Generation of human ILC3 from allogeneic and autologous CD34+ hematopoietic progenitors toward adoptive transfer.

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.

Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease.

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.

Cell-free DNA levels are increased in acute graft-versus-host disease.

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.

Heme oxygenase-1: Equally important in allogeneic hematopoietic stem cell transplantation and organ transplantation?

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.

Age-related distribution and dynamics of T-cells in blood and lymphoid tissues of goats.

Neonatal mammals have increased disease susceptibility and sub-optimal vaccine responses. This raises problems in both humans and farm animals. The high prevalence of paratuberculosis in goats and the lack of an effective vaccine against it have a strong impact on the dairy sector, and calls for vaccines optimized for the neonatal immune system. We characterized the composition of the T-cell pool in neonatal kids and adult goats and quantified their turnover rates using in vivo deuterium labelling. From birth to adulthood, CD4+ T-cells were the predominant subset in the thymus and lymph nodes, while spleen and bone marrow contained mainly CD8+ lymphocytes. In blood, CD4+ T-cells were the predominant subset during the neonatal period, while CD8+ T-cells predominated in adults. We observed that thymic mass and cellularity increased during the first 5 months after birth, but decreased later in life. Deuterium labelling revealed that T-cell turnover rates in neonatal kids are considerably higher than in adult animals.

Short Lifespans of Memory T-cells in Bone Marrow, Blood, and Lymph Nodes Suggest That T-cell Memory Is Maintained by Continuous Self-Renewal of Recirculating Cells.

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived, non-circulating memory T-cells, it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study, we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates-and thereby expected lifespans-of memory T-cells from BM, blood and lymph nodes (LN). While the fraction of Ki-67 positive cells, a snapshot marker for recent cell division, was higher in memory T-cells from blood compared to BM and LN, in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery, and are continuously recirculating between the blood, BM, and LN.