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.

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote ex vivo Expansion of Hematopoietic Stem and Progenitor Cells.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

The Metastatic Bone Marrow Niche in Neuroblastoma: Altered Phenotype and Function of Mesenchymal Stromal Cells.

Background: The bone marrow (BM) is the main site of metastases and relapse in patients with neuroblastoma (NB). BM-residing mesenchymal stromal cells (MSCs) were shown to promote tumor cell survival and chemoresistance. Here we characterize the MSC compartment of the metastatic NB BM niche. Methods: Fresh BM of 62 NB patients (all stages), and control fetal and adult BM were studied by flow cytometry using well-established MSC-markers (CD34-, CD45-, CD90+, CD105+), and CD146 and CD271 subtype-markers. FACS-sorted BM MSCs and tumor cells were validated by qPCR. Moreover, isolated MSCs were tested for multilineage differentiation and Colony-forming-unit-fibroblasts (CFU-Fs) capacity. Results: Metastatic BM contains a higher number of MSCs (p < 0.05) with increased differentiation capacity towards the osteoblast lineage. Diagnostic BM contains a MSC-subtype (CD146+CD271-), only detected in BM of patients with metastatic-NB, determined by flow cytometry. FACS-sorting clearly discriminated MSC(-subtypes) and NB fractions, validated by mRNA and DNA qPCR. Overall, the CD146+CD271- subtype decreased during therapy and was detected again in the majority of patients at relapse. Conclusions: We demonstrate that the neuroblastoma BM-MSC compartment is different in quantity and functionality and contains a metastatic-niche-specific MSC-subtype. Ultimately, the MSCs contribution to tumor progression could provide targets with potential for eradicating resistant metastatic disease.

TGFBI Expressed by Bone Marrow Niche Cells and Hematopoietic Stem and Progenitor Cells Regulates Hematopoiesis.

The interactions of hematopoietic stem and progenitor cells (HSPCs) with extracellular matrix (ECM) components and cells from the bone marrow (BM) microenvironment control their homeostasis. Regenerative BM conditions can induce expression of the ECM protein transforming growth factor beta-induced gene H3 (TGFBI or BIGH3) in murine HSPCs. In this study, we examined how increased or reduced TGFBI expression in human HSPCs and BM mesenchymal stromal cells (MSCs) affects HSPC maintenance, differentiation, and migration. HSPCs that overexpressed TGFBI showed accelerated megakaryopoiesis, whereas granulocyte differentiation and proliferation of granulocyte, erythrocyte, and monocyte cultures were reduced. In addition, both upregulation and downregulation of TGFBI expression impaired HSPC colony-forming capacity of HSPCs. Interestingly, the colony-forming capacity of HSPCs with reduced TGFBI levels was increased after long-term co-culture with MSCs, as measured by long-term culture-colony forming cell (LTC-CFC) formation. Moreover, TGFBI downregulation in HSPCs resulted in increased cobblestone area-forming cell (CAFC) frequency, a measure for hematopoietic stem cell (HSC) capacity. Concordantly, TGFBI upregulation in HSPCs resulted in a decrease of CAFC and LTC-CFC frequency. These results indicate that reduced TGFBI levels in HSPCs enhanced HSC maintenance, but only in the presence of MSCs. In addition, reduced levels of TGFBI in MSCs affected MSC/HSPC interaction, as observed by an increased migration of HSPCs under the stromal layer. In conclusion, tight regulation of TGFBI expression in the BM niche is essential for balanced HSPC proliferation and differentiation.

Mesenchymal Stromal Cells Stimulate the Proliferation and IL-22 Production of Group 3 Innate Lymphoid Cells.

Infusion of mesenchymal stromal cells (MSCs) is a promising and increasingly applied therapy for patients who suffer from a variety of inflammatory diseases, including graft-versus-host disease (GvHD), a common and life-threatening complication after allogeneic hematopoietic stem cell transplantation. The therapeutic effect of MSCs is mainly ascribed to their ability to suppress T cells and to support tissue repair. However, clinical response rates in patients with GvHD are limited to 50%, and the determinants for MSC responsiveness are unknown. We recently reported that high frequencies of activated group 3 innate lymphoid cells (ILC3s) before and after allogeneic hematopoietic stem cell transplantation were associated with a lower risk of GvHD. This may be related to IL-22 production by ILC3s, a cytokine important for intestinal epithelial cell homeostasis. In this study, we investigated whether ILC3s may contribute to the therapeutic effect of MSCs by studying the interaction between MSCs and ILC3s in vitro. ILC3s isolated from human tonsils were cocultured with human bone marrow-derived MSCs for 5 d in the presence of IL-2. Coculture with MSCs enhanced the proliferation and IL-22 production of ILC3s. Reciprocally, ILC3s promoted ICAM-1 and VCAM-1 expression on MSCs. For both directions, the activation was mainly mediated by cell-cell contact and by MSC-derived IL-7 and likely by aryl hydrocarbon receptor ligands. Thus, in addition to inhibiting the proliferation of alloreactive T cells, MSCs also promote the expansion and IL-22 production of ILC3s, which may contribute to healthy homeostasis and wound repair in the treatment of various inflammatory conditions in the intestine, including GvHD.

Interferon-Gamma Impairs Maintenance and Alters Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells.

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.

Different Balance of Wnt Signaling in Adult and Fetal Bone Marrow-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271(bright)CD146(-) and CD271(bright)CD146(+) subsets correlates with donor age. The main subset in adults was CD271(bright)CD146(-), whereas the CD271(bright)CD146(+) population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271(-)CD146(+) cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration.

Detailed understanding of mesenchymal stromal cells (MSC) migration is imperative for future cellular therapies. To identify genes involved in the process of MSC migration, we generated gene expression profiles of migrating and nonmigrating fetal bone marrow MSC (FBMSC). Only 12 genes showed differential expression in migrating versus nonmigrating FBMSC. The nuclear receptors Nur77 and Nurr1 showed the highest expression in migratory MSC. Nur77 and Nurr1 are members of NR4A nuclear orphan receptor family, and we found that their expression is rapidly increased upon exposure of FBMSC to the migratory stimuli stromal-derived factor-1α (SDF-1α) and platelet-derived growth factor-BB. Lentiviral expression of Nur77 or Nurr1 resulted in enhanced migration of FBMSC toward SDF-1α compared with mock-transduced FBMSC. Analysis of the cell cycle, known to be involved in MSC migration, revealed that expression of Nur77 and Nurr1 decreases the proportion of cells in S-phase compared with control cells. Further, gain-of-function experiments showed increased hepatocyte growth factor expression and interleukin (IL)-6 and IL-8 production in MSC. Despite the altered cytokine profile, FBMSC expressing Nur77 or Nurr1 maintained the capacity to inhibit T-cell proliferation in a mixed lymphocyte reaction. Our results demonstrate that Nur77 and Nurr1 promote FBMSC migration. Modulation of Nur77 and Nurr1 activity may therefore offer perspectives to enhance the migratory potential of FBMSC which may specifically regulate the local immune response.

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency-but only in the presence of H435-IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435-IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435-IgG3 to be a candidate for monoclonal antibody therapies.

Cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are potential cells for cellular therapies, in which the recruitment and migration of MSC towards injured tissue is crucial. Our data show that culture-expanded MSC from fetal lung and bone marrow, adult bone marrow and adipose tissue contained a small percentage of migrating cells in vitro, but the optimal stimulus was different. Overall, fetal lung-MSC had the highest migratory capacity. As fetal bone marrow-MSC had lower migratory potential than fetal lung-MSC, the tissue of origin may determine the migratory capacity of MSC. No additive effect in migration towards combined stimuli was observed, which suggests only one migratory MSC fraction. Interestingly, actin rearrangement and increased paxillin phosphorylation were observed in most MSC upon stromal cell-derived factor-1alpha or platelet-derived growth factor-BB stimulation, indicating that this mechanism involved in responding to migratory cues is not restricted to migratory MSC. Migratory MSC maintained differentiation and migration potential, and contained significantly less cells in S- and G2/M-phase than their non-migrating counterpart. In conclusion, our results suggest that MSC from various sources have different migratory capacities, depending on the tissue of origin. Similar to haematopoietic stem cells, cell cycle contributes to MSC migration, which offers perspectives for modulation of MSC to enhance efficacy of future cellular therapies.

The presence of activated CD4(+) T cells is essential for the formation of colony-forming unit-endothelial cells by CD14(+) cells.

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

FcgammaRIIIb and complement component C7 codeficiency in a patient with recurrence of fulminant meningococcal septic shock.

Individuals with deficiencies of the late components of complement exhibit a susceptibility to the recurrence of meningococcal disease with a usually mild clinical presentation. We report the recurrence of fulminant meningococcal disease in a complement component C7-deficient patient. We found a total deficiency of FcgammaRIIIb on neutrophils, which could partially explain the unusually severe clinical presentation.

A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcgammaRIIA and FcgammaIIIA genes.

A single recombinant immunoglobulin G1 (IgG1) anti-RhD antibody (MonoRho) was compared with a currently used polyclonal anti-RhD product (Rhophylac) in a phase 1 study for safety, efficacy of Rhesus D (RhD)-positive red blood cell (RBC) clearance, and prevention of RhD immunization in RhD-negative men challenged with 15 mL RhD-positive RBCs. Both the polyclonal product and recombinant anti-RhD effectively cleared RhD-positive RBCs after intravenous and intramuscular injection. The recombinant anti-RhD demonstrated a slower clearance rate compared with the polyclonal anti-RhD. There was no dose response, and there was considerable variation among subjects who received the same dose of recombinant anti-RhD. Interestingly, RhD-positive RBC clearance rates were strongly associated with Fcgamma receptor IIA (FcgammaRIIA) and FcgammaIIIA but not with FcgammaIIIB polymorphisms. Subjects homozygous for FcgammaRIIA-131H or FcgammaRIIIA-158V allotypes showed a faster clearance rate compared with both the heterozygote and the corresponding alternative homozygote allotypes. A similar but less marked trend was seen for the polyclonal anti-RhD. Despite the variation in clearance rates there was no evidence of anti-RhD alloantibodies in any of the subjects at +6 months after the RBC challenge.