Potential application of mesenchymal stromal cells as a new therapeutic approach in acute respiratory distress syndrome and pulmonary fibrosis.

While the COVID-19 outbreak and its complications are still under investigation, post-inflammatory pulmonary fibrosis (PF) has already been described as a long-term sequela of acute respiratory distress syndrome (ARDS) secondary to SARS-CoV2 infection. However, therapeutical strategies for patients with ARDS and PF are still limited and do not significantly extend lifespan. So far, lung transplantation remains the only definitive treatment for end-stage PF. Over the last years, numerous preclinical and clinical studies have shown that allogeneic mesenchymal stromal cells (MSCs) might represent a promising therapeutical approach in several lung disorders, and their potential for ARDS treatment and PF prevention has been investigated during the COVID-19 pandemic. From April 2020 to April 2022, we treated six adult patients with moderate COVID-19-related ARDS in a late proliferative stage with up to two same-dose infusions of third-party allogeneic bone marrow-derived MSCs (BM-MSCs), administered intravenously 15 days apart. No major adverse events were registered. Four patients completed the treatment and reached ICU discharge, while two received only one dose of MSCs due to multiorgan dysfunction syndrome (MODS) and subsequent death. All four survivors showed improved gas exchanges (PaO2/FiO2 ratio > 200), contrary to the others. Furthermore, LDH trends after MSCs significantly differed between survivors and the deceased. Although further investigations and shared protocols are still needed, the safety of MSC therapy has been recurrently shown, and its potential in treating ARDS and preventing PF might represent a new therapeutic strategy.

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.

Allogeneic Mesenchymal Stromal Cells as a Global Pediatric Prospective Approach in the Treatment of Respiratory Failure Associated with Surfactant Protein C Dysfunction.

Mesenchymal stromal cells (MSCs) have been proposed as a new therapeutic strategy to treat congenital and acquired respiratory system diseases. We describe a case report of an 18-month-old male patient with progressive chronic respiratory failure, associated with mutations of the surfactant protein C gene (SFTPC) due to c.289G > T variant p.Gly97Ser (rs927644577) and c.176A > G variant (p.His59Arg), submitted to repeated intravenous infusions of allogeneic bone marrow (BM) MSCs. The clinical condition of the patient was monitored. Immunologic studies before and during MSC treatment were performed. No adverse events related to the MSC infusions were recorded. Throughout the MSC treatment period, the patient showed a growth recovery. Starting the second infusion, the patient experienced an improvement in his respiratory condition, with progressive adaptation to mechanical ventilation. After the third infusion, five hours/die of spontaneous breathing was shown, and after infusion IV, spontaneous ventilation for 24/24 h was recorded. A gradual decrease of lymphocytes and cell subpopulations was observed. No variations in the in vitro T cell response to PHA were determined by MSC treatment as well as for the in vitro B cell response. A decrease in IFN-γ, TNF-α, and IL-10 levels was also detected. Even though we cannot exclude an improvement of pulmonary function due to the physiological maturation, the well-known action of MSCs in the repair of lung tissue, together with the sequence of events observed in our patient, may support the therapeutic role of MSCs in this clinical condition. However, further investigations are necessary to confirm the result and long-term follow-up will be mandatory to confirm the benefits on the pulmonary condition.

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.

Human Umbilical Cord Mesenchymal Stem Cell-Based in vitro Model for Neurotoxicity Testing.

Neurotoxicity (NT) testing for regulatory purposes is based on in vivo animal testing. There is general consensus, however, about the need for the development of alternative methodologies to allow researchers to more rapidly and cost effectively screen large numbers of chemicals for their potential to cause NT, or to investigate their mode of action. In vitro assays are considered an important source of information for making regulatory decisions, and human cell-based systems are recommended as one of the most relevant models in toxicity testing, to reduce uncertainty in the extrapolation of results from animal-based models. Human neuronal models range from various neuroblastoma cell lines to stem cell-derived systems, including those derived from mesenchymal stem/stromal cells (hMSC). hMSCs exhibit numerous advantages, including the fact that they can be obtained in high yield from healthy human adult tissues, can be cultured with a minimal laboratory setup and without genetic manipulations, are able of continuous and repeated self-renewal, are nontumorigenic, and can form large populations of stably differentiated cells representative of different tissues, including neuronal cells. hMSCs derived from human umbilical cord (hUC) in particular possess several prominent advantages, including a painless, non-invasive, and ethically acceptable collection procedure, simple and convenient preparation, and high proliferation capacity. In addition, hMSCs can be efficiently differentiated into neuron-like cells (hNLCs), which can then be used for the assessment of neuronal toxicity of potential neurotoxic compounds in humans. Here, we describe a step-by-step procedure to use hMSCs from the umbilical cord for in vitro neurotoxicity testing. First, we describe how to isolate, amplify, and store hMSCs derived from the umbilical cord. We then outline the steps to transdifferentiate these cells into hNLCs, and then use the hNLCs for neurotoxicity testing by employing multiple common cytotoxicity assays after treatment with test compounds. The approach follows the most updated guidance on using human cell-based systems. These protocols will allow investigators to implement an alternative system for obtaining primary NLCs of human origin, and support advancement in neurotoxicity research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and maintenance of human mesenchymal stem/stromal cells (hMSCs) obtained from the umbilical cord lining membrane Basic Protocol 2: Transdifferentiation of hMSCs into neuron-like cells (hNLCs) and basic neurotoxicity assessment.

Bone Marrow Microenvironment in Light-Chain Amyloidosis: In Vitro Expansion and Characterization of Mesenchymal Stromal Cells.

Immunoglobulin light-chain amyloidosis (AL) is caused by misfolded light chains produced by a small B cell clone. Mesenchymal stromal cells (MSCs) have been reported to affect plasma cell behavior. We aimed to characterize bone marrow (BM)-MSCs from AL patients, considering functional aspects, such as proliferation, differentiation, and immunomodulatory capacities. MSCs were in vitro expanded from the BM of 57 AL patients and 14 healthy donors (HDs). MSC surface markers were analyzed by flow cytometry, osteogenic and adipogenic differentiation capacities were in vitro evaluated, and co-culture experiments were performed in order to investigate MSC immunomodulatory properties towards the ALMC-2 cell line and HD peripheral blood mononuclear cells (PBMCs). AL-MSCs were comparable to HD-MSCs for morphology, immune-phenotype, and differentiation capacities. AL-MSCs showed a reduced proliferation rate, entering senescence at earlier passages than HD-MSCs. The AL-MSC modulatory effect on the plasma-cell line or circulating plasma cells was comparable to that of HD-MSCs. To our knowledge, this is the first study providing a comprehensive characterization of AL-MSCs. It remains to be defined if the observed abnormalities are the consequence of or are involved in the disease pathogenesis. BM microenvironment components in AL may represent the targets for the prevention/treatment of the disease in personalized therapies.

Human mesenchymal stromal cells do not express ACE2 and TMPRSS2 and are not permissive to SARS-CoV-2 infection.

Anti-inflammatory and immune-modulatory therapies have been proposed for the treatment of COVID-19 and its most serious complications. Among others, the use of mesenchymal stromal cells (MSCs) is under investigation given their well-documented anti-inflammatory and immunomodulatory properties. However, some critical issues regarding the possibility that MSCs could be infected by the virus have been raised. Angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) are the main host cell factors for the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), entry, but so far it is unclear if human MSCs do or do not express these two proteins. To elucidate these important aspects, we evaluated if human MSCs from both fetal and adult tissues constitutively express ACE2 and TMPRSS2 and, most importantly, if they can be infected by SARS-CoV-2. We evaluated human MSCs derived from amnios, cord blood, cord tissue, adipose tissue, and bone marrow. ACE2 and TMPRSS2 were expressed by the SARS-CoV-2-permissive human pulmonary Calu-3 cell line but not by all the MSCs tested. MSCs were then exposed to SARS-CoV-2 wild strain without evidence of cytopathic effect. Moreover, we also excluded that the MSCs could be infected without showing lytic effects since their conditioned medium after SARS-CoV-2 exposure did not contain viral particles. Our data, demonstrating that MSCs derived from different human tissues are not permissive to SARS-CoV-2 infection, support the safety of MSCs as potential therapy for COVID-19.

Clinical Applications of Mesenchymal Stem/Stromal Cell Derived Extracellular Vesicles: Therapeutic Potential of an Acellular Product.

In the last decade, the secreting activity of mesenchymal stem/stromal cells (MSCs) has been widely investigated, due to its possible therapeutic role. In fact, MSCs release extracellular vesicles (EVs) containing relevant biomolecules such as mRNAs, microRNAs, bioactive lipids, and signaling receptors, able to restore physiological conditions where regenerative or anti-inflammatory actions are needed. An actual advantage would come from the therapeutic use of EVs with respect to MSCs, avoiding the possible immune rejection, the lung entrapment, improving the safety, and allowing the crossing of biological barriers. A number of concerns still have to be solved regarding the mechanisms determining the beneficial effect of MSC-EVs, the possible alteration of their properties as a consequence of the isolation/purification methods, and/or the best approach for a large-scale production for clinical use. Most of the preclinical studies have been successful, reporting for MSC-EVs a protecting role in acute kidney injury following ischemia reperfusion, a potent anti-inflammatory and anti-fibrotic effects by reducing disease associated inflammation and fibrosis in lung and liver, and the modulation of both innate and adaptive immune responses in graft versus host disease (GVHD) as well as autoimmune diseases. However, the translation of MSC-EVs to the clinical stage is still at the initial phase. Herein, we discuss the therapeutic potential of an acellular product such as MSC derived EVs (MSC-EVs) in acute and chronic pathologies.

Tissue Engineered Esophageal Patch by Mesenchymal Stromal Cells: Optimization of Electrospun Patch Engineering.

Aim of work was to locate a simple, reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited, namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size, pore orientation, porosity, and pore distribution were produced using two different techniques, such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity, seeding efficiency, and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method, as these conditions successfully improved the cellularization of polymeric patches. Furthermore, the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results, it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover, the protocol turned out to be simple, repeatable, and reproducible.

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.

In vitro toxicity screening of magnetite nanoparticles by applying mesenchymal stem cells derived from human umbilical cord lining.

Despite the growing interest in nanoparticles (NPs), their toxicity has not yet been defined and the development of new strategies and predictive models are required. Human stem cells (SCs) offer a promising and innovative cell-based model. Among SCs, mesenchymal SCs (MSCs) derived from cord lining membrane (CL) may represent a new species-specific tool for establishing efficient platforms for primary screening and toxicity/safety testing of NPs. Superparamagnetic iron oxide NPs, including magnetite (Fe3 O4 NPs), have aroused great public health and scientific concerns despite their extensive uses. In this study, CL-MSCs were characterized and applied for in vitro toxicity screening of Fe3 O4 NPs. Cytotoxicity, internalization/uptake, differentiation and proliferative capacity were evaluated after exposure to different Fe3 O4 NP concentrations. Data were compared with those obtained from bone marrow (BM)-MSCs. We observed, at early passages (P3), that: (1) cytotoxicity occurred at 10 µg/mL in CL-MSCs and 100 µg/mL in BM-MSCs (no differences in toxicity, between CL- and BM-MSCs, were observed at higher dosage, 100-300 µg/mL); (2) cell density decrease and monolayer features loss were affected at ≥50 µg/mL in CL-MSCs only; and (3) NP uptake was concentration-dependent in both MSCs. After 100 µg/mL Fe3 O4 NP exposures, the capacity of proliferation was decreased (P5-P9) in CL-MSCs without morphology alteration. Moreover, a progressive decrease of intracellular Fe3 O4 NPs was observed over culture time. Antigen surface expression and multilineage differentiation were not influenced. These findings suggest that CL-MSCs could be used as a reliable cell-based model for Fe3 O4 NP toxicity screening evaluation and support the use of this approach for improving the confidence degree on the safety of NPs to predict health outcomes.

The spleen of patients with myelofibrosis harbors defective mesenchymal stromal cells.

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.

Neutrophil CD64 expression: a reliable diagnostic marker of infection in advanced cancer patients?

Infection and sepsis are major health problems in cancer patients. There is a need for the identification and validation of biomarkers to improve their early diagnosis and treatment. Emerging evidence showed that neutrophil CD64 is a highly sensitive and specific marker for systemic infection and sepsis in critically ill patients with various diseases but data on patients bearing solid tumors are still lacking. Using a dedicated flow cytometric assay we evaluated neutrophil CD64 expression in patients with advanced cancer without active infections to verify if it could be utilized as a reliable biomarker of early infections also in oncologic patients.

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.

Genomic alterations in human umbilical cord-derived mesenchymal stromal cells call for stringent quality control before any possible therapeutic approach.

BACKGROUND AIMS: The umbilical cord (UC) is a promising source of mesenchymal stromal cells (MSCs). UC-MSCs display very similar in vitro characteristics to bone marrow-MSCs and could represent a valuable alternative for cell-based therapies. However, it is still unclear whether UC-MSCs are prone or not to the acquisition of genomic imbalances during in vitro expansion. METHODS: With the use of array-comparative genomic hybridization, we compared copy number variations of early (P2-P3) and late (>P5) passages of in vitro-expanded UC-MSCs. RESULTS: In two of 11 long-term UC-MSCs cultures, we observed the appearance of clones carrying genomic imbalances, which generated genetic mosaicism at intermediate passages. Although still able to reach the senescence phase, the cells carrying the genomic imbalance acquired a proliferative advantage, as demonstrated by the increase in frequency during long-term culture. CONCLUSIONS: Altogether, our results suggest that UC-MSC-based clinical protocols should be designed with caution; their clinical use should be preceded by array-comparative genomic hybridization screening for the acquisition of genomic imbalances during in vitro expansion.

Kinetics of effector functions and phenotype of virus-specific and γδ T lymphocytes in primary human cytomegalovirus infection during pregnancy.

The T-cell response to human cytomegalovirus (HCMV) primary infection was analyzed in 27 pregnant women during the first year after primary HCMV infection. Pregnant women with remote HCMV infection were enrolled as controls. Interferon-γ-producing T cells were readily detected at levels comparable (CD4(+)) or higher (CD8(+)) than controls, whereas the CD4(+) and CD8(+) lymphoproliferative response as well as IL-2 production was significantly reduced with respect to controls for at least 9 months after infection. In addition, CD45RA re-expression as well as cytotoxic T lymphocyte activity and perforin expression were the major components of the adaptive CD4(+) and CD8(+) T-cell immune response, while Vδ2(-) γδ T-cell expansion in response to HCMV infection followed kinetics similar to that of CD8(+) T cells. Reduced CD45RA re-expression directly correlated with HCMV transmission to the fetus, thus providing an important prognostic parameter.

B lymphocyte reconstitution after hematopoietic stem cell transplantation: functional immaturity and slow recovery of memory CD27+ B cells.

OBJECTIVE: Functional recovery of B lymphocytes after hematopoietic stem cell transplantation (HSCT) can take up to 2 years. HSCT recipients may obtain protective titers of pathogen-specific antibody through vaccination, but optimal timing of reimmunization remains to be defined. PATIENTS AND METHODS: In this study, we evaluated the reconstitution of B-cell number and activity in 139 children given HSCT, by B-cell subset phenotyping and in vitro immunoglobulin (Ig) production. RESULTS: Patients were longitudinally studied at 3, 6, 12, and 18 to 24 months after transplantation. At all time points, recipients displayed a significantly higher percentage of naive (IgD+CD27-) B cells and showed significantly lower production of stimulated in vitro Ig as compared to healthy controls. Moreover, during follow-up, we observed an increase in the proportion of patients who had CD27+ B subsets and who were able to mount in vitro Ig production greater than the 5th percentile. CONCLUSION: Similar to what has been described in adults, most children lack memory B cells and produce low amounts of Ig. However, the number of B cells, as well as their function, gradually recovered over time and the spread of data we observed suggests that the reimmunization schedule should be individualized for each patient. It remains to be defined in a prospective clinical study the time point at which a patient should start reimmunization. A reasonable hypothesis to be explored is the time point at which a percentage of memory B cells greater than the 5th percentile of normal controls is reached.