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Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1- KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium-induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a “corrupted” BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P

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.

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., In a preclinical model of Alzheimer's disease, characterized by neuronal damage and a high rate of inflammation (left), the intranasal (IN) administration of extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) operates in dampening inflammation (by reducing microglia activation) and in inducing neuroprotective effects (by decreasing spine loss) (right). These data suggest the possibility that the IN route administration of MSC‐EVs might accelerate the chance of a translational exploitation of MSC‐EVs toward therapy.

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.

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-b 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.

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.

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.

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 × 106/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.

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.

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. Copyright © 2013 by Lippincott Williams & Wilkins.

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.

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.

Background. Immune mechanisms of extracorporeal photochernotherapy (ECP) in refractory/resistant graft-versus-host disease (GvHD) are complex. We have previously analyzed the role of CD4(+)CD25(+)Foxp3(+) 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 CD4(+)CD25(+)Foxp3(+)GITR(+)CD62L(+)CCR7(+) 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.

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.

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-ζ, anti-CD19-DAP10, anti-CD19-4-1BB-ζ, and anti-CD19-CD28-ζ. 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-ζ 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)-α, TNF-β, IL-5, IL-6, and IL-8 elicited by coculture with ALL cells. Notably, anti-CD19-4-1BB-ζ 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. © 2007 ISEH - Society for Hematology and Stem Cells.

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.

Adoptive T-cell immunotherapy may provide complementary therapy for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this study, we have analyzed the functional characteristics of anti-BCP-ALL effector T cells generated by co-culturing T lymphocytes and dendritic cells (DC) from allogeneic human stem cell transplantation (HSCT) donors. After 21-day co-culture with DC pulsed with CD40L+ apoptotic BCP-ALL blasts, T cells presented with both effector and central memory phenotype, and showed high and specific cytotoxic activity against leukemic cells (average lysis = 77%), mostly mediated by CD8+ T cells. Noticeably, growth of CD4 T cells was maintained (45% of total cells), which actively produced Th1 cytokines (IFN-gamma, TNF-alpha, IL-2), but not IL-4, IL-5 and IL-10. Anti-BCP-ALL T cells expressed CD49d and CXCR4 (implicated in the recruitment to bone marrow), and CD62L and CCR7 (involved in the migration to lymphoid organs). In accordance with this profile, T cells significantly migrated in response to the chemokines CXCL12 and CCL19. In conclusion, stimulation of T cells with CD40L+BCP-ALL cells-loaded DC not only elicited the generation of potent and specific anti-leukemic cytotoxic effectors, but also the differentiation of specific and functional Th-1 CD4 lymphocytes. These effectors are fully equipped to reach leukemia-infiltrated tissues and have characteristics to support and orchestrate the anti-tumor immune-response.

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.

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. © 2013 Elsevier Ltd. All rights reserved.

Mesenchymal stem cells (MSCs) are self-renewable, multipotent progenitor cells able to differentiate into various mesodermal lineages.1 MSCs are present in basically all tissues, and have a pivotal role in tissue repair and in local control of inflammation.

Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs. PTX3 knockout MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis. We demonstrated that PTX3-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1+) in the wound bed. Moreover, wounds treated with PTX3-/- MSCs were characterized by an excessive accumulation of fibrin at the 2nd day after injury. Accordingly, PTX3-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3-/- MSCs failed to close the ulcers in PTX3-/- mice. In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair. Disclosures: No relevant conflicts of interest to declare.

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34+-sorted cells, SDS-MSCs were able to sustain CD34+ cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients. © 2012 Macmillan Publishers Limited All rights reserved.

Mesenchymal stromal cells (MSC) are tested in clinical trials to treat graft versus host disease (GvHD) after stem cell transplantation (SCT).In vitrostudies 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.

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.

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.

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.

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-ζ-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.

Abstract 743 Background: Acute Graft versus host disease (aGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). Conventional treatment with high dose steroids fails to achieve a complete and sustained response in more than 50% of patients. Several second line treatments have been described but none of these can be considered superior or a standard of care (Paul J. Martin et al, BBMT 2012). Among these treatments, the use of third party mesenchymal stromal cells (MSC) has been proposed (LeBlanc et al, Lancet 2008). In this study, we assessed the safety and efficacy of third party human MSC, in a prospective, multicenter, phase I study (EudraCT 2008–007869-23). Methods: Forty-seven patients with steroid-resistant, acute or chronic grade II-IV GvHD were enrolled into this study. Human MSC were obtained from bone marrow harvests of healthy donors and expanded in vitro using serum free medium supplemented with human platelet lysate (Capelli C et al, BMT, 2007; Capelli C. et al, Cytotherapy 2009). In vitro expanded MSC were produced in two officially authorized Cell Factories and tested in four Italian Hematology Units. The primary endpoint of this study was the safety. Secondary endpoints were the response of GvHD (evaluated 28 days after the last MSC infusion), as well as the overall survival and transplant-related deaths. Blood samples were periodically collected before and after MSC infusion to measure plasma levels of IL2Ralpha by ELISA, as previously described by our group (Dander E et al, Leukemia 2012). Results: Between August 2009, and June 2012, 47 patients (16 children, 31 adults, median age 25.5 years, range 1 to 67) were treated. The median dose of infused MSC was 1.5×106 cells per kg bodyweight. Enrolled patients presented with aGvHD in 37 cases, chronic overlap syndrome in 7 cases, and chronic classic GvHD in 3 cases. Fifteen pts had grade II GvHD, 23 grade III and 9 grade IV, according to NIH criteria. In 17 cases GvHD involved a single organ, in 24 cases 2, and in 6 cases 3 organs. Prior to MSC infusion 22 patients had received only high dose steroids, 12 patients received one cycle of pentostatin (1 mg/kg bodyweight for 3 days, Schmitt T. et al BMT, 2011: 46 580–585), while 13 received other conventional immunosuppressants. Patients received a median of 3 MSC infusions (range 1 to 8). No side effects were registered immediately after MSC infusion and no complications were lately referred as MSC-related. Overall, in 30 patients (63.8%) a clinical response of GvHD was registered. Thirteen of these patients (27.6%) had a complete response and 17 (36.1%) a partial response to treatment. Twenty-two of the 30 responding patients did not require further lines of immunosuppression after MSC infusion. Response was significantly more likely in patients exhibiting grade II GvHD versus those exhibiting more severe gradings (87.5% vs. 51.6%, p = 0.02) and in patients receiving MSC in a time interval of 30 days from the onset of GvHD (75.9% vs. 43.7%, p= 0.05). Current median follow up for this cohort is 200 days (range 30–1066). Responders show a significant lower transplant-related mortality (10.0% vs. 88.2%, p Measurements of plasmatic levels of IL2Ralpha, when comparing responders vs non-responders patients, showed a statistically significant difference in terms of fold decrease of the marker (p=0.027), corroborating clinical results. Similarly, a significant trend of fold decrease change (p=0.058) was observed when comparing responding patients receiving MSC within or after 30 days from the onset of the disease, in line with clinical results. Conclusions: This study confirms that human MSC prepared in academic cell therapy facilities may represent a safe and effective treatment of patients with steroid-refractory GvHD. Plasmatic inflammatory markers may help in evaluating and monitoring of clinical response. The sequential or combined administration of MSC and other immunosuppressants, such as pentostatin, is equally safe and feasible and deserves further investigation. We suggest to consider the use of MSC promptly, as early as possible, after steroid failure. Disclosures: No relevant conflicts of interest to declare.

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.

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 × 106) or PTX3−/−-MSCs (1 × 106). 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. Electronic supplementary material The online version of this article (doi:10.1186/s40635-017-0126-5) contains supplementary material, which is available to authorized users.