Efficient In Vitro Generation of IL-22-Secreting ILC3 From CD34+ Hematopoietic Progenitors in a Human Mesenchymal Stem Cell Niche

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34+ hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro-generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer

HCMV Infection in a Mesenchymal Stem Cell Niche: Differential Impact on the Development of NK Cells versus ILC3

Human cytomegalovirus (HCMV) is highly prevalent in most populations worldwide and has a major influence on shaping the human immune system. Natural killer (NK) cells are important antiviral effectors that adapt to HCMV infection by expansion of virus-specific effector/memory cells. The impact of HCMV infection on the development of NK cells and innate lymphoid cells (ILC) in general is less well understood. In this context, we have recently established a novel in vitro platform to study human NK cell development in a stem cell niche based on human bone marrow-derived mesenchymal stem cells (MSC). Here, the system was modified by infecting MSC with HCMV to study the influence of virus infection on NK/ILC development. We show that cord blood-derived hematopoietic progenitor cells are successfully differentiated into mature CD56+CD94+NKG2A+ NK cells on HCMV-infected MSC with significant higher anti-viral cytokine production compared to NK cells developing on non-infected MSC. Furthermore, the generation of ILC3, characterized by expression of the signature transcription factor RAR-related orphan receptor gamma (RORγt) and the production of IL-22, was strongly impaired by HCMV infection. These observations are clinically relevant, given that ILC3 are associated with protection from graft-versus-host disease (GvHD) following stem cell transplantation and HCMV reactivation in turn is associated with increased incidence of GvHD

HLA-E expression constitutes a novel determinant for ALL disease monitoring following hematopoietic stem cell transplantation

Design!#!Prospective diagnostic study.!##!Objectives!#!Primary imaging-based diagnosis of spinal cord tumor-suspected lesions is often challenging. The identification of the definite entity is crucial for dedicated treatment and therefore reduction of morbidity. The aim of this trial was to investigate specific quantitative signal patterns to differentiate unclear intramedullary tumor-suspected lesions based on diffusion tensor imaging (DTI).!##!Setting!#!Medical Center - University of Freiburg, Germany.!##!Methods!#!Forty patients with an unclear tumor-suspected lesion of the spinal cord prospectively underwent DTI. Primary diagnosis was determined by histological or clinical work-up or remained indeterminate with follow-up. DTI metrics (FA/ADC) were evaluated at the central lesion area, lesion margin, edema, and normal spinal cord and compared between different diagnostic groups (ependymomas, other spinal cord tumors, inflammations).!##!Results!#!Mean DTI metrics for all spinal cord tumors (n = 18) showed significantly reduced FA and increased ADC values compared to inflammatory lesions (n = 8) at the lesion margin (p < 0.001, p = 0.001) and reduced FA at the central lesion area (p < 0.001). There were no significant differences comparing the neoplastic subgroups of ependymomas (n = 10) and other spinal cord tumors (n = 8), but remaining differences for both compared to the inflammation subgroup. We found significant higher ADC (p = 0.040) and a trend to decreased FA (p = 0.081) for ependymomas compared to inflammations at the edema.!##!Conclusion!#!Even if distinct differentiation of ependymomas from other spinal cord neoplasms was not possible based on quantitative DTI metrics, FA and ADC were feasible to separate inflammatory lesions. This may avoid unnecessary surgery in patients with unclear intramedullary tumor-suspected lesions

DataSheet_1_Efficient In Vitro Generation of IL-22-Secreting ILC3 From CD34+ Hematopoietic Progenitors in a Human Mesenchymal Stem Cell Niche.pdf

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34+ hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro-generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer.</p