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Clarification of human blood ILC subtype interrelatedness and discovery of amphiregulin production by human NK cells shed light on HIV-1 pathogenesis

ABSTRACT: Human blood innate lymphoid cells (ILCs), which include ILCs and natural killer (NK) cells, derive from a common CD117+ILC precursor (ILCP). Yet, the relationship among the ILC subsets remains unclear. Bulk and single cell RNA-Seq and ATAC-Seq showed that blood ILC subsets cluster into ILC2s, ILCPs, a mixed cluster of CD56dim and CD56– NK cells, and a separate cluster of CD56hiNK cells that shares features with both ILCs and CD56dim/–NK cells. In surprising contrast to mice, tissue repair protein amphiregulin (AREG) was produced by human NK cells, with even higher levels in CD56hiNK cells than in ILCs. AREG expression in NK cells was driven by TCF7/WNT signaling and inhibited by TGFB1, a cytokine elevated in HIV-1+ people. Knockout of RUNX3, a WNT antagonist acting downstream of TGFB1, increased AREG production. Consistent with these findings, AREG+NK cells were decreased in people living with HIV-1. Additionally, functionally defective CD56–NK cells expanded in HIV-1+ people, in inverse correlation with CD56dimNK cells, ILCs, and CD4+T cells. Experiments in tissue culture and in humanized mice showed that CD56–NK cells derive from the epigenetically similar CD56dimNK cells, and that stimulation of MTOR by CD4+T cells or exogenous IL-2 prevents their expansion. These findings clarify how ILC subsets are related to each other and provide insight into how HIV-1 infection disrupts ILC homeostasis and contributes to pathology

ORGANISM(S): Homo sapiens

PROVIDER: GSE203002 | GEO | 2023/06/07

REPOSITORIES: GEO

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Project description:Understanding Natural Killer (NK) cell anatomical distribution is key to dissect the role of these unconventional lymphocytes in physiological and disease conditions. In mouse, NK cells have been detected in various lymphoid and non-lymphoid organs, while in humans the current knowledge of NK cell distribution at steady state is mainly restricted to lymphoid tissues. The translation to humans of findings obtained in mice is facilitated by the identification of NK cell markers conserved between these two species. The Natural Cytotoxicity Receptor (NCR) NKp46 is a marker of the NK cell lineage evolutionary conserved in mammals. In mice, NKp46 is also present on rare T cell subsets and on a subset of gut Innate Lymphoid Cells (ILCs) expressing the retinoic acid receptor-related orphan receptor gammat (RORgammat) transcription factor. Here, we documented the distribution and the phenotype of human NKp46+ cells in lymphoid and non-lymphoid tissues isolated from healthy donors. Human NKp46+ cells were found in splenic red pulp, in lymph nodes, in lungs and gut lamina propria, thus mirroring mouse NKp46+ cell distribution. We identified a novel cell subset of CD56dimNKp46low cells that includes RORgammat+ILCs with a lineage-CD94-CD117brightCD127bright phenotype.We also included data regarding the genome-wide transcriptional profiles of human healthy colonic NK cells and RORgammat+ILCs.The use of NKp46 thus contributes to establish the basis for analyzing quantitative and qualitative changes of NK cell and ILC subsets in human diseases. Human colonic CD56-NKp46lowCD117brightCD127bright cells = LTi cells and CD56+NKp46+CD117-CD127- cells =NK cells were isolated from macroscopically unaffected areas of colon of patients with colon cancer. Indicated populations were sorted and immediately lysed in RLT buffer supplemented with 10% beta-mercaptoethanol (Quiagen, France). Lysates of three distinct donors were pooled and RNA was isolated by using RNAeasy Microkit (Quiagen, France). Duplicates were performed for each cell type. cRNA were obtained after double amplification using the MessageAmp II aRNA Amplification Kit (Ambion, France). cRNA were then hybridized on Human Genome HG_U133 +2.0 Affymetrix chips. Chip images were generated using Affymetrix AGCC 3.2 software, then expression data were extracted and normalized using Affymetrix Expression console 1.1 with the algorithm RMA. Data obtained were expressed as log2.

Project description:Human Natural Killer (NK) cells comprise two main subsets, CD56bright and CD56dim cells, that differ in function, phenotype and tissue localization. To further dissect the heterogeneity of CD56dim cells, we have performed transcriptome analysis and functional ex vivo characterization of human NK cell subsets according to the expression of markers related to differentiation, migration or competence. Here, we show for the first time that the ability to respond to cytokines or to activating receptors is mutually exclusive in almost all NK cells with the exception of CD56dim CD62L+ cells. Indeed, only these cells combine the ability to produce interferon (IFN)-gamma after cytokines and proliferate in vivo during viral infection with the capacity to kill and produce cytokines upon engagement of activating receptors. Therefore, CD56dim CD62L+ cells represent a unique subset of polyfunctional NK cells. Ex vivo analysis of their function, phenotype, telomere length, frequencies during ageing as well as transfer experiments of NK cell subsets into immunodeficient mice suggest that CD56dim CD62L+ cells represent an intermediate stage of NK cell maturation, which after restimulation can accomplish multiple tasks and further develop into terminally differentiated effectors. Gene expression profiles of FACSAria sorted CD3- CD56bright CD62L+, CD3- CD56dim CD62L+ and CD3- CD56dim CD62L- NK cells from human peripheral blood of three donors were compared using Affymetrix GeneChip Human Genome HG-U133_Plus_2. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 Âµg cRNA hybridized in triplicates for each of the three groups to the GeneChip arrays. Group1: CD3- CD56bright CD62L+,.Group2: CD3- CD56dim CD62L+, Group3: CD3- CD56dim CD62L-. Lists of differentially regulated genes were created using High Performance Chip Data Analysis (HPCDA) with Bioretis database (http://www.bioretis-analysis.de). Worldwide data sharing is possible via Bioretis, please ask the authors.

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Clarification of human blood ILC subtype interrelatedness and discovery of amphiregulin production by human NK cells shed light on HIV-1 pathogenesis

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