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Background Aims: The success of allogeneic hematopoietic cell transplantation (HCT) as therapy for hematologic conditions is negatively impacted by the occurrence of graft-versus-host disease (GVHD). Tissue damage, caused, for example, by chemotherapy and radiotherapy, is a key factor in GVHD pathogenesis. Innate lymphoid cells (ILCs) are important mediators of tissue repair and homeostasis. The presence of ILCs before, and enhanced ILC reconstitution after, allogeneic HCT is associated with a reduced risk to develop mucositis and GVHD. However, ILC reconstitution after allogeneic HCT is slow and often incomplete. A way to replenish the pool of ILC relies on the differentiation of hematopoietic progenitor cells (HPCs) into ILC., Methods: We developed an ex vivo stromal cell-containing culture system to study the capacity of HPCs to differentiate into all mature helper ILC subsets., Results: ILC development depended on the source of HPCs. ILCs developed at high frequencies from umbilical cord blood- and fetal liver-derived HPC and at low frequencies when HPCs were obtained from allogeneic or autologous adult HCT grafts or healthy adult bone marrow. Although all helper ILC subsets could be generated from adult HPC sources, development of tissue protective ILC2 and NKp44 + ILC3 was notoriously difficult., Conclusions: Our data suggest that slow ILC recovery after allogeneic HCT may be related to an intrinsic incapability of adult HPC to develop into ILC., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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: 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.)