( n  = 2) or at day -1 pretransplant ( n  = 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 ex vivo 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).

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., Competing Interests: The authors declare no conflicts of interest.

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., (Copyright © 2017. Published by Elsevier B.V.)

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., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., (Copyright © 2015. Published by Elsevier B.V.)

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., (Copyright© Ferrata Storti Foundation.)

Cytokine-induced killer (CIK) cells are in-vitro-expanded T lymphocytes that represent a heterogeneous population. A large majority of CIK cells are CD3(+)CD56(+), and this population has been shown to confer a cytotoxic effect against tumor targets. The scope of this work was to study whether CD56 has a direct role in CIK-mediated cytotoxicity. Blocking of CD56 with the anti-CD56 monoclonal antibody GPR165 significantly reduced CIK-mediated lysis of three CD56(+) hematopoietic tumor cell lines (AML-NS8, NB4, and KCL22), whereas no effect was observed on three CD56(-) hematopoietic tumor cell lines (K562, REH, and MOLT-4). Knockdown of CD56 in CIK cells by short interfering RNA made the cells less cytotoxic against a CD56(+) target, and knockdown of CD56 in target cells with lentiviral short hairpin RNA significantly altered their susceptibility to CIK-mediated lysis. Our data suggest that homophilic interaction between CD56 molecules may occur in tumor-cell recognition, leading to CIK-mediated cell death., (Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100., (Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3(+)CD56(+) CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.

Despite advances in graft-versus-host-disease (GVHD) treatment, it is estimated that overall survival (OS) at 2 years for hematopoietic cell transplantation (HCT) recipients who experience steroid-resistant GVHD is 10%. Among recent therapeutic approaches for GVHD treatment, mesenchymal stromal cells (MSCs) hold a key position. We describe a multicenter experience of 11 pediatric patients diagnosed with acute or chronic GVHD (aGVHD, cGVHD) treated for compassionate use with GMP-grade unrelated HLA-disparate donors' bone marrow-derived MSCs, expanded in platelet-lysate (PL)-containing medium. Eleven patients (aged 4-15 years) received intravenous (i.v.) MSCs for aGVHD or cGVHD, which was resistant to multiple lines of immunosuppression. The median dose was 1.2 x 10(6)/kg (range: 0.7-3.7 x 10(6)/kg). No acute side effects were observed, and no late side effects were reported at a median follow-up of 8 months (range: 4-18 months). Overall response was obtained in 71.4% of patients, with complete response in 23.8% of cases. None of our patients presented GVHD progression upon MSC administration, but 4 patients presented GVHD recurrence 2 to 5 months after infusion. Two patients developed chronic limited GVHD. This study underlines the safety of PL-expanded MSC use in children. MSC efficacy seems to be greater in aGVHD than in cGVHD, even after failure of multiple lines of immunosuppression., (Copyright (c) 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: The histone deacetylase inhibitor ITF2357 has potent cytotoxic activity in multiple myeloma in vitro and has entered clinical trials for this disease., Design and Methods: In order to gain an overall view of the activity of ITF2357 and identify specific pathways that may be modulated by the drug, we performed gene expression profiling of the KMS18 multiple myeloma cell line treated with the drug. The modulation of several genes and their biological consequence were verified in a panel of multiple myeloma cell lines and cells freshly isolated from patients by using polymerase chain reaction analysis and western blotting., Results: Out of 38,500 human genes, we identified 140 and 574 up-regulated genes and 102 and 556 down-modulated genes at 2 and 6 h, respectively, with a significant presence of genes related to transcription regulation at 2 h and to cell cycling and apoptosis at 6 h. Several of the identified genes are particularly relevant to the biology of multiple myeloma and it was confirmed that ITF2357 also modulated their encoded proteins in different multiple myeloma cell lines. In particular, ITF2357 down-modulated the interleukin-6 receptor alpha (CD126) transcript and protein in both cell lines and freshly isolated patients' cells, whereas it did not significantly modify interleukin-6 receptor beta (CD130) expression. The decrease in CD126 expression was accompanied by decreased signaling by interleukin-6 receptor, as measured by STAT3 phosphorylation in the presence and absence of inter-leukin-6. Finally, the drug significantly down-modulated the MIRHG1 transcript and its associated microRNA, miR-19a and miR-19b, known to have oncogenic activity in multiple myeloma., Conclusions: ITF2357 inhibits several signaling pathways involved in myeloma cell growth and survival.

Background and Objectives: Cytokine-induced killer (CIK) cells have shown anti-leukemic activity and little graft-versus-host disease (GVHD) in several animal models. The safety of these cells in autologous settings has been shown. We performed a phase I study of allogeneic (donor's) CIK cells in patients relapsing after allogeneic haematopoietic stem cell transplantation (HSCT)., Design and Methods: Eleven patients with acute myelogenous leukemia (n=4), Hodgkin's disease (n=3), chronic myelomonocytic leukemia, (n=1), pre-B acute lymphoblastic leukemia (n=1) and myelodysplasia (n=2), all of whom had relapsed after sibling (n=6) or matched unrelated donor (n=5) HSCT, entered this study., Results: Before CIK administration, six patients had received other salvage treatments including chemotherapy (n=5), radiotherapy (n=1) and unmanipulated donor lymphocytes (n=6) without any significant tumor response. The median number of CIK infusions was two (range 1-7) and the median number of total CIK cells was 12.4x106/kg (range 7.2-87.4). The infusions were well tolerated and no acute or late infusion-related reactions were recorded. Acute GVHD (grade I and II) was observed in four patients, 30 days after the last CIK infusion, and progressed into extensive chronic GVHD in two cases. Disease progression and death occurred in six patients. One patient had stable disease, one had hematologic improvement and three achieved complete responses., Interpretation and Conclusions: This study shows that the production of allogeneic CIK cells is feasible under clinical-grade conditions, well tolerated and may contribute to clinical responses.

Background and Objectives: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro., Design and Methods: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays., Results: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines, complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(p<0.0001). In t(12;21) samples, efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels, suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H, blocking the CD55 and CD59 complement inhibitors, and more effectively by combining campath-1H with fludarabine., Interpretation and Conclusions: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL, alone or in combination with cytotoxic drugs.

Background and Objectives: We have set up a murine B lymphoma model stably expressing human CD20 and homing in lymph nodes in immunocompetent mice to study the mechanism of action of rituximab., Design and Methods: The B lymphoma line 38C13 was stably transduced with the human CD20 cDNA by retroviral infection and injected into syngeneic mice., Results: The transduced 38C13-CD20(+) cells stably expressed human CD20 on 100% of cells. Rituximab alone did not inhibit 38C13-CD20+ cell growth but relocalized the human CD20 into lipid rafts and induced complement-mediated lysis in vitro. Inoculation of 4x10(3) 38C13-CD20(+) intravenously into syngeneic mice led to the development of tumor masses in the spleen, bone marrow and lymph nodes, detectable from day 15 by polymerase chain reaction (PCR) analysis, and with a median survival of 21-24 days. Treatment with 250 mg rituximab i.p. given 1-10 days after tumor inoculation cured 100% of animals, with disappearance of tumor documented by immunohistochemistry and PCR analysis. Depletion of both NK cells and neutrophils did not affect the therapeutic activity of rituximab in vivo. Similarly, removal of phagocytic macrophages using clodronate-liposomes did not modify the capacity of rituximab to control tumor growth. In contrast, the protective activity of the antibody was completely abolished after complement depletion with cobra venom factor. Complement was also required when cells were inoculated subcutaneously in nude mice., Interpretation and Conclusions: These data demonstrate that complement is required for the therapeutic activity of rituximab in vivo in a murine model of B-cell lymphoma, independently of its localization in lymph nodes or subcutaneously.

Background and Objectives: The therapeutic antibody alemtuzumab is directed against the CD52 molecule and is used for the treatment of B-cell lymphocytic leukemia (B-CLL). We investigated the mechanism of action of this antibody in vitro against different neoplastic B cells and compared it to the anti-CD20 antibody rituximab., Design and Methods: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells using human serum as the source of complement. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated by chromium release assays, using peripheral blood mononuclear cells as effector cells, before and after 2 days of culture with interleukin-2 (IL-2)., Results: Alemtuzumab lysed cells from the 23 B-CLL samples through complement activation (mean 80%) much more efficiently than rituximab did (mean 16%), presumably because of the higher expression of CD52 than of CD20. All other leukemic B cells, including 1 prolymphocytic leukemia, 2 hairy cell leukemias and 6 B-non Hodgkin's lymphomas were effective targets for both antibodies, with 88% and 85% mean lysis, respectively. Both CD52 and CD20 were highly expressed in these cells. In contrast, most neoplastic B cell samples were poorly lysed through ADCC using freshly isolated peripheral blood mononuclear cells as effectors with either monoclonal antibody and regardless of target antigen levels. ADCC was, however, significantly increased in all cases by culturing the effector cells with IL-2 for 2 days., Interpretation and Conclusions: Complement-mediated lysis is likely to be an important mechanism of action of alemtuzumab in B-CLL and combination with IL-2 may increase this antibody's efficacy through ADCC. Mature neoplastic B cells other than B-CLL express high levels of CD52 and are good targets for alemtuzumab-mediated cytotoxicity.

Background and Objectives: One of the few measurable clinical results obtained by the use of somatic cells in onco hematology is the clear-cut effect donor leukocyte infusions (DLI) in patients with chronic myeloid leukemia (CML) who relapse after an allogeneic bone marrow transplantation (BMT). From then on much research has focused on the use of cells to treat different aspects of oncologic diseases from leukemia relapse to development in BMT recipients., Methods and Information Sources: In this review we critically and schematically summarize the cell-based therapies which have led to a clinical application and recapitulate the results., Results and State of the Art: Although the overall numbers of successfully treated patients is small, therapy has been shown to be safe and effective in a variety of clinical contexts in oncohematology., Perspectives: Preliminary data will have to be validated in well designed clinical trials with cells generated by reproducible methods and in accreditated structures working according to Good Manufacturing Practices (GMP).

Allogeneic bone marrow transplantation as a cure for leukaemia and lymphoma is limited by the development of graft-vs-host disease (GVHD), an immunological reaction of the donor's T lymphocytes against the host's normal tissues. One therapeutic option to treat GVHD is the transfer of 'suicide' genes into the donor's T lymphocytes to render them susceptible to prodrug administration. This procedure should permit the elimination of unwanted T lymphocytes in GVHD. The main genes proposed for such a strategy will be described in this chapter, together with the advantages and limitations found during preclinical and clinical studies to date.

Background and Objectives: CD20 has been proposed as a novel suicide gene system for the treatment of graft-versus-host disease (GVHD), a fatal complication of allogeneic bone marrow transplantation: indeed expression of the human non-immunogenic exogenous CD20 protein allows positive immunoselection of transduced cells as well as their killing in vitro with rituximab. Lentiviral vectors are promising tools in the field of gene therapy. We therefore searched for a lentivector giving good efficiency of transduction of human T lymphocytes activated by the sole addition of interleukin (IL)-2 and high expression levels of the CD20 transgene., Design and Methods: The T cell line CEM and peripheral T lymphocytes activated by phytohemagglutinin (PHA) and/or IL-2 were transduced with two different vectors carrying the CD20 transgene driven by either the phosphoglycerate kinase (PGK) or elongation factor 1alpha (EF1alpha) promoter, and using different multiplicities of infection (MOIs)., Results: Both the PGK- and EF1alpha-CD20 vectors allowed efficient transduction of the CEM cell line and PHA-activated T cells, reaching 99 and 90% in the different targets, respectively. However EF1alpha-CD20 led to much higher expression levels of the transgene (mean fluorescence intensity 588-618 compared to 53 for PGK-CD20). Furthermore lymphocytes activated with IL-2 alone could be efficiently transduced with EF1alpha-CD20, reaching 10-25% positivity for CD20 (mean fluorescence intensity 409-424), allowing adequate immunoselection and strong complement-mediated lysis., Interpretation and Conclusions: EF1alpha-CD20 may represent a good candidate vector for gene therapy with the CD20 suicide system in the setting of allogeneic bone marrow transplants.

Background and Objectives: We analyzed the sensitivity of freshly isolated neoplastic B cells to rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC), using different effector cells., Design and Methods: ADCC was performed by 51Cr release assays in vitro, using peripheral blood mononuclear cells, IL-2-activated or expanded NK cells, neutrophils or macrophages as effector cells. B lymphoma lines and freshly isolated leukemic samples were used as targets., Results: NK cells, but PMN or macrophages mediated rituximab dependent cellular cytotoxicity against two B lymphoma lines. Purified NK cells (95% CD56+/CD16+) reached 70% lysis at the highest E:T ratio. By contrast, all freshly isolated B leukemia or lymphoma cases, including 5 chronic lymphocytic leukemia, 1 B-prolymphocytic leukemia, 1 mantle cell lymphoma, 2 marginal zone lymhomas and 2 follicular lymphomas were poorly lysed by ADCC in the same conditions and regardless of CD20 expression levels, reaching a mean of 4% and 27% maximal lysis with PBMC or purified NK cells, respectively. Interestingly, short term IL-2 cultured PBMC, containing 10 % activated NK cells, as well as long-term expanded NK cells, containing 80-95% activated NK cells, became strong ADCC effector cells with rituximab and lysed all leukemic samples to a mean of 57% and 67% at the highest E:T ratio, respectively., Interpretation and Conclusions: Primary leukemic cells are more resistant than cell lines to rituximab- and NK cell-mediated ADCC but short-term exposure to IL-2 or long-term expansion of NK cells in vitro may provide effective tools to improve the therapeutic activity of rituximab.