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Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34 + HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34 + HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown., Objective: The aim of this study is to investigate the role of cfDNA as a marker of aGvHD., Methods: We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction., Results: In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells., Conclusion: We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Allogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD., Objective: To investigate whether the presence of mature ILCs within G-CSF-mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT., Study Design: We analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3 - lineage - CD45 + CD127 + CD161 + lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples., Results: Remission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF-mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44 + ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44 + ILC3 signature groups., Conclusion: The presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The intracellular enzyme heme oxygenase-1 (HO-1) is responsible for the degradation of cell-free (cf) heme. Cfheme, released upon cell damage and cell death from hemoglobin, mitochondria and myoglobin, functions as a powerful damage-associated molecular pattern (DAMP). Indeed, cfheme plays a role in a myriad of diseases characterized by (systemic) inflammation, and its rapid degradation by HO-1 is pivotal to maintain homeostasis. In the past decade, HO-1 has been extensively studied for its potential protective role in different transplantation settings, including allogeneic hematopoietic stem cell transplantation (HSCT), solid organ transplantation and pancreatic islet transplantation. These studies have shown that HO-1 can be induced by a wide range of molecules, and that induction of HO-1 has the potential to significantly reduce the incidence and severity of transplantation-related complications such as graft-versus-host disease (GvHD) and ischemia/reperfusion injury (IRI). As such, further investigation into the use of HO-1-inducing agents in human transplantation settings to facilitate the potential use of these agents in the clinic is warranted. In this review, we summarize the literature of the past 10 years on the role of HO-1 in allogeneic HSCT, solid organ transplantation (focusing on kidney and liver) and pancreatic islet transplantation. Furthermore, we provide a hypothesis about the way that HO-1 is able to provide protection against acute GvHD after allogeneic HSCT. A total of 48 research articles and 17 review articles were included in this review., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

Background: Although new therapeutic strategies have been developed to control Crohn's disease, medical treatment for refractory cases is not able to prevent extensive and/or repeat surgery. Recently, several cases have been reported of successful remission induction in Crohn's disease patients by means of hematopoietic stem cell transplantation (HSCT). Here we report our long-term (4 to 6 years) outcome in three patients., Patients: Three patients (two male, one female) with active severe Crohn's disease were planned to undergo autologous HSCT. All patients were intolerant or refractory to conventional therapies, including anti-TNFα antibodies. Patients either refused surgery or surgery was considered not to be a feasible alternative due to the extensive disease involvement of the small intestine., Methods: Peripheral blood stem cells were mobilized using a single infusion of cyclophosphamide 4 g/m(2), followed on day 4 by subcutaneous injections with G-CSF 5 μg/kg twice daily until leukapheresis. CD34+ cells were isolated after leukapheresis by magnetic cell sorting. In two of the three patients a second round of stem cell mobilization using G-CSF only was required, either because of low yield or because of insufficient recovery after CD34 selection. Prior to transplantation, immune ablation was achieved using cyclophosphamide 50mg/kg/day (4 days), antithymocyte globulin 30 mg/kg/day (3 days) and prednisolone 500 mg (3 days). Endoscopy, barium small bowel enteroclysis and MRI enterography were performed., Results: All three patients successfully completed stem cell mobilization, and two of them subsequently underwent conditioning and autologous HSCT with CD34+ cell selection. Treatment was well tolerated, with acceptable toxicity. Now, 5 and 6 years post-transplantation, these patients are in remission under treatment. The third patient went into remission after mobilization and therefore she decided not to undergo conditioning and HSCT transplantation. After a successful pregnancy she relapsed two years later. Since then, she suffers from refractory Crohn's disease for which we are now reconsidering conditioning and transplantation., Conclusion: Autologous HSCT appears to be safe and can be an alternative strategy for Crohn's disease patients with severe and therapy resistant disease., (Copyright © 2011 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

To investigate whether the migratory ability of peripheral blood-derived CD34+ cells of patients undergoing autologous peripheral blood stem cell transplantation is related to the homing efficiency of these cells, the migration in vitro of these cells was determined and correlated with in vivo hematopoietic recovery. Large inter-individual differences of the in vitro migratory ability of the CD34+ cells were observed, ranging from 1.1% to 16.4% for spontaneous migration and 6.2% to 40.8% for SDF-1-induced (100 ng/mL) migration. Significantly faster hematologic recovery was observed in those patients who received transplanted CD34+ cells that showed high spontaneous and SDF-1-induced migration in vitro (P <.05). Moreover, CD34+ cells from healthy G-CSF-mobilized donors exhibited significantly higher spontaneous and SDF-1-induced (P <.01) migration than CD34+ cells from patients mobilized with chemotherapy and G-CSF. The lower migratory capacity in vitro of patient-derived CD34+ cells was not due to lower expression of CXCR-4 but probably reflected decreased motogenic behavior of the cells. These results indicate that the migratory capacity of the cells is important for hematopoietic recovery. The data suggest that the engraftment potential of autologous stem cells is more or less impaired by treatment before or during the mobilization procedure and might possibly be restored by in vitro manipulation of the cells. In addition, an exponential relation between CXCR-4 expression and number of CD34+ cells that mobilized to the peripheral blood was found (P <.001), suggesting that CXCR-4 expression plays a role in the mobilization of CD34+ cells.