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Macrophages populate the embryo early in gestation, but their role in development is not well defined. In particular, specification and function of macrophages in intestinal development remain little explored. To study this event in the human developmental context, we derived and combined human intestinal organoid and macrophages from pluripotent stem cells. Macrophages migrate into the organoid, proliferate, and occupy the emerging microanatomical niches of epithelial crypts and ganglia. They also acquire a transcriptomic profile similar to that of fetal intestinal macrophages and display tissue macrophage behaviors, such as recruitment to tissue injury. Using this model, we show that macrophages reduce glycolysis in mesenchymal cells and limit tissue growth without affecting tissue architecture, in contrast to the pro-growth effect of enteric neurons. In short, we engineered an intestinal tissue model populated with macrophages, and we suggest that resident macrophages contribute to the regulation of metabolism and growth of the developing intestine., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Background: T lymphocytes exhibit numerous alterations in relapsing-remitting (RRMS), secondary progressive (SPMS), and primary progressive multiple sclerosis (PPMS). The NKG2D pathway has been involved in MS pathology. NKG2D is a co-activating receptor on subsets of CD4 + and most CD8 + T lymphocytes. The ligands of NKG2D are expressed at low levels in normal tissues but are elevated in MS postmortem brain tissues compared with controls. Whether the NKG2D pathway shows specific changes in different forms of MS remains unclear., Methods: We performed unsupervised and supervised flow cytometry analysis to characterize peripheral blood T lymphocytes from RRMS, SPMS, and PPMS patients and healthy controls (HC). We used an in vitro microscopy approach to assess the role of NKG2D in the interactions between human CD8 + T lymphocytes and human astrocytes., Results: Specific CD8 + , CD4 + , and CD4 - CD8 - T cell populations exhibited altered frequency in MS patients' subgroups. The proportion of NKG2D + T lymphocytes declined with age in PPMS patients but not in RRMS and HC. This reduced percentage of NKG2D + cells was due to lower abundance of γδ and αβ CD4 - CD8 - T lymphocytes in PPMS patients. NKG2D + T lymphocytes were significantly less abundant in RRMS than in HC; this was caused by a decreased frequency of CD4 - CD8 - and CD8 + T lymphocytes and was not linked to age. Blocking NKG2D increased the motility of CD8 + T lymphocytes co-cultured with astrocytes expressing NKG2D ligand. Moreover, preventing NKG2D from interacting with its ligands increased the proportion of CD8 + T lymphocytes exhibiting a kinapse-like behavior characterized by short-term interaction while reducing those displaying a long-lasting synapse-like behavior. These results support that NKG2D participates in the establishment of long-term interactions between activated CD8 + T lymphocytes and astrocytes., Conclusion: Our data demonstrate specific alterations in NKG2D + T lymphocytes in MS patients' subgroups and suggest that NKG2D contributes to the interactions between human CD8 + T lymphocytes and human astrocytes., Competing Interests: Declaration of interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Interleukin-27 (IL-27) can trigger both pro- and anti-inflammatory responses. This cytokine is elevated in the central nervous system (CNS) of multiple sclerosis (MS) patients, but how it influences neuroinflammatory processes remains unclear. As astrocytes express the receptor for IL-27, we sought to determine how these glial cells respond to this cytokine and whether such exposure alters their interactions with infiltrating activated T lymphocytes. To determine whether inflammation shapes the impact of IL-27, we compared the effects of this cytokine in non-inflamed and inflamed conditions induced by an IL-1β exposure., Main Body: Transcriptomic analysis of IL-27-exposed human astrocytes showed an upregulation of multiple immune genes. Human astrocytes increased the secretion of chemokines (CXCL9, CXCL10, and CXCL11) and the surface expression of proteins (PD-L1, HLA-E, and ICAM-1) following IL-27 exposure. To assess whether exposure of astrocytes to IL-27 influences the profile of activated T lymphocytes infiltrating the CNS, we used an astrocyte/T lymphocyte co-culture model. Activated human CD4 + or CD8 + T lymphocytes were co-cultured with astrocytes that have been either untreated or pre-exposed to IL‑27 or IL-1β. After 24 h, we analyzed T lymphocytes by flow cytometry for transcription factors and immune molecules. The contact with IL-27-exposed astrocytes increased the percentages of T-bet, Eomes, CD95, IL-18Rα, ICAM-1, and PD-L1 expressing CD4 + and CD8 + T lymphocytes and reduced the proportion of CXCR3-positive CD8 + T lymphocytes. Human CD8 + T lymphocytes co-cultured with human IL-27-treated astrocytes exhibited higher motility than when in contact with untreated astrocytes. These results suggested a preponderance of kinapse-like over synapse-like interactions between CD8 + T lymphocytes and IL-27-treated astrocytes. Finally, CD8 + T lymphocytes from MS patients showed higher motility in contact with IL-27-exposed astrocytes compared to healthy donors' cells., Conclusion: Our results establish that IL-27 alters the immune functions of human astrocytes and shapes the profile and motility of encountered T lymphocytes, especially CD8 + T lymphocytes from MS patients., (© 2022. The Author(s).)

Modeling the tumor-immune cell interactions in humanized mice is complex and limits drug development. Here, we generated easily accessible tumor models by transforming either primary skin fibroblasts or induced pluripotent stem cell-derived cell lines injected in immune-deficient mice reconstituted with human autologous immune cells. Our results showed that fibroblastic, hepatic, or neural tumors were all efficiently infiltrated and partially or totally rejected by autologous immune cells in humanized mice. Characterization of tumor-immune infiltrates revealed high expression levels of the dysfunction markers Tim3 and PD-1 in T cells and an enrichment in regulatory T cells, suggesting rapid establishment of immunomodulatory phenotypes. Inhibition of PD-1 by Nivolumab in humanized mice resulted in increased immune cell infiltration and a slight decrease in tumor growth. We expect that these versatile and accessible cancer models will facilitate preclinical studies and the evaluation of autologous cancer immunotherapies across a range of different tumor cell types., Competing Interests: The authors have no conflicts of interest to disclose. C.R. and M.P. are co-founders, shareholders, and officers of the regenerative medicine company Morphocell Technologies, Inc., (© 2021 The Authors.)

Background and Objectives: We posit the involvement of the natural killer group 2D (NKG2D) pathway in multiple sclerosis (MS) pathology via the presence of specific NKG2D ligands (NKG2DLs). We aim to evaluate the expression of NKG2DLs in the CNS and CSF of patients with MS and to identify cellular stressors inducing the expression of UL16-binding protein 4 (ULBP4), the only detectable NKG2DL. Finally, we evaluate the impact of ULBP4 on functions such as cytokine production and motility by CD8 + T lymphocytes, a subset largely expressing NKG2D, the cognate receptor., Methods: Human postmortem brain samples and CSF from patients with MS and controls were used to evaluate NKG2DL expression. In vitro assays using primary cultures of human astrocytes and neurons were performed to identify stressors inducing ULBP4 expression. Human CD8 + T lymphocytes from MS donors and age/sex-matched healthy controls were isolated to evaluate the functional impact of soluble ULBP4., Results: We detected mRNA coding for the 8 identified human NKG2DLs in brain samples from patients with MS and controls, but only ULBP4 protein expression was detectable by Western blot. ULBP4 levels were greater in patients with MS, particularly in active and chronic active lesions and normal-appearing white matter, compared with normal-appearing gray matter from MS donors and white and gray matter from controls. Soluble ULBP4 was also detected in CSF of patients with MS and controls, but a smaller shed/soluble form of 25 kDa was significantly elevated in CSF from female patients with MS compared with controls and male patients with MS. Our data indicate that soluble ULBP4 affects various functions of CD8 + T lymphocytes. First, it enhanced the production of the proinflammatory cytokines GM-CSF and interferon-γ (IFNγ). Second, it increased CD8 + T lymphocyte motility and favored a kinapse-like behavior when cultured in the presence of human astrocytes. CD8 + T lymphocytes from patients with MS were especially altered by the presence of soluble ULBP4 compared with healthy controls., Discussion: Our study provides new evidence for the involvement of NKG2D and its ligand ULBP4 in MS pathology. Our results point to ULBP4 as a viable target to specifically block 1 component of the NKG2D pathway without altering immune surveillance involving other NKG2DL., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)