Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Third-party virus-specific T cells for the treatment of double-stranded DNA viral reactivation and posttransplant lymphoproliferative disease after solid organ transplant.

Reactivation or primary infection with double-stranded DNA viruses is common in recipients of solid organ transplants (SOTs) and is associated with significant morbidity and mortality. Treatment with conventional antiviral medications is limited by toxicities, resistance, and a lack of effective options for adenovirus (ADV) and BK polyomavirus (BKPyV). Virus-specific T cells (VSTs) have been shown to be an effective treatment for infections with ADV, BKPyV, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Most of these studies have been conducted in stem cell recipients, and no large studies have been published in the SOT population to date. In this study, we report on the outcome of quadrivalent third-party VST infusions in 98 recipients of SOTs in the context of an open-label phase 2 trial. The 98 patients received a total of 181 infusions, with a median of 2 infusions per patient. The overall response rate was 45% for BKPyV, 65% for cytomegalovirus, 68% for ADV, and 61% for Epstein-Barr virus. Twenty percent of patients with posttransplant lymphoproliferative disorder had a complete response and 40% of patients had a partial response. All the VST infusions were well tolerated. We conclude that VSTs are safe and effective in the treatment of viral infections in SOT recipients.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

The Natural History of BK Polyomavirus and the Host Immune Response After Stem Cell Transplantation.

BACKGROUND: BK polyomavirus (BKPyV) is associated with symptomatic hemorrhagic cystitis after hematopoietic cell transplantation (HCT). Little is known about the host immune response, effectiveness of antiviral treatment, or impact of asymptomatic replication on long-term kidney function. METHODS: In children and young adults undergoing allogeneic HCT, we quantified BKPyV viruria and viremia (pre-HCT and at Months 1-4, 8, 12, and 24 post-HCT) and tested associations of peak viremia ≥10 000 or viruria ≥109 copies/mL with estimated kidney function (glomerular filtration rate, eGFR) and overall survival at 2 years posttransplant. We examined the factors associated with viral clearance by Month 4, including BKPyV-specific T cells by enzyme-linked immune absorbent spot at Month 3 and cidofovir use. RESULTS: We prospectively enrolled 193 participants (median age 10 years) and found that 18% had viremia ≥10 000 copies/mL and 45% had viruria ≥109 copies/mL in the first 3 months post-HCT. Among the 147 participants without cystitis (asymptomatic), 58 (40%) had any viremia. In the entire cohort and asymptomatic subset, having viremia ≥10 000 copies/mL was associated with a lower creatinine/cystatin C eGFR at 2 years post-HCT. Viremia ≥10 000 copies/mL was associated with a higher risk of death (adjusted hazard ratio, 2.2; 95% confidence interval, 1.1-4.2). Clearing viremia was associated with detectable BKPyV-specific T cells and having viremia <10 000 copies/mL, but not cidofovir exposure. CONCLUSIONS: Screening for BKPyV viremia after HCT identifies asymptomatic patients at risk for kidney disease and reduced survival. These data suggest potential changes to clinical practice, including prospective monitoring for BKPyV viremia to test virus-specific T cells to prevent or treat BKPyV replication.

CD38brightCD8+ T Cells Associated with the Development of Acute GVHD Are Activated, Proliferating, and Cytotoxic Trafficking Cells.

We have previously reported that a peripheral blood absolute CD38brightCD8+ effector memory T cell (TEM) population expansion of >35 cells/µL predicts the development of acute graft-versus-host disease (GVHD). We hypothesized that these T cells are activated, proliferating, and cytotoxic trafficking cells that are not a response to viral reactivation and may be involved in acute GVHD. We characterized peripheral blood T cell populations at the time of maximum CD38brightCD8+ TEM expansion in patients from our originally reported pediatric allogeneic hematopoietic cell transplantation recipient cohort. Samples were incubated with fluorochrome-conjugated antibodies directed against CD3, CD8, CD38, HLA-DR (T cell activation), Ki-67 (T cell proliferation), granzyme B (marker of cytotoxic T cells), CLA (skin trafficking), CCR5 (visceral trafficking), and CXCR6 (liver trafficking). We also incubated samples with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide pools and measured IFN-γ production by flow cytometry and performed EBV and CMV tetramer staining. Higher median proportions of cell expression of HLA-DR, Ki-67, granzyme B, CLA, CCR5, and CXCR6 were observed for CD38brightCD8+ T cells compared with CD38nonbrightCD8+ T cells in patients with acute GVHD (P < .05) but not in patients without acute GVHD (P not significant). No IFN-γ production was observed after incubation with CMV and EBV peptide pools. EBV-specific tetramer populations of 6.85% and 3.17% were detected in 2 patients with acute GVHD, whereas a CMV-specific tetramer population of 3.77% was detected in 1 patient with acute GVHD. No EBV- or CMV-specific tetramer populations were detected in any patient without acute GVHD. We conclude that CD38brightCD8+ T cells associated with the development of acute GVHD are activated, proliferating, and cytotoxic trafficking cells that do not appear to respond to CMV or EBV reactivation. Further studies are needed to determine whether these cells are directly involved in acute GVHD pathogenesis.

EBV-directed viral-specific T-lymphocyte therapy for the treatment of EBV-driven lymphoma in two patients with primary immunodeficiency and DNA repair defects.

Children with ataxia telangiectasia (AT), a primary immunodeficiency caused by mutations in ATM, which is critical for repairing DNA defects, are at risk for the development of hematologic malignancy, frequently driven by infection with Epstein-Barr virus (EBV). Conventional chemotherapy is poorly tolerated by patients with AT, with excessive toxicity even when doses are reduced. Here, we report on two patients with AT and EBV-positive neoplasms who were treated with EBV-targeted viral-specific T cells (VST). One patient had a prolonged complete response to VSTs while the other had a partial response. Therapy was well tolerated without infusion toxicity or graft-versus-host disease.

Pulmonary macrophage transplantation therapy.

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor-ß-deficient (Csf2rb(-/-)) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms.

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.

Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

A toxicology study of Csf2ra complementation and pulmonary macrophage transplantation therapy of hereditary PAP in mice.

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, and established a no adverse effect level and 10-fold safety margin.

Third-Party and Patient-Specific Donor-Derived Virus-Specific T Cells Demonstrate Similar Efficacy and Safety for Management of Viral Infections after Hematopoietic Stem Cell Transplantation in Children and Young Adults.

Infections with double-stranded DNA viruses are a common complication after hematopoietic stem cell transplantation (HSCT) and cause significant morbidity and mortality in the post-transplantation period. Both donor-derived (DD) and third-party (TP) virus-specific T cells (VSTs) have shown efficacy and safety in viral management following HSCT in children and young adults. Owing to a greater degree of HLA matching between the recipient and stem cell donor, DD VSTs potentially persist longer in circulation compared to TP VSTs, because they are collected from a well-matched donor. However, TP VSTs are more easily accessible, particularly for smaller transplantation centers that do not have VST manufacturing capabilities, and more economical than creating a customized product for each transplant recipient. We conducted the present study to compare clinical efficacy and safety outcomes for DD VSTs and TP VSTs in a large cohort of pediatric and young adult HSCT recipients and to determine whether DD VSTs are associated with improved outcomes owing to potentially longer persistence in the recipient's circulation. This retrospective cohort study included 145 patients who received VSTs at Cincinnati Children's Hospital Medical Center (CCHMC) between 2017 and 2021 for the treatment of adenovirus, BK virus, cytomegalovirus, and/or Epstein-Barr virus. Viruses were detected using quantitative polymerase chain reaction. Patients received VSTs on a DD (NCT02048332) or TP (NCT02532452) protocol, and VST products for both protocols were manufactured in an identical fashion. The primary study outcome was clinical response to VSTs, evaluated 4 weeks after VST administration, defined as decrease in viral load to under the inclusion thresholds, or resolution of symptoms of invasive viral infection, without the need for additional conventional antiviral medication following VST administration. Secondary outcomes included graft-versus-host-disease, transplant-associated thrombotic microangiopathy, renal function, hospital length of stay, and overall survival at 30 days and 100 days after VST administration and 1 year after HSCT. Statistical analysis was performed using the Fisher exact test or chi-square test. An unpaired t test was used to compare continuous variables. The study group comprised 77 patients in the DD cohort and 68 patients in the TP cohort. Eighteen patients in the TP cohort underwent HSCT at CCHMC, and the other 50 underwent HSCT at other institutions and presented to CCHMC solely for VST administration. There was no statistically significant difference in clinical response rates between DD and TP cohorts (65.6% versus 62.7%; odds ratio [OR], 1.162; 95% confidence interval [CI], .619 to 2.164; P = .747). There were no significant differences in secondary outcomes between the 2 cohorts. The percentage of patients requiring multiple infusions for a clinical response did not differ significantly between the DD and TP cohorts (38.2% versus 32.5%; OR, .780; 95% CI, .345 to 1.805; P = .666). We found no significant difference in clinical response rate between DD VSTs and TP VSTs and a similar safety profile. Our data suggest that TP VSTs may be sufficient to control viral infection until immune reconstitution occurs despite the potential for more rapid VST clearance compared to DD VSTs. The lack of significant differences between DD VSTs and TP VSTs is an important finding, indicating that it is not necessary for every transplant center to manufacture customized DD VSTs, and that TP VSTs are a satisfactory substitute.

Dynamic regulation of platelet-derived growth factor receptor α expression in alveolar fibroblasts during realveolarization.

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.

Off-the-Shelf Third-Party Virus-Specific T Cell Therapy to Treat JC Polyomavirus Infection in Hematopoietic Stem Cell Transplantation Recipients.

Progressive multifocal leukoencephalopathy (PML) is a progressive and generally fatal demyelinating neurologic disease that occurs in profoundly immunocompromised patients due to infection with the human polyomavirus JC virus (JCPyV). Treatment options are limited and are largely focused on restoring T cell immunity, and outcomes are historically poor. Control of JCPyV in the setting of an immunocompromised patient by adoptive transfer of third-party virus specific T cells (VSTs) has been described in a small number of cases. To investigate treatment response and outcomes in recipients of hematopoietic stem cell transplantation (HSCT) with PML treated with third-party VSTs directed against the BK virus, a highly homologous polyoma virus that shares immunogenic epitopes with JCPyV. A retrospective chart review was performed on 4 patients who received VSTs for the treatment of PML at Cincinnati Children's Hospital Medical Center since 2019. VSTs were administered safely, with no cases of graft-versus-host disease and no infusion reactions. One patient who was treated almost immediately after diagnosis was able to clear JCPyV from blood and cerebrospinal fluid, with resultant stabilization of neurologic decline. IFN-γ enzyme-linked immunospot (ELISpot) analysis demonstrated VSTs in the peripheral blood following infusion. Response was maintained through repeat infusions. Three other patients, all of whom had a longer delay between diagnosis and infusion, exhibited progressive neurologic decline despite varying degrees of improvement in viral load. PML is a rare but often fatal complication following HSCT for which few treatment options are available. BK-directed, JCPyV cross-reactive VSTs are a safe and viable therapeutic option, and prompt administration should be considered once PML is diagnosed. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Scheduled administration of virus-specific T cells for viral prophylaxis after pediatric allogeneic stem cell transplant.

Infections with double-stranded DNA viruses are a significant cause of morbidity and mortality in pediatric patients following allogeneic hematopoietic stem cell transplantation (HSCT). Virus-specific T-cell therapies (VSTs) have been shown to be an effective treatment for infections with adenovirus, BK virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). To date, prophylactic regimens to prevent or mitigate these infections using conventional antiviral medications provide suboptimal response rates. Here we report on a clinical trial (NCT03883906) performed to assess the feasibility of rapid manufacturing and early infusion of quadrivalent VSTs generated from stem cell donors ("donor-derived VSTs") into allogeneic HSCT recipients with minimal or absent viremia. Patients were eligible to receive scheduled VSTs as early as 21 days after stem cell infusion. Twenty-three patients received scheduled VSTs. Twenty of 23 patients had no viremia at the time of infusion, while 3 patients had very low-level BK viremia. Two developed clinically significant graft-versus-host disease (GVHD), although this incidence was not outside of expected incidence early after HSCT, and both were successfully treated with systemic corticosteroids (n = 2). Five patients were deemed treatment failures. Three developed subsequent significant viremia/viral disease (n = 3). Eighteen patients did not fail treatment, 7 of whom did not develop any viremia, while 11 developed low-level, self-limited viremia that resolved without further intervention. No infusion reactions occurred. In conclusion, scheduled VSTs appear to be safe and potentially effective at limiting serious complications from viral infections after allogeneic transplantation. A randomized study comparing this scheduled approach to the use of VSTs to treat active viremia is ongoing.

Genetic modification of airway progenitors after lentiviral gene delivery to the amniotic fluid of murine fetuses.

Lentiviral vectors with the firefly luciferase or enhanced green fluorescent protein (EGFP) transgenes were delivered to the amniotic fluid of murine fetuses at Embryonic Day (E) 14.5 or E16.5. Whole-body imaging of luciferase recipients after birth demonstrated transgene expression in the peritoneal and thoracic regions. Organ imaging showed luciferase expression in lung, skin, stomach, and/or intestine. Histological immunofluorescence analysis of EGFP recipients demonstrated that small clusters (≤ three cells) of EGFP-positive epithelial cells were present in the large and small airways of recipients at up to 7 months (n = 11). There was no difference in the frequency of transgene expression in mice injected at E14.5 or E16.5 in respiratory or nonrespiratory organs. Analysis of the bronchoalveolar duct junctions on tissue sections of recipient mice identified multiple EGFP-positive epithelial cells. Cells coexpressing EGFP, Clara cell 10-kd protein, and surfactant protein C (SPC) were also found in lungs, consistent with the transduction of bronchoalveolar stem cells. Next, naphthalene lung injury in both luciferase and EGFP recipients was performed to determine whether transduced cells could contribute to tissue repair. In luciferase recipients, the whole-body luciferase signal increased 2- to 20-fold at 2 weeks after naphthalene treatment. Remarkably, immunohistological analysis of the lungs of EGFP recipients after lung injury repair demonstrated repopulation of airways with long stretches of EGFP-positive epithelial cells (n = 4). Collectively, these data demonstrate that lentiviral gene delivery to the amniotic fluid of murine fetuses genetically modifies long-lived epithelial progenitors capable of contributing to lung injury repair.

Recovery of multipotent progenitors from the peripheral blood of patients requiring extracorporeal membrane oxygenation support.

RATIONALE: Studies have demonstrated that bone marrow-derived cells can be recruited to injured lungs through an unknown mechanism. We hypothesize that marrow progenitors are mobilized into the circulation of patients with cardiac and/or respiratory failure, and may then traffic to and incorporate into the sites of tissue injury. OBJECTIVES: To determine whether progenitor populations are increased in the blood of patients with severe acute cardiorespiratory failure placed on extracorporeal membrane oxygenation (ECMO). METHODS: Mononuclear cells from ECMO, umbilical cord, and control blood samples were evaluated in colony-forming assays for hematopoietic, mesenchymal, and epithelial cells. Progenitors were identified by proliferative and differentiative capacities, and confirmed by the expression of lineage-specific markers. MEASUREMENTS AND MAIN RESULTS: Significantly higher levels of hematopoietic progenitors were observed in ECMO (n = 41) samples than neonatal intensive care unit (n = 16) or pediatric intensive care unit controls (n = 14). Hematopoietic progenitor mobilization increased with time on ECMO support. Mesenchymal progenitors (MSC) were recovered from 18/58 ECMO samples with rapid sample processing (< 4 h) critical to their recovery. MSC were not recovered from normal controls. ECMO-derived MSC had osteogenic, chondrogenic, and adipogenic differentiation potential. The recovery of MSC did not influence survival outcome (61%). Epithelial progenitors were observed in eight ECMO samples but not in control samples. Their presence was associated with a lower survival trend (38%). CONCLUSIONS: Hematopoietic, mesenchymal, and epithelial progenitors were mobilized into the circulation of patients on ECMO. This may reflect a response to severe cardiopulmonary injury, blood-foreign surface interactions with the ECMO circuit, and/or hemodilution.

Virus-specific T cells for adenovirus infection after stem cell transplantation are highly effective and class II HLA restricted.

Infection with adenoviruses is a common and significant complication in pediatric patients after allogeneic hematopoietic stem cell transplantation. Treatment options with traditional antivirals are limited by poor efficacy and significant toxicities. T-cell reconstitution is critical for the management of adenoviral infections, but it generally takes place months after transplantation. Ex vivo-generated virus-specific T cells (VSTs) are an alternative approach for viral control and can be rapidly generated from either a stem cell donor or a healthy third-party donor. In the context of a single-center phase 1/2 clinical trial, we treated 30 patients with a total of 43 infusions of VSTs for adenoviremia and/or adenoviral disease. Seven patients received donor-derived VSTs, 21 patients received third-party VSTs, and 2 received VSTs from both donor sources. Clinical responses were observed in 81% of patients, with a complete response in 58%. Epitope prediction and potential epitope identification for common HLA molecules helped elucidate HLA restriction in a subset of patients receiving third-party products. Intracellular interferon-γ expression in T cells in response to single peptides and response to cell lines stably transfected with a single HLA molecule demonstrated HLA-restricted CD4+ T-cell response, and these results correlated with clinical outcomes. Taken together, these data suggest that VSTs are a highly safe and effective therapy for the management of adenoviral infection in immunocompromised hosts. The trials were registered at www.clinicaltrials.gov as #NCT02048332 and #NCT02532452.

Bone marrow progenitor cells repair rat hepatic sinusoidal endothelial cells after liver injury.

BACKGROUND & AIMS: Damage to hepatic sinusoidal endothelial cells (SECs) initiates sinusoidal obstruction syndrome (SOS), which is most commonly a consequence of myeloablative chemoirradiation or ingestion of pyrrolizidine alkaloids such as monocrotaline (Mct). This study examines whether SECs are of bone marrow origin, whether bone marrow repair can be a determinant of severity of liver injury, and whether treatment with progenitor cells is beneficial. METHODS: Mct-treated female rats received infusion of male whole bone marrow or CD133(+) cells at the peak of sinusoidal injury. The Y chromosome was identified in isolated SECs by fluorescent in situ hybridization. Bone marrow suppression was induced by irradiation of both lower extremities with shielding of the abdomen. RESULTS: SECs in uninjured liver have both hematopoietic (CD45, CD33) and endothelial (CD31) markers. After Mct-induced SOS, infusion of bone marrow-derived CD133(+) progenitor cells replaces more than one quarter of SECs. All CD133(+) cells recovered from the SEC fraction after injury are CD45(+). CD133(+)/CD45(+) progenitors also repaired central vein endothelium. Mct suppresses CD133(+)/CD45(+) progenitors in bone marrow by 50% and in the circulation by 97%. Irradiation-induced bone marrow suppression elicited SOS from a subtoxic dose of Mct, whereas infusion of bone marrow during the necrotic phase of SOS nearly eradicates histologic features of SOS. CONCLUSIONS: SECs have both hematopoietic and endothelial markers. Bone marrow-derived CD133(+)/CD45(+) progenitors replace SECs and central vein endothelial cells after injury. Toxicity to bone marrow progenitors impairs repair and contributes to the pathogenesis of SOS, whereas timely infusion of bone marrow has therapeutic benefit.

Molecular characterization of the human NANOG protein.

NANOG is a key transcriptional regulator of pluripotent stem cell (PSC) self-renewal. NANOG occupies promoters that are active and others that are repressed during self-renewal; however, the mechanisms by which NANOG regulates transcriptional repression and activation are unknown. We hypothesized that individual protein domains of NANOG control its interactions with both the promoters and its coregulators. We performed a detailed characterization of the functional domains in the human (h) NANOG protein, using a panel of deletion-mutant and point-mutant constructs. We determined that six amino acids in the homeodomain ((136)YKQVKT(141)) are sufficient for the nuclear localization of hNANOG. We also determined that the tryptophan-rich region (W) of hNANOG contains a CRM1-independent signal for nuclear export, suggesting a possible cellular shuttling behavior that has not been reported for hNANOG. We also show that at least four tryptophans are required for nuclear export. We also determined that similar to murine (m) NANOG, the W region of hNANOG contains a homodimerization domain. Finally, in vitro transactivation analyses identified distinct regions that enhance or diminish activity at gene promoters that are active during self-renewal. Specifically, the N-terminal region interferes with transcription and removal of this region that produced a "super-active" hNANOG with enhanced transcriptional activity. We also confirmed that the transcriptional activator in hNANOG is contained in the C-terminal region, similar to murine NANOG. In summary, this study has characterized the structure and function of hNANOG protein leading to an increased understanding of the mechanism by which hNANOG regulates both transcriptional activation and repression during PSC self-renewal.

Virus-specific T-cell therapy to treat BK polyomavirus infection in bone marrow and solid organ transplant recipients.

BK polyomavirus (BKPyV) infection is a major complication of hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT). Treatment options are limited, poorly effective, and have significant toxicities. Cellular therapy using T cells directed against BKPyV is an emerging therapy, and we report efficacy in controlling BKPyV-associated disease in highly immunocompromised patients. Virus-specific T cells (VSTs) against BKPyV were manufactured using either blood from the patient's stem cell donor (donor-derived VSTs) or from unrelated donors (third-party VSTs). VSTs were used to treat BKPyV in 38 HSCT recipients and 3 SOT recipients between June 2017 and December 2019. Overall response rate was 86% in patients treated for BK viremia, 100% in patients treated for hemorrhagic cystitis, and 87% in patients treated for both BK viremia and hemorrhagic cystitis. No infusional toxicity, de novo graft-versus-host disease, or rejection of the organ occurred attributable to the VST infusion. BKPyV-specific immune responses were demonstrated by interferon-γ production by peripheral blood mononuclear cells postinfusion in response to BKPyV antigens. VSTs are a safe and potentially effective strategy to treat BKPyV and associated symptoms in recipients of HSCT and SOT. Cellular therapy should be considered for all patients with BKPyV and underlying immune suppression at risk of complications. This trial was registered at www.clinicaltrials.gov as #NCT02532452.