Publications by authors named "Erica Dander"

29 Publications

  • Page 1 of 1

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

Br J Haematol 2021 Mar 13. Epub 2021 Mar 13.

Centro Ricerca Tettamanti, Pediatric Dep, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14 CD16 monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.
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http://dx.doi.org/10.1111/bjh.17330DOI Listing
March 2021

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Cancers (Basel) 2020 Nov 5;12(11). Epub 2020 Nov 5.

Department of Medicine and Surgery, University of Parma, 43126 Parma, Italy.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.
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http://dx.doi.org/10.3390/cancers12113267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694402PMC
November 2020

Heterogeneity of the bone marrow niche in patients with myeloproliferative neoplasms: ActivinA secretion by mesenchymal stromal cells correlates with the degree of marrow fibrosis.

Ann Hematol 2021 Jan 21;100(1):105-116. Epub 2020 Oct 21.

Centro Ricerca M. Tettamanti, Department of Pediatrics, University of Milano-Bicocca, Monza, Italy.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.
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http://dx.doi.org/10.1007/s00277-020-04306-wDOI Listing
January 2021

Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of Alzheimer's disease.

Stem Cells Transl Med 2020 09 4;9(9):1068-1084. Epub 2020 Jun 4.

School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.

The critical role of neuroinflammation in favoring and accelerating the pathogenic process in Alzheimer's disease (AD) increased the need to target the cerebral innate immune cells as a potential therapeutic strategy to slow down the disease progression. In this scenario, mesenchymal stem cells (MSCs) have risen considerable interest thanks to their immunomodulatory properties, which have been largely ascribed to the release of extracellular vesicles (EVs), namely exosomes and microvesicles. Indeed, the beneficial effects of MSC-EVs in regulating the inflammatory response have been reported in different AD mouse models, upon chronic intravenous or intracerebroventricular administration. In this study, we use the triple-transgenic 3xTg mice showing for the first time that the intranasal route of administration of EVs, derived from cytokine-preconditioned MSCs, was able to induce immunomodulatory and neuroprotective effects in AD. MSC-EVs reached the brain, where they dampened the activation of microglia cells and increased dendritic spine density. MSC-EVs polarized in vitro murine primary microglia toward an anti-inflammatory phenotype suggesting that the neuroprotective effects observed in transgenic mice could result from a positive modulation of the inflammatory status. The possibility to administer MSC-EVs through a noninvasive route and the demonstration of their anti-inflammatory efficacy might accelerate the chance of a translational exploitation of MSC-EVs in AD.
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http://dx.doi.org/10.1002/sctm.19-0327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445021PMC
September 2020

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells.

Int J Mol Sci 2020 Mar 10;21(5). Epub 2020 Mar 10.

Department of Medicine and Surgery, University of Parma, 43125 Parma, Italy.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.
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http://dx.doi.org/10.3390/ijms21051899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084684PMC
March 2020

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

Br J Haematol 2020 07 2;190(2):262-273. Epub 2020 Mar 2.

Centro Ricerca Tettamanti, University of Milano-Bicocca, MBBM Foundation, Monza, Italy.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILβ pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILβ-stimulated BM-MSC strongly attract E/R Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34 IL7R progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILβ compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.
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http://dx.doi.org/10.1111/bjh.16523DOI Listing
July 2020

Activin A contributes to the definition of a pro-oncogenic bone marrow microenvironment in t(12;21) preleukemia.

Exp Hematol 2019 05 28;73:7-12.e4. Epub 2019 Feb 28.

Centro Ricerca Tettamanti, Pediatric Department, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy. Electronic address:

The TEL-AML1 fusion gene, generated by the t(12;21) chromosome translocation, arises in a progenitor/stem cell and could induce clonal expansion of a persistent preleukemic B-cell clone which, on acquisition of secondary alterations, may turn into full-blown leukemia. During infections, deregulated cytokine signaling, including transforming growth factor β (TGF-β), can further accelerate this process by creating a protumoral bone marrow (BM) microenvironment. Here, we show that activin A, a member of the TGF-β family induced under inflammatory conditions, inhibits the proliferation of normal progenitor B cells but not that of preleukemic TEL-AML1-positive clones, thereby providing a selective advantage to the latter. Finally, we find that activin A inhibits BM-derived mesenchymal stromal cell-mediated secretion of CXCL12, a major chemoattractant in the BM compartment, thereby contributing to shape a leukemia-promoting environment. Overall, our findings indicate that activin A, in concert with TGF-β, could play an important role in the creation of a pro-oncogenic BM microenvironment and provide novel mechanistic insights into TEL-AML1-associated leukemogenesis.
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http://dx.doi.org/10.1016/j.exphem.2019.02.006DOI Listing
May 2019

ActivinA: a new leukemia-promoting factor conferring migratory advantage to B-cell precursor-acute lymphoblastic leukemic cells.

Haematologica 2019 03 27;104(3):533-545. Epub 2018 Sep 27.

Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-β family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34 cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.
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http://dx.doi.org/10.3324/haematol.2018.188664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395324PMC
March 2019

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells.

Front Immunol 2018 1;9:1207. Epub 2018 Jun 1.

Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN the production of soluble factors with a major role played by Prostaglandin E (PGE). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c) are a small percentage of BM cells, we demonstrated colocalization of CD271 MSCs with CD1c DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c cells in the proximity of CD271 MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.
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http://dx.doi.org/10.3389/fimmu.2018.01207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992779PMC
August 2019

Mesenchymal stromal cells from Shwachman-Diamond syndrome patients fail to recreate a bone marrow niche in vivo and exhibit impaired angiogenesis.

Br J Haematol 2018 07 16;182(1):114-124. Epub 2018 May 16.

Paediatric Department, Centro Ricerca Tettamanti, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy.

Shwachman-Diamond syndrome (SDS) is a rare multi-organ recessive disease mainly characterised by pancreatic insufficiency, skeletal defects, short stature and bone marrow failure (BMF). As in many other BMF syndromes, SDS patients are predisposed to develop a number of haematopoietic malignancies, particularly myelodysplastic syndrome and acute myeloid leukaemia. However, the mechanism of cancer predisposition in SDS patients is only partially understood. In light of the emerging role of mesenchymal stromal cells (MSCs) in the regulation of bone marrow homeostasis, we assessed the ability of MSCs derived from SDS patients (SDS-MSCs) to recreate a functional bone marrow niche, taking advantage of a murine heterotopic MSC transplant model. We show that the ability of semi-cartilaginous pellets (SCPs) derived from SDS-MSCs to generate complete heterotopic ossicles in vivo is severely impaired in comparison with HD-MSC-derived SCPs. Specifically, after in vitro angiogenic stimuli, SDS-MSCs showed a defective ability to form correct networks, capillary tubes and vessels and displayed a marked decrease in VEGFA expression. Altogether, these findings unveil a novel mechanism of SDS-mediated haematopoietic dysfunction based on hampered ability of SDS-MSCs to support angiogenesis. Overall, MSCs could represent a new appealing therapeutic target to treat dysfunctional haematopoiesis in paediatric SDS patients.
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http://dx.doi.org/10.1111/bjh.15388DOI Listing
July 2018

Intraperitoneal adoptive transfer of mesenchymal stem cells enhances recovery from acid aspiration acute lung injury in mice.

Intensive Care Med Exp 2017 Dec 6;5(1):13. Epub 2017 Mar 6.

Department of Anesthesia, Critical Care and Emergency, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.

Background: Mesenchymal stem cells (MSCs) might act as fine-tuners of inflammation during acute lung injury. We assessed the effects of adoptive transfer of MSCs in acid aspiration acute lung injury and explored the role of long pentraxin PTX3.

Methods: We conducted a prospective experimental interventional study on wild-type (WT) and PTX3-deficient (PTX3) mice. Acute lung injury was induced in WT and PTX3 mice by instillation of hydrochloric acid into the right bronchus. One hour later, animals received intraperitoneal sterile phosphate-buffered saline (PBS), WT-MSCs (1 × 10) or PTX3-MSCs (1 × 10). Twenty-four hours after injury, we measured the effects of treatments on arterial blood gases, wet/dry lung weight (W/D), CT scan analysis of lung collapse, neutrophils, TNFα and CXCL1 in bronchoalveolar lavage, and plasma PTX3. D-dimer was assayed in 1 week and OH-proline in 2 weeks to track the fibrotic evolution.

Results: In 24 h, in comparison to PBS, WT-MSCs improved oxygenation and reduced W/D and alveolar collapse. These effects were associated with decreased concentrations of alveolar neutrophils and cytokines. WT-MSCs increased D-dimer concentration and decreased OH-proline levels, too. Treatment with PTX3-MSCs ameliorated oxygenation, W/D, and alveolar TNFα, though to a lesser extent than WT-MSCs. PTX3-MSCs did not improve lung collapse, neutrophil count, CXCL1, D-dimer, and OH-proline concentrations. The protective effects of WT-MSCs were dampened by lack of endogenous PTX3, too.

Conclusions: In acid aspiration acute lung injury, MSCs improve pulmonary function and limit fibrosis by fine-tuning inflammation. The role of PTX3 in determining MSCs' effects might merit further scrutiny.
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http://dx.doi.org/10.1186/s40635-017-0126-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339261PMC
December 2017

Mesenchymal stromal cell-secreted chemerin is a novel immunomodulatory molecule driving the migration of ChemR23-expressing cells.

Cytotherapy 2017 02 6;19(2):200-210. Epub 2016 Dec 6.

Centro di Ricerca Tettamanti, Clinica Pediatrica, Università di Milano-Bicocca, Monza, Italy. Electronic address:

Background: Mesenchymal stromal cells (MSCs) are multipotent cells characterized by broad immunomodulatory properties exploited for the treatment of inflammatory disorders. However, the efficacy of MSC-based therapy is highly variable and tightly linked to MSC culture conditions and treatment schedule. Thus, the identification of novel key molecules regulating MSC immunomodulatory activities in vivo might constitute a crucial step toward the optimization of currently available clinical protocols. In this regard, herein, we sought to determine whether the newly identified chemotactic protein, chemerin, plays a role in MSC-mediated regulation of inflammation.

Methods: Chemerin production by human MSCs was investigated under different culture conditions using enzyme-linked immunosorbent assay (ELISA). After purification, MSC-secreted chemerin was identified using mass spectrometry analysis and the biological activity of secreted isoforms was evaluated using migration assay.

Results: Bone marrow-derived MSCs secrete chemerin and express its receptors ChemR23 and CCRL2. Chemerin production is dependent on culture conditions and increases upon stimulation with inflammatory cytokines. In particular, platelet lysate (PL)-MSCs produce higher levels of chemerin compared with fetal bovine serum (FBS)-MSCs. Furthermore, chemerin is secreted by MSCs as an inactive precursor, which can be converted into its active form by exogenous chemerin-activating serine and cysteine proteases.

Discussion: Our data indicate that, in response to various inflammatory stimuli, MSCs secrete high amounts of inactive chemerin, which can then be activated by inflammation-induced tissue proteases. In light of these initial findings, we propose that further analysis of chemerin functions in vivo might constitute a crucial step toward optimizing MSC-based therapy for inflammatory diseases.
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http://dx.doi.org/10.1016/j.jcyt.2016.11.006DOI Listing
February 2017

Pentraxin 3 plasma levels at graft-versus-host disease onset predict disease severity and response to therapy in children given haematopoietic stem cell transplantation.

Oncotarget 2016 Dec;7(50):82123-82138

"M. Tettamanti" Research Center, Pediatric Department, University of Milano-Bicocca, Monza, Italy.

Acute Graft-versus-Host Disease (GvHD) remains a major complication of allogeneic haematopoietic stem cell transplantation, with a significant proportion of patients failing to respond to first-line systemic corticosteroids. Reliable biomarkers predicting disease severity and response to treatment are warranted to improve its management. Thus, we sought to determine whether pentraxin 3 (PTX3), an acute-phase protein produced locally at the site of inflammation, could represent a novel acute GvHD biomarker. Using a murine model of the disease, we found increased PTX3 plasma levels after irradiation and at GvHD onset. Similarly, plasma PTX3 was enhanced in 115 pediatric patients on day of transplantation, likely due to conditioning, and at GvHD onset in patients experiencing clinical symptoms of the disease. PTX3 was also found increased in skin and colon biopsies from patients with active disease. Furthermore, PTX3 plasma levels at GvHD onset were predictive of disease outcome since they resulted significantly higher in both severe and therapy-unresponsive patients. Multiple injections of rhPTX3 in the murine model of GvHD did not influence the disease course. Taken together, our results indicate that PTX3 constitutes a biomarker of GvHD severity and therapy response useful to tailor treatment intensity according to early risk-stratification of GvHD patients.
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http://dx.doi.org/10.18632/oncotarget.13488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347680PMC
December 2016

Mesenchymal Stromal Cell-Derived PTX3 Promotes Wound Healing via Fibrin Remodeling.

J Invest Dermatol 2016 Jan;136(1):293-300

Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM/San Gerardo Hospital, Monza, Italy. Electronic address:

Although mesenchymal stromal cells (MSCs) can promote wound healing in different clinical settings, the underlying mechanism of MSC-mediated tissue repair has yet to be determined. Because a nonredundant role of pentraxin 3 (PTX3) in tissue repair and remodeling has been recently described, here we sought to determine whether MSC-derived PTX3 might play a role in wound healing. Using a murine model of skin repair, we found that Ptx3-deficient (Ptx3(-/-)) MSCs delayed wound closure and reduced granulation tissue formation compared with wt MSCs. At day 2, confocal microscopy revealed a dramatic reduction in green fluorescent protein (GFP)-expressing Ptx3(-/-) MSCs recruited to the wound, where they appeared to be not only poorly organized in bundles but also scattered in the extracellular matrix. These findings were further confirmed by quantitative biochemical analysis of GFP content in wound extracts. Furthermore, Ptx3(-/-) MSC-treated skins displayed increased levels of fibrin and lower levels of D-dimer, suggesting delayed fibrin-rich matrix remodeling compared with control skins. Consistently, both pericellular fibrinolysis and migration through fibrin were found to be severely affected in Ptx3(-/-) MSCs. Overall, our findings identify an essential role of MSC-derived PTX3 in wound repair underscoring the beneficial potential of MSC-based therapy in the management of intractable wounds.
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http://dx.doi.org/10.1038/JID.2015.346DOI Listing
January 2016

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

Biol Blood Marrow Transplant 2014 Mar 7;20(3):375-81. Epub 2013 Dec 7.

HSCT Pediatric Unit and Laboratory of Cell Therapy "S. Verri", San Gerardo Hospital, Monza, Italy.

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.
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http://dx.doi.org/10.1016/j.bbmt.2013.11.033DOI Listing
March 2014

Immunosuppression does not affect human bone marrow mesenchymal stromal cell efficacy after transplantation in traumatized mice brain.

Neuropharmacology 2014 Apr 15;79:119-26. Epub 2013 Nov 15.

IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Department of Neuroscience, Milan, Italy.

The need for immunosuppression after allo/xenogenic mesenchymal stromal cell (MSC) transplantation is debated. This study compared the long-term effects of human (h) bone marrow MSC transplant in immunocompetent or immunosuppressed traumatic brain injured (TBI) mice. C57Bl/6 male mice were subjected to TBI or sham surgery followed 24 h later by an intracerebroventricular infusion of phosphate buffer saline (PBS, control) or hMSC (150,000/5 μl). Immunocompetent and cyclosporin A immunosuppressed (CsA) mice were analyzed for gene expression at 72 h, functional deficits and histological analysis at five weeks. Gene expression analysis showed the effectiveness of immunosuppression (INFγ reduction in CsA treated groups), with no evidence of early rejection (no changes of MHCII and CD86 in all TBI groups) and selective induction of T-reg (increase of Foxp3) only in the TBI hMSC group. Five weeks after TBI, hMSC had comparable efficacy, with functional recovery (on both sensorimotor and cognitive deficits) and structural protection (contusion volume, vessel rescue effect, gliotic scar reduction, induction of neurogenesis) in immunosuppressed and immunocompetent mice. Therefore, long-term hMSC efficacy in TBI is not dependent on immunosuppressive treatment. These findings could have important clinical implication since immunosuppression in acute TBI patients may increase their risk of infection and not be tolerated.
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http://dx.doi.org/10.1016/j.neuropharm.2013.11.001DOI Listing
April 2014

The effects of propofol and ketamine on the cytokine levels of children with acute lymphoblastic leukemia.

J Pediatr Hematol Oncol 2013 Oct;35(7):e296-300

*Department of Anesthesia, McGill University Health Centre, Montreal Children Hospital, Montreal, QC, Canada §Department of Anaesthesia and Intensive Care, Anadolu Medical Center, Kocaeli, Turkey ‡Dipartimento di Medicina Esperimentale, Università Milano Bicocca, Milan †U.O. Anestesia e Rianimazione I ∥Centro di Ricerca "M. Tettamanti" ¶Clinica Pediatrica, Dipartimento Materno Infantile, Ospedale San Gerardo, Monza, Italy.

The immune system of children with acute lymphoblastic leukemia (ALL) is affected by both the underlying disease and the chemotherapy. Children with ALL receive sedation for diagnostic and therapeutic procedures, which may contribute to immune competence alteration. The effects of propofol-ketamine combination on the immune system of children with ALL have not been investigated. This cohort study was designed to assess the immunomodulatory activity of the propofol-ketamine combination on proinflammatory and anti-inflammatory cytokines of children with ALL undergoing painful procedures. We enrolled 20 children with ALL undergoing bone marrow aspiration (BMA) and lumbar puncture with methotrexate. All children received sedation with IV ketamine (0.5 mg/kg) and propofol (3±2 mg/kg). Plasma concentration of cytokines interleukin (IL)-1β, IL-2, IL-6, IL-10, IL-8, IL-12p70, and interferon-γ before sedation for BMA was represented as T0, during lumbar puncture with methotrexate sedation 6 hours after T0 was represented as T1, and 24 hours after BMA was represented as T2. Sedation with propofol-ketamine combination did not modify the plasma concentration of the most measured cytokines and the T helper 1/2 ratio in children with ALL. There was a significant reduction in IL-8 concentration 24 hours after BMA associated with the concomitant administration of steroids and methotrexate. These data suggest that sedation with propofol-ketamine combination may not affect the immediate outcome of children with ALL.
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http://dx.doi.org/10.1097/MPH.0b013e31829bc92fDOI Listing
October 2013

Mesenchymal stromal cells do not increase the risk of viral reactivation nor the severity of viral events in recipients of allogeneic stem cell transplantation.

Stem Cells Int 2012 30;2012:690236. Epub 2012 May 30.

Clinica Pediatrica, Università degli Studi di Milano Bicocca, Ospedale San Gerardo, 20900 Monza, Italy.

Mesenchymal stromal cells (MSC) are tested in clinical trials to treat graft versus host disease (GvHD) after stem cell transplantation (SCT). In vitro studies demonstrated MSC's broad immunosuppressive activity. As infections represent a major risk after SCT, it is important to understand the role of MSC in this context. We analyzed 24 patients (pts) receiving MSC for GvHD in our Unit between 2009 and 2011. We recorded viral reactivations as measured in whole blood with polymerase chain reaction for 100 days following MSC administration. In patients with a documented viral reactivation in the first 3 days following MSCs infusion the frequency of virus-specific IFNgamma-producing cells was determined through enzyme-linked immunospot assay. In our cohort of patients viral reactivation after MSC infusion occurred in 45% of the cases, which did not significantly differ from the incidence in a historical cohort of patients affected by steroid resistant GvHD and treated with conventional immunosuppression. No patient presented severe form of infection. Two cases could be checked for immunological response to viral stimulus and demonstrated virus specific T-cytotoxic lymphocyte activity. In our experience MSC infusion did not prove to trigger more frequent or severer viral reactivations in the post transplantation setting.
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http://dx.doi.org/10.1155/2012/690236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369546PMC
August 2012

Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population.

Biol Blood Marrow Transplant 2010 Sep 27;16(9):1293-301. Epub 2010 Mar 27.

Clinica Pediatrica, Università Milano Bicocca, Ospedale San Gerardo, Via Donizetti 106, Monza, MI, Italy.

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.
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http://dx.doi.org/10.1016/j.bbmt.2010.03.017DOI Listing
September 2010

Interleukin-17-producing T-helper cells as new potential player mediating graft-versus-host disease in patients undergoing allogeneic stem-cell transplantation.

Transplantation 2009 Dec;88(11):1261-72

Centro Ricerca M. Tettamanti, Clinica Pediatrica Università degli Studi di Milano Bicocca, Monza, Italy.

Objectives: Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients.

Methods: Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA.

Results: An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17+/interferon (IFN)-gamma- and IL-17+/IFN-gamma+ subsets and expressed IL-23 receptor. Interestingly, IFN-gamma+ TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD.

Conclusions: These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.
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http://dx.doi.org/10.1097/TP.0b013e3181bc267eDOI Listing
December 2009

Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex.

J Virol 2010 Jan 4;84(2):1005-13. Epub 2009 Nov 4.

Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland.

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC(90)] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.
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http://dx.doi.org/10.1128/JVI.01809-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798344PMC
January 2010

Regulatory T cells and extracorporeal photochemotherapy: correlation with clinical response and decreased frequency of proinflammatory T cells.

Transplantation 2009 May;87(9):1422-5

Pediatric Department, Laboratory of Cell Therapy Stefano Verri, University of Milano-Bicocca, San Gerardo Hospital, Monza (Milan), Italy.

Background: Immune mechanisms of extracorporeal photochemotherapy (ECP) in refractory/resistant graft-versus-host disease (GvHD) are complex. We have previously analyzed the role of CD4CD25Foxp3 regulatory T cells (T-regs).

Methods: In the current study, we have enlarged the size of the population (n=27; chronic GvHD=18, acute GvHD=9) for a median follow-up of 24 months. T-regs were monitored for CD4, CD25, glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L, CCR7, Foxp3, and STAT-5. Immune analysis by interleukin (IL)-17 Elispot was carried out on circulating T-helper CD4 cells secreting IL-17, a subset of T cells considered relevant in the pathogenesis of GvHD.

Results: We confirm that ECP is accompanied by a significant increase of CD4CD25Foxp3GITRCD62LCCR7 T-regs. Sorted T-regs show augmented phosphorylation of STAT-5. Only ECP-responding patients demonstrate a raise of circulating T-regs, being mostly affected by chronic GvHD. Moreover, this phenomenon corresponds to a diminished secretion of IL-17.

Discussion: In conclusion, our study shows that T-regs represent important immune mediators of the clinical benefits of ECP in patients affected by GvHD.
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http://dx.doi.org/10.1097/TP.0b013e3181a27a5dDOI Listing
May 2009

Isolation of monocytes from leukapheretic products for large-scale GMP-grade generation of cytomegalovirus-specific T-cell lines by means of an automated elutriation device.

Transfusion 2008 Aug 29;48(8):1644-9. Epub 2008 May 29.

Dipartimento di Patologia Clinica, Servizio di Immunoematologia e Trasfusionale, Unità di Aferesi e Nuove Tecnologie Trasfusionali, Ospedale San Gerardo de' Tintori, Monza (MI), Italy.

Background: Dendritic cells (DC) act as antigen-presenting cells in immune response-mediated mechanisms against malignant cells and/or viral or fungal pathogens. CD14+ monocytes have been so far isolated by techniques of plastic adherence or by using immunomagnetic methods. Here the effectiveness of a commercially available cell separation system (Elutra, Gambro BCT) in the separation of monocytes and the large-scale production of cytomegalovirus (CMV)-specific T-cell lines were investigated.

Study Design And Methods: Six mononuclear cell (MNC) collections were processed with the Elutra system. Monocyte-enriched fraction was differentiated into DCs by addition of granulocyte-macrophage-colony-stimulating factor and interleukin (IL)-4. After 6 days of culture, DCs were matured in the presence of interferon (IFN)-gamma, IFN-alpha, IL-1beta, tumor necrosis factor-alpha, and poly(I:C) and pulsed with a pool of 48 MHC Class I and II-binding CMV peptides. Lymphocytes were then stimulated with mature autologous CMV peptide-pulsed DCs.

Results: After elutriation, the mean monocyte yield was 0.89 x 10(9) +/- 0.65 x 10(9), with a 51.0 +/- 31.6 percent recovery and a 51.1 +/- 35.4 percent purity. A significant correlation was observed when basal monocyte content was related to the postelutriation recovery (p < 0.0116). More than 60 percent of plated monocytes were differentiated into DCs, which after pulsing with CMV peptides, were able to stimulate a robust enrichment in CMV antigen-specific T cells in all tested samples (mean percentage of pentamer-positive CD8+ cells, 35% compared to the initial 2%).

Conclusion: Our findings might be helpful for an appropriate MNC collection, to maximize the efficiency of the elutriation system and subsequently obtain an optimal monocyte-enriched yield for further DC generation and T-cell stimulation.
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http://dx.doi.org/10.1111/j.1537-2995.2008.01756.xDOI Listing
August 2008

Characterization of migratory activity and cytokine profile of helper and cytotoxic CMV-specific T-cell lines expanded by a selective peptide library.

Exp Hematol 2008 Apr 8;36(4):473-85. Epub 2008 Feb 8.

Centro Ricerca "M. Tettamanti," Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Monza, Italy.

Objective: Reconstitution of cellular immunity by infusion of cytomegalovirus (CMV)-specific T lymphocytes is an attractive alternative to drugs currently used to control CMV reactivation in immunocompromised patients. For this purpose, we established a method for generating both anti-CMV CD4 and CD8 T cells following Good Manufacturing Practice indications, and we extensively characterized their immune functions.

Materials And Methods: For generating CD4 and CD8 CMV-specific lymphocytes, T cells from 11 CMV-seropositive donors were stimulated three times with dendritic cells (DC) pulsed with a library of selected CMV peptides, recognized by >85% of the Caucasian population. At the end of the culture, T cells were analyzed for their specificity, cytotoxicity, chemotactic migration, proliferation, and cytokine production.

Results: T cells were successfully expanded and enriched in CMV-specific subsets with an effector memory or an effector memory CD45 RA(+) phenotype. CMV-specific T-cell lines showed specific cytotoxicity (average lysis: 47%) against CMV peptides-pulsed DCs, and were depleted of auto- and alloreactivity. Moreover, the ability to proliferate following antigenic stimulation and the presence of functional CD4 lymphocytes producing Th1 and Th2 cytokines can ensure long-term antiviral immunity after in vivo injection. CMV-specific T lymphocytes also proved to be fully equipped to reach CMV-infected tissues, because they expressed CD49d and CCR1, CXCR3, CXCR4, necessary to recruit effector cells to inflamed sites. In accordance with this profile, they significantly migrated towards inflammatory chemokines and towards the supernatant collected from inflamed lung fibroblasts, frequently involved in CMV pathology.

Conclusion: This strategy allows expansion of effector T cells capable to exert CD8 and CD4-mediated immune functions and, thus, is suitable for clinical use.
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http://dx.doi.org/10.1016/j.exphem.2007.12.007DOI Listing
April 2008

Enhancement of the anti-leukemic activity of cytokine induced killer cells with an anti-CD19 chimeric receptor delivering a 4-1BB-zeta activating signal.

Exp Hematol 2007 Sep 25;35(9):1388-97. Epub 2007 Jul 25.

Centro Ricerca M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Monza, Italy.

Objective: There is growing interest in the use of cytokine-induced killer (CIK) cells in cancer therapy. In this study, we sought to maximize the antileukemic activity of anti-CD19 receptor-modified CIK cells against B-lineage acute lymphoblastic leukemia (ALL).

Materials And Methods: CIK cells were transduced with retroviral vectors carrying different types of anti-CD19 chimeric receptors: anti-CD19-zeta, anti-CD19-DAP10, anti-CD19-4-1BB-zeta, and anti-CD19-CD28-zeta. A truncated form of the receptor was used as a control. Transduced CIK cells were then analyzed for their cytotoxic activity against ALL cells and for their capability to proliferate and to release cytokines after ALL encounter.

Results: CIK cells were efficiently transduced with all the anti-CD19 retroviral vectors. Anti-CD19 receptor expression conferred powerful killing activity against ALL cells. However, there were clear advantages when receptors containing the co-stimulatory molecules 4-1BB or CD28 were transduced. Such cells had significantly more potent cytotoxicity than cells expressing the anti-CD19-zeta or anti-CD19-DAP10. Moreover, the presence of 4-1BB or CD28 in the receptor increased the production of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, TNF-beta, IL-5, IL-6, and IL-8 elicited by coculture with ALL cells. Notably, anti-CD19-4-1BB-zeta CIK cells secreted particularly low levels of interleukin-10 and proliferated strongly after contact with ALL cells.

Conclusions: Anti-CD19 chimeric receptors delivering primary and costimulatory signals render CIK cells powerfully cytotoxic against ALL cells and induce secretion of immunostimulatory cytokines and proliferation. These results support the testing of genetically modified CIK cells in clinical trials.
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http://dx.doi.org/10.1016/j.exphem.2007.05.018DOI Listing
September 2007

Repeated infusions of donor-derived cytokine-induced killer cells in patients relapsing after allogeneic stem cell transplantation: a phase I study.

Haematologica 2007 Jul;92(7):952-9

Laboratory of Cellular and Gene Therapy G. Lanzani, Ospedali Riuniti di Bergamo, Bergamo, Italy.

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.
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http://dx.doi.org/10.3324/haematol.11132DOI Listing
July 2007

Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies.

Haematologica 2007 Mar;92(3):381-8

Centro di Ricerca Matilde Tettamanti, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Monza, Milan, Italy.

Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy.
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http://dx.doi.org/10.3324/haematol.10873DOI Listing
March 2007

Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy.

Exp Hematol 2006 Sep;34(9):1219-29

Centro Ricerca M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Monza, Italy.

Objective: Cytokine-induced killer (CIK) cells are ex vivo expanded cells enriched in CD3(+)CD56(+) natural killer T (NKT) cells with major histocompatibility-unrestricted cytotoxicity against several tumoral targets, except B-lineage acute lymphoblastic leukemia (B-ALL). We redirected CIK cells cytotoxicity toward B-ALL with a chimeric receptor specific for the CD19 antigen and then explored if modified-CIK cells maintain the same chemotactic properties of freshly isolated NKT cells, whose trafficking machinery reflects their preferential localization into the sites of B-ALL infiltration.

Material And Methods: CIK cells were expanded ex vivo for 21 days and analyzed for expression of adhesion molecules and chemokine receptors regulating adhesion and homing toward leukemia-infiltrated tissues. CIK cells were then transduced with the anti-CD19-zeta-internal ribosomal entry site-green fluorescent protein retroviral vector and characterized for their cytotoxicity against B-ALL cells in a (51)Cr-release assay and for their trafficking properties, including chemotactic activity, adhesion and transendothelial migration, and metalloproteases-dependent invasion of Matrigel.

Results: Similarly to freshly isolated NKT cells, CD49d and CD11a were highly expressed on CIK cells. Moreover, CIK cells expressed CXCR4, CCR6, and CCR7 (mean expression 72%, 60%, and 32%, respectively), presenting chemotactic activity toward their respective ligands. Anti-CD19 chimeric receptor-modified CIK cells became cytotoxic against B-ALL cells (mean lysis, 60%) and showed, after exposure to a CXCL12 gradient, high capacity to adhere and transmigrate through endothelial cells and to invade Matrigel.

Conclusion: The potential capacity to localize into leukemia-infiltrated tissues of anti-CD19 chimeric receptor-redirected CIK cells, together with their ability to efficiently kill B-ALL cells, suggests that modified-CIK cells represent a valuable tool for leukemia immunotherapy.
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http://dx.doi.org/10.1016/j.exphem.2006.05.004DOI Listing
September 2006

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice.

EMBO J 2004 Sep 19;23(17):3505-15. Epub 2004 Aug 19.

Istituto Ricerche Farmacologiche Mario Negri, Milan, Italy.

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.
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http://dx.doi.org/10.1038/sj.emboj.7600361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516633PMC
September 2004