Expression of the inhibitory Ly49E receptor is not critically involved in the immune response against cutaneous, pulmonary or liver tumours

Natural killer (NK) lymphocytes are part of the innate immune system and are important in immune protection against tumourigenesis. NK cells display a broad repertoire of activating and inhibitory cell surface receptors that regulate NK cell activity. The Ly49 family of NK receptors is composed of several members that recognize major histocompatibility complex class I (MHC-I) or MHC-I-related molecules. Ly49E is a unique inhibitory member, being triggered by the non-MHC-I-related protein urokinase plasminogen activator (uPA) in contrast to the known MHC-I-triggering of the other inhibitory Ly49 receptors. Ly49E also has an uncommon expression pattern on NK cells, including high expression on liver DX5-NK cells. Furthermore, Ly49E is the only Ly49 member expressed by epidermal gamma delta T cells. As gamma delta T cells and/or NK cells have been shown to be involved in the regulation of cutaneous, pulmonary and liver malignancies, and as uPA is involved in tumourigenesis, we investigated the role of the inhibitory Ly49E receptor in the anti-tumour immune response. We demonstrate that, although Ly49E is highly expressed on epidermal gamma delta T cells and liver NK cells, this receptor does not play a major role in the control of skin tumour formation or in lung and liver tumour development

Ly49E Expression on CD8αα-expressing intestinal intraepithelial lymphocytes plays no detectable role in the development and progression of experimentally induced inflammatory bowel diseases

The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8 alpha alpha-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8 alpha alpha-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNF Delta ARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8 alpha alpha-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases

A murine intestinal intraepithelial NKp46-negative innate lymphoid cell population characterized by group 1 properties

The Ly49E receptor is preferentially expressed on murine innate-like lymphocytes, such as epidermal V gamma 3 T cells, intestinal intraepithelial CD8 alpha alpha(+) T lymphocytes, and CD49a(+) liver natural killer (NK) cells. As the latter have recently been shown to be distinct from conventional NK cells and have innate lymphoid cell type 1 (ILC1) properties, we investigated Ly49E expression on intestinal ILC populations. Here, we show that Ly49E expression is very low on known ILC populations, but it can be used to define a previously unrecognized intraepithelial innate lymphoid population. This Ly49E-positive population is negative for NKp46 and CD8 alpha alpha, expresses CD49a and CD103, and requires T-bet expression and IL-15 signaling for differentiation and/or survival. Transcriptome analysis reveals a group 1 ILC gene profile, different from NK cells, iCD8 alpha cells, and intraepithelial ILC1. Importantly, NKp46(-)CD8 alpha alpha(-)Ly49E(+) cells produce interferon (IFN)-gamma, suggesting that this previously unrecognized population may contribute to Th1-mediated immunity

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and regulate other immune cells by cytokine secretion. Whereas murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells (hESC) and by expressing the dominant-negative ETS1 p27 isoform in cord blood (CB) hematopoietic progenitor cells (HPCs), we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation, i.e. E4BP4, TXNIP, TBET, GATA3, HOBIT and BLIMP1. In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation

IRF2 is required for development and functional maturation of human NK cells

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells

The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression

Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: Apc(Min/+)-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer

Abundant stage-dependent Ly49E expression by liver NK cells is not essential for their differentiation and function

The NKR Ly49E has several unique characteristics. Unlike most NKRs, Ly49E is highly expressed on fetal NK cells, whereas expression is decreased on bone marrow-derived NK cells in adult mice. To investigate a possible role for Ly49E in NK cell differentiation and function, we have generated an Ly49E KO mouse. Our results show that bone marrow and splenic NK cells are present in normal numbers in Ly49E KO mice, expressing an unaltered panel of NKRs and differentiation markers. Furthermore, cytokine production and cytotoxicity by these cells are unaffected. Surprisingly, WT DX5(-) liver NK cells express high Ly49E levels in fetal and adult mice. Ly49E(+)DX5(-) liver NK cells transferred into Rag-2(-/-)/gc(-/-) mice maintain high Ly49E expression in the liver and differentiate into DX5(+) NK cells in spleen and bone marrow. Ly49E expression is not crucial for liver NK cell differentiation during ontogeny, as the DX5(-)/DX5(+) ratio, the NKR repertoire, and the granzyme B and TRAIL levels are comparable in Ly49E KO versus WT mice, except for lower TRAIL expression on DX5(-) liver NK cells in 20-day-old mice. The TRAIL-, perforin-, and FasL-mediated cytolysis by liver NK cells is unaffected in Ly49E KO mice. Collectively, we show that in addition to high Ly49E expression on fetal NK cells versus low Ly49E expression on conventional NK cells in adult life, Ly49E remains highly expressed on DX5(-) liver NK cells. However, Ly49E expression does not have a crucial role in differentiation and/or function of these NK cells

Differential Ly49e expression pathways in resting versus TCR-activated intraepithelial γδ T cells

The Ly49 NK receptor family in mice is composed of several members that recognize MHC class I (MHC-I) or MHC-I-related molecules. We and others have shown before that Ly49E is a unique member, with a different expression pattern on NK cells and being triggered by the non-MHC-I-related protein urokinase plasminogen activator. Among the entire Ly49 receptor family, Ly49E is the only Ly49 member expressed by epidermal-localized gamma delta T cells and their fetal thymic TCR gamma delta precursors, and it is the most abundantly expressed member on intestinal intraepithelial gamma delta T cell lymphocytes. In this study, we provide mechanistic insights into the regulation of Ly49e expression in gamma delta T cells. First, we demonstrate that TCR-mediated activation of intraepithelial gamma delta T cells significantly increases Ly49E expression. This results from de novo Ly49E expression and is highly selective, because no other Ly49 family members are induced. TCR-mediated Ly49E induction is a conserved feature of skin-and gut-residing intraepithelial-localized gamma delta T cell subsets, whereas it is not observed in spleen gamma delta T cells. By investigating Ly49e promoter activities and lymphotoxin (LT) alpha beta dependency in resting versus TCR-activated intraepithelial gamma delta T cells, we reveal two separate regulatory pathways for Ly49E expression, as follows: a LT alpha beta-dependent pathway leading to basal Ly49E expression in resting cells that is induced by Pro2-mediated Ly49e transcription, and a LT alpha beta-independent pathway leading to elevated, Pro3-driven Ly49E expression in TCR-stimulated cells

TXNIP promotes human NK cell development but is dispensable for NK cell functionality

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation