Production of donor-derived cytotoxic T lymphocytes with potent anti-leukemia activity for adoptive immunotherapy in high-risk pediatric patients given haploidentical hematopoietic stem cell transplantation.

BACKGROUND AIMS: Somatic cell therapy based on the infusion of donor-derived cytotoxic T lymphocytes (CTL) able to recognize patients' leukemia blasts (LB) is a promising approach to control leukemia relapse after allogeneic HSCT. The success of this approach strongly depends on the ex vivo generation of high-quality donor-derived anti-leukemia CTL in compliance with Good Manufacturing Practices (GMP). We previously described a procedure for generating large numbers of donor-derived anti-leukemia CTL through stimulation of CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with apoptotic LB in the presence of interleukin (IL)-12, IL-7 and IL-15. Here we report that the use of IFN-DC and the addition of IFNα2b during the priming phase significantly improve the generation of an efficient anti-leukemia T cells response in vitro. METHODS: Using this approach, 20 high-risk pediatric patients given haploidentical HSCT for high-risk acute leukemia were enrolled and 51 batches of advanced therapy medical products (ATMP), anti-leukemia CTL, were produced. RESULTS: Quality controls demonstrated that all batches were sterile, free of mycoplasma and conformed to acceptable endotoxin levels. Genotype analysis confirmed the molecular identity of the ATMP based on the starting biological material used for their production. The majority of ATMP were CD3+/CD8+ cells, with a memory/terminal activated phenotype, including T-central memory populations. ATMP were viable after thawing, and most ATMP batches displayed efficient capacity to lyse patients' LB and to secrete interferon-γ and tumor necrosis factor-α. CONCLUSIONS: These results demonstrated that our protocol is highly reproducible and allows the generation of large numbers of immunologically safe and functional anti-leukemia CTL with a high level of standardization.

Circulating Mesenchymal Stromal Cells in Patients with Infantile Hemangioma: Evaluation of Their Functional Capacity and Gene Expression Profile.

We previously published that in patients with infantile hemangioma (IH) at the onset (T0) colony forming unit-fibroblasts (CFU-Fs) are present in in vitro cultures from PB. Herein, we characterize these CFU-Fs and investigate their potential role in IH pathogenesis, before and after propranolol therapy. The CFU-F phenotype (by flow cytometry), their differentiation capacity and ability to support angiogenesis (by in vitro cultures) and their gene expression (by RT-PCR) were evaluated. We found that CFU-Fs are actual circulating MSCs (cMSCs). In patients at T0, cMSCs had reduced adipogenic potential, supported the formation of tube-like structures in vitro and showed either inflammatory (IL1ß and ESM1) or angiogenic (F3) gene expression higher than that of cMSCs from CTRLs. In patients receiving one-year propranolol therapy, the cMSC differentiation in adipocytes improved, while their support in in vitro tube-like formation was lost; no difference was found between patient and CTRL cMSC gene expressions. In conclusion, in patients with IH at T0 the cMSC reduced adipogenic potential, their support in angiogenic activity and the inflammatory/angiogenic gene expression may fuel the tumor growth. One-year propranolol therapy modifies this picture, suggesting cMSCs as one of the drug targets.

New therapeutic approaches in pediatric diseases: Mesenchymal stromal cell and mesenchymal stromal cell-derived extracellular vesicles as new drugs.

Mesenchymal Stromal Cell (MSC) clinical applications have been widely reported and their therapeutic potential has been documented in several diseases. MSCs can be isolated from several human tissues and easily expanded in vitro, they are able to differentiate in a variety of cell lineages, and they are known to interact with most immunological cells, showing immunosuppressive and tissue repair properties. Their therapeutic efficacy is closely associated with the release of bioactive molecules, namely Extracellular Vesicles (EVs), effective as their parental cells. EVs isolated from MSCs act by fusing with target cell membrane and releasing their content, showing a great potential for the treatment of injured tissues and organs, and for the modulation of the host immune system. EV-based therapies provide, as major advantages, the possibility to cross the epithelium and blood barrier and their activity is not influenced by the surrounding environment. In the present review, we deal with pre-clinical reports and clinical trials to provide data in support of MSC and EV clinical efficacy with particular focus on neonatal and pediatric diseases. Considering pre-clinical and clinical data so far available, it is likely that cell-based and cell-free therapies could become an important therapeutic approach for the treatment of several pediatric diseases.

Mesenchymal Stromal Cell on Liver Decellularised Extracellular Matrix for Tissue Engineering.

BACKGROUND: In end-stage chronic liver disease, transplantation represents the only curative option. However, the shortage of donors results in the death of many patients. To overcome this gap, it is mandatory to develop new therapeutic options. In the present study, we decellularised pig livers and reseeded them with allogeneic porcine mesenchymal stromal cells (pMSCs) to understand whether extracellular matrix (ECM) can influence and/or promote differentiation into hepatocyte-like cells (HLCs). METHODS: After decellularisation with SDS, the integrity of ECM-scaffolds was examined by histological staining, immunofluorescence and scanning electron microscope. DNA quantification was used to assess decellularisation. pMSCs were plated on scaffolds by static seeding and maintained in in vitro culture for 21 days. At 3, 7, 14 and 21 days, seeded ECM scaffolds were evaluated for cellular adhesion and growth. Moreover, the expression of specific hepatic genes was performed by RT-PCR. RESULTS: The applied decellularisation/recellularisation protocol was effective. The number of seeded pMSCs increased over the culture time points. Gene expression analysis of seeded pMSCs displayed a weak induction due to ECM towards HLCs. CONCLUSIONS: These results suggest that ECM may address pMSCs to differentiate in hepatocyte-like cells. However, only contact with liver-ECM is not enough to induce complete differentiation.

Proliferation Pattern of Pediatric Tumor-Derived Mesenchymal Stromal Cells and Role in Cancer Dormancy: A Perspective of Study for Surgical Strategy.

The explanation for cancer recurrence still remains to be fully elucidated. Moreover, tumor dormancy, which is a process whereby cells enter reversible G0 cell cycle arrest, appears to be a critical step in this phenomenon. We evaluated the cell cycle proliferation pattern in pediatric tumor-derived mesenchymal stromal cells (MSCs), in order to provide a better understanding of the complex mechanisms underlying cancer dormancy. Specimens were obtained from 14 pediatric patients diagnosed with solid tumors and submitted to surgery. Morphology, phenotype, differentiation, immunological capacity, and proliferative growth of tumor MSCs were studied. Flow cytometric analysis was performed to evaluate the cell percentage of each cell cycle phase. Healthy donor bone marrow-derived mesenchymal stromal cells (BM-MSCs) were employed as controls. It was noted that the DNA profile of proliferating BM-MSC was different from that of tumor MSCs. All BM-MSCs expressed the typical DNA profile of proliferating cells, while in all tumor MSC samples, ≥70% of the cells were detected in the G0/G1 phase. In particular, seven tumor MSC samples displayed intermediate cell cycle behavior, and the other seven tumor MSC samples exhibited a slow cell cycle. The increased number of tumor MSCs in the G0-G1 phase compared with BM-MSCs supports a role for quiescent MSCs in tumor dormancy regulation. Understanding the mechanisms that promote dormant cell cycle arrest is essential in identifying predictive markers of recurrence and to promote a dedicated surgical planning.

MKRN3 and KISS1R mutations in precocious and early puberty.

BACKGROUND: Pubertal timing is known to be influenced by interactions among various genetic, nutritional, environmental and socio-economic factors, although the ultimate mechanisms underlying the increase in pulsatile GnRH secretion at puberty have yet to be fully elucidated. The aim of our research was to verify the role of KISSR1 (previously named GPR54) and MKRN3 genes on pubertal timing. METHODS: We analyzed the DNA sequence of these genes in 13 girls affected by central precocious puberty (CPP) who showed onset of puberty before 8 years of age, and in 6 girls affected by early puberty (EP) between 8 and 10 years of age. RESULTS: Direct sequencing of the KISS1R (GPR54) gene revealed two SNPs. One SNP is a missense variant (rs 350,132) that has been previously reported in connection to CPP in Korean girls. The other variant that we found in the GPR54 gene (rs764046557) was a missense SNP located in exon 5 at position 209 of the aminoacid. We identified this variant in only one CPP patient. Automatic sequencing of MKRN3 in all patients revealed three variants in eight subjects. In 6 out of 19 (31.5%) patients (3/13 CPP patients and 3/6 EP patients) we found the synonymous variant c.663C > T (rs2239669). Another synonymous variant (rs140467331) was found in one of our CPP patients, as well as one missense variant (rs760981395) in another CPP patient. CONCLUSION: In conclusion, we identified sequence variations of the KISS1R and MKRN3 genes, two of the most frequent genetic causes of ICPP. Our results suggest that these variants might be inducible factors in the pathogenesis of CPP.

Allogeneic mesenchymal stromal cells: Novel therapeutic option for mutated FLNA-associated respiratory failure in the pediatric setting.

BACKGROUND: Mesenchymal stromal cell (MSC)-mediated therapeutic effects have been observed in the treatment of lung diseases. For the first time, this treatment was used as rescue therapy in a pediatric patient with a life-threatening respiratory syndrome associated with the filamin A (FLNA) gene mutation. METHODS: A child with a new pathogenic variant of the FLNA gene c.7391_7403del (p.Val2464AlafsTer5), at the age of 18 months, due to serious and irreversible chronic respiratory failure, was treated with repeated intravenous infusions of allogeneic bone marrow (BM)-MSCs. The child's respiratory condition was monitored. Immunologic studies before each MSC treatment were performed. RESULTS: No acute adverse events related to the MSC infusions were observed. After the second infusion, the child's respiratory condition progressively improved, with reduced necessity for mechanical ventilation support. A thorax computed tomography (CT) scan showed bilateral recovery of the basal parenchyma, anatomical-functional alignment and aerial penetration improvement. After the first MSC administration, an increase in Th17 and FoxP3+ T percentages in the peripheral blood was observed. After the second MSC infusion, a significant rise in the Treg/Th17 ratio was noted, as well as an increased percentage of CD20+ /CD19+ B lymphocytes and augmented PHA-induced proliferation. DISCUSSION: MSC infusions are a promising therapeutic modality for patients in respiratory failure, as observed in this pediatric patient with an FLNA mutation. MSCs may have an immunomodulatory effect and thus mitigate lung injury; although in this case, MSC antimicrobial effects may have synergistically impacted the clinical improvements. Further investigations are planned to establish the safety and efficacy of this treatment option for interstitial lung diseases in children.

GH-IGF-1 Axis in Children with Cystic Fibrosis.

OBJECTIVE: To verify whether growth hormone receptor (GHR) gene expression plays a role in growth of children with cystic fibrosis (CF), as a consequence of the chronic inflammatory condition and malnutrition. DESIGN: We enrolled 49 prepubertal patients (24 males and 25 females) affected by CF in a stable clinical condition, 19 of whom had been diagnosed through newborn screening and 30 following presentation of symptoms. Patients had no significant comorbidity affecting growth or cystic fibrosis transmembrane conductance regulator (CFTR)-related diabetes requiring insulin therapy. Blood was collected during two follow-up visits to measure insulin-like growth factor (IGF-I), growth hormone-binding protein (GHBP), and GHR gene expression. Recruited as a control group were 52 healthy children, sex- and age-matched, were recruited as a control group. METHODS: We compared body mass index (BMI), height, weight, IGF-I, GHBP, and GHR gene expression values (evaluated by Chemiluminescent Immunometric assay; ELISA and real-time PCR, respectively) in CF patients diagnosed through newborn screening (NBS) or by symptoms (late diagnosis [LD]) and in healthy controls. RESULTS: BMI increased significantly in patients between the time of diagnosis and check-up (P<0.001), particularly in the LD group; median value was lower at diagnosis and significantly higher (P<0.001) at follow-up visits compared to controls. At initial evaluation, higher levels of IGF-I (not statistically significant) were found in both the NBS group and the LD group compared to the control group. At the second evaluation, significantly higher levels of IGF-I (P=0.003) were found in both the NBS and LD groups compared to controls; GHR mRNA expression had significantly increased (P=0.013) in LD patients compared with the first evaluation and was significantly higher in the NBS and LD groups than in controls. GHBP values had significantly increased (P=0.047) in the NBS group after one year of therapy compared to first visit levels and were significantly higher (P<0,0001) in the NBS and LD groups compared to controls. CONCLUSION: In our LD patients during childhood, we observed good auxological values and a GH/IGF-I axis function within normal range for the factor evaluated. However, earlier diagnosis through NBS might further minimize and prevent growth retardation, by reducing the duration of symptoms before treatment.

Generation of donor-derived Wilms tumor antigen 1-specific cytotoxic T lymphocytes with potent anti-leukemia activity for somatic cell therapy in children given haploidentical stem cell transplantation: a feasibility pre-clinical study.

BACKGROUND: The Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors. AIM: The aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT). METHODS: For WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way. RESULTS: WT1-specific CTLs, displaying high-level cytotoxicity against patients' leukemia blasts and negligible activity against patients' non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity. DISCUSSION: Results of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.

Pre- and post-transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia.

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 × 10-3 and ≥1 × 10-3 , respectively, versus 73% in MRD-negative patients (P < 0·001). This effect was maintained in different disease remissions, but low-level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post-HSCT1 and post-HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre-emptive immuno-therapy.

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy.

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation.

Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse.

Use of alternative donors/sources of hematopoietic stem cells (HSC), such as cord blood (CB) or HLA-haploidentical (Haplo)-related donors, is associated with a significant delay in immune reconstitution after transplantation. Long-term T-cell immune reconstitution largely relies on the generation of new T cells in the recipient thymus, which can be evaluated through signal joint (sj) and beta T-cell-Receptor Excision Circles (TREC) quantification. We studied two groups of 33 and 24 children receiving, respectively, HSC Transplantation (HSCT) from an HLA-haploidentical family donor or an unrelated CB donor, for both malignant (46) and non-malignant disorders (11). Relative and absolute sj and beta-TREC values indicated comparable thymic function reconstitution at 3 and 6 months after the allograft in both groups. Compared to children with non-malignant disorders, those with hematological malignancies had significantly lower pre-transplantation TREC counts. Patients who relapsed after HSCT had a significantly less efficient thymic function both before and 6 months after HSCT with especially low beta-TREC values, this finding suggesting an impact of early intra-thymic T-cell differentiation on the occurrence of leukemia relapse.