We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)–like receptors (KIR) ligand-mismatched donors. We ...showed that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype, as well as through phenotypic and functional analyses of NK cells, both at the polyclonal and clonal level. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether, these results may have important clinical implications for the selection of optimal donors for haplo-HSCT.
Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more ...than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related malignancy expressing EBV antigens that are possible targets of cell therapy, including latent membrane protein 2 (LMP2). We ...conducted a clinical trial of EBV-targeted cell therapy with autologous virus-specific cytotoxic T lymphocytes (CTLs) for NPC refractory to conventional treatments.
Ten patients with EBV-related stage IV NPC in progression after conventional radiotherapy and chemotherapy received intravenously autologous EBV-specific CTLs reactivated and expanded ex vivo from peripheral blood lymphocytes through stimulation with EBV-transformed autologous B-lymphoblastoid cell lines (LCL). Toxicity, specific cellular immune responses, and clinical tumor responses were evaluated.
EBV-specific CTLs could be generated in all patients and were predominantly CD3+/CD8+ T lymphocytes displaying specific killing of autologous EBV-LCL, autologous NPC cells as well as autologous targets bearing the EBV antigen LMP2. Patients received two to 23 infusions of EBV-specific CTLs that were well tolerated with the exception of grade 1 to 2 inflammatory reactions at the tumor site in two cases. Control of disease progression was obtained in six of 10 patients (two with partial response and four with stable disease). Analysis of interferon-gamma-producing cells demonstrated an increased frequency of EBV-specific immunity, with appearance of LMP2-specific responses in four patients, of whom three had clinical benefit.
Cell therapy with EBV-targeted autologous CTLs is safe, induces LMP-2-specific immunologic responses, and is associated with objective responses and control of disease progression in patients with stage IV NPC resistant to conventional treatments.
Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their ...immunomodulatory effects, which could be influenced by both the cell source and culture conditions.
We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells.
The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G.
Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.
Homing of intraarterially infused rat mesenchymal stem cells (rMSCs) in the brain of rats bearing a 6‐hydroxydopamine (6‐OHDA)‐induced lesion of the nigrostriatal tract was evaluated to establish ...whether the toxin‐induced damage enables MSC passage across the blood‐brain barrier (BBB) or if transient BBB disruption is necessary. Appreciable delivery of rMSCs to the brain of lesioned animals occurred only after mannitol pretreatment. rMSC infusion induced progressive normalization of the pathological response to apomorphine administration.
Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for Parkinson's disease (PD) and systemic administration of these cells has been tested in preclinical and clinical studies. However, no information on survival and actual capacity of MSCs to reach the brain has been provided. In this study, we evaluated homing of intraarterially infused rat MSCs (rMSCs) in the brain of rats bearing a 6‐hydroxydopamine (6‐OHDA)‐induced lesion of the nigrostriatal tract, to establish whether the toxin‐induced damage is sufficient to grant MSC passage across the blood‐brain barrier (BBB) or if a transient BBB disruption is necessary. The rMSC distribution in peripheral organs and the effects of cell infusion on neurodegenerative process and motor deficits were also investigated. rMSCs were infused 14 days after 6‐OHDA injection. A hyperosmolar solution of mannitol was used to transiently permeabilize the BBB. Behavioral impairment was assessed by adjusting step test and response to apomorphine. Animals were sacrificed 7 and 28 days after cell infusion. Our work shows that appreciable delivery of rMSCs to the brain of 6‐OHDA‐lesioned animals can be obtained only after mannitol pretreatment. A notable percentage of infused cells accumulated in peripheral organs. Infusion of rMSCs did not modify the progression of 6‐OHDA‐induced damage or the motor impairment at the stepping test, but induced progressive normalization of the pathological response (contralateral turning) to apomorphine administration. These findings suggest that many aspects should be further investigated before considering any translation of MSC systemic administration into the clinical setting for PD treatment.
Significance
This study demonstrates that mesenchymal stem cells infused through the carotid artery do not efficiently cross the blood‐brain barrier in rats with a Parkinson's disease‐like degeneration of nigrostriatal neurons, unless a permeabilizing agent (e.g., mannitol) is used. The infusion did not reduce the neuronal damage and associated motor impairment, but abolished the motor abnormalities these animals typically show when challenged with a dopaminergic agonist. Therefore, although arterially infused mesenchymal stem cells did not show neurorestorative effects in this study's Parkinson's disease model, they appeared to normalize the pathological responsiveness of striatal neurons to dopaminergic stimulation. This capability should be further explored in future studies.
Type II refractory celiac disease (RCD), as defined according to the amount of aberrant intraepithelial lymphocytes, is a condition characterized by severe malabsorption syndrome and poor prognosis, ...with no effective treatment. Based on the regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs), we investigated the feasibility, safety, and efficacy of serial infusions of autologous bone marrow-derived MSCs in a 51-year-old woman with type II RCD. Mesenchymal stem cells were isolated, expanded, and characterized following standard protocols. Monitoring of the patient's malabsorption indexes, mucosal architecture, and percentage of aberrant intraepithelial lymphocytes was scheduled for the time of enrollment, at each infusion, and after 6 months. Determination of mucosal expression of interleukin (IL)-15 and its receptor was also performed. Expansion of MSCs was feasible, and the patient underwent 4 systemic infusions of 2 × 10(6) MSCs/kg body weight 4 months apart, without adverse effects. During the treatment period, she experienced gradual and durable amelioration of her general condition, with normalization of stool frequency, body mass index, laboratory test results, and mucosal architecture. Remarkably, the expression of IL-15 and its receptor almost completely disappeared. Thus, treatment of RCD with serial MSC infusions seems promising, leading to recovery from the life-threatening condition while blocking the IL-15 pathogenic pathway.
Abstract We report the first case of renal antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis treated with autologous mesenchymal stromal cells (MSCs). A 73-year-old man was admitted to ...the hospital for malaise, weight loss, and oliguria. His serum creatinine level was 2.7 mg/dL but it rapidly increased to 7.8 mg/dL; urinalysis showed proteinuria and hematuria, and the ANCA to myeloperoxidase with a perinuclear pattern (pANCA) titer was high (132 IU/mL). Renal biopsy showed necrotizing crescentic glomerulonephritis. Standard immunosuppressive therapy (cyclophosphamide and corticosteroids) was ineffective. Rituximab therapy was started, but it was discontinued after the third dose to minimize the risk of systemic spread of a severe oral Candida infection and to prevent superinfections that were facilitated by leukopenia. The patient received autologous MSCs, 1.5 × 106 cells/kg body weight, intravenously. After 7 days, his serum creatinine level decreased to 2.2 mg/dL, pANCA titer decreased to 75 IU/mL, and urinalysis findings normalized. Eight months later, he received a second MSC infusion because his serum creatinine level increased. In 1 week, his creatinine level decreased to 1.9 mg/dL and his pANCA titer decreased to 14 IU/mL. Immunosuppressive therapy was subsequently withdrawn. At the last follow-up visit, 12 months after the second MSC infusion, the patient remained in clinical remission without any therapy. Infusion of MSCs induced expansion of the T-lymphocyte subset expressing a regulatory T-cell phenotype (CD4+ CD25+ Foxp3+ ) and a notable reduction in interferon-γ, interleukin 6, and tumor necrosis factor serum levels.
Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with beta-thalassemia given a bone marrow transplant. We investigated the ...relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for beta-thalassemia.
Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens.
All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again.
Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with beta-thalassemia given a cord blood transplant from a relative.
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