YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

CXCL16 and CXCR6 are upregulated in psoriasis and mediate cutaneous recruitment of human CD8+ T cells.

Psoriatic skin lesions are characterized by an inflammatory infiltrate, consisting of dendritic cells, monocytes, and both CD4(+) and CD8(+) T lymphocytes. Although the chemokines involved in the migration of CD4(+) T cells into psoriatic skin are well characterized, those regulating CD8(+) T-cell recruitment are less understood. We found that the percentages of peripheral blood CD8(+) T cells expressing CXCR6 were higher in psoriatic patients than in healthy or atopic individuals. In addition, CXCR6 expression in psoriatic patients was more abundant in the CD8(+) than in the CD4(+) T-cell compartment. CXCR6 mRNA expression was also stronger in skin CD8(+) T cells than in the corresponding blood-derived counterparts. Immunofluorescence analysis revealed profound upregulation of the CXCR6 ligand CXCL16 by monocytes, keratinocytes, and dendritic cells in psoriatic skin compared with healthy or atopic dermatitis skin. In line with this, CXCR6(+) CD8(+) T cells also were most prevalent in psoriatic skin. Furthermore, CXCL16 induced Ca(2+) influx and chemotactic migration of psoriatic skin-derived CD8(+) T cells in vitro. Most importantly, CXCL16 potently recruited human CD8(+) T cells to human skin grafts previously transplanted onto SCID mice in vivo. These investigations indicate that CXCL16-CXCR6 interactions mediate homing of CD8(+) T cells into human skin, and thereby contribute to psoriasis pathogenesis.

Triggering the succinate receptor GPR91 on dendritic cells enhances immunity.

Succinate acts as an extracellular mediator signaling through the G protein-coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid-specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

IL-12 instructs skin homing of human Th2 cells.

Distinct pattern of homing receptors determines the tissue preference for T cells to exert their effector functions. This homing competence is mostly determined early during T cell activation of naive T cells. In contrast, mechanisms governing the acquisition of particular homing receptors by T cells of the memory phenotype remain enigmatic. Th2 cell-mediated allergic diseases tend to flare during infections despite that these infections prime APCs to produce the prototypic Th1 cell-differentiating cytokine IL-12. In this study, we investigate the effect of IL-12 on the regulation of cutaneous lymphocyte Ag (CLA) on differentiated Th2 cells and consequences of this expression for allergic inflammation. Upon activation with IL-12, CLA- Th2 cells rapidly up-regulated IL-12Rbeta2 chain, alpha(1-3)-fucosyltransferase VII, and CLA molecules. IL-12-mediated CLA expression on Th2 cells was functional because it mediated rolling of these Th2 cells on E-selectin in vitro and migration into human skin grafts in SCID mice. CLA induction occurred immediately after exposure to IL-12 and was independent of IFN-gamma expression. In accordance, the transcription factor mediating IFN-gamma expression, T-bet, does not directly affect CLA expression. However, CLA expression was further enhanced after IL-12 treatment of T-bet+ -transfected Th2 cells in agreement with an increased IL-12 responsiveness of these cells caused by T-bet. The finding that IL-12 conferred skin-homing potential to already differentiated Th2 cells before inducing a switch in their cytokine production profile may explain the observed exacerbation of allergic skin diseases following bacterial infections.

Regulation of human T helper cell differentiation by antigen-presenting cells: the bee venom phospholipase A2 model.

Whereas some individuals develop immunity to bee sting and mount protective IgG4- mediated antibody responses to bee venom phospholipase A2 (PLA), others produce large amounts of PLA-specific IgE antibodies and become allergic to this, otherwise, innocuous antigen. PLA-specific IgE responses are the result of imbalanced T helper (Th)2-cell differentiation. There are multiple mechanisms driving the differentiation of naive CD4+ T cells into Th1- or Th2-cell phenotypes. Most of them are linked to the conditions occurring during initial or repeated encounters with the allergen, in the context of an antigen-presenting cell (APC). The different types of APC and their availability to display particular cytokine production profiles, pattern recognition receptors, costimulatory molecules and specific HLA haplotypes are key determinants for human Th1- and Th2-cell polarization.

CCL18 is expressed in atopic dermatitis and mediates skin homing of human memory T cells.

CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA(+) T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

Sustained T-bet expression confers polarized human TH2 cells with TH1-like cytokine production and migratory capacities.

BACKGROUND: The transcription factor T-bet mediates IFN-gamma production by T(H)1 cells and suppresses T(H)2 cytokine production when ectopically expressed in polarized murine T(H)2 cells. Thus T-bet-mediated inhibition of T(H)2 cytokine production might be beneficial for the treatment of allergic diseases like asthma or atopic dermatitis. OBJECTIVE: We sought to investigate the effects of ectopic T-bet expression in highly polarized human T(H)2 cells obtained from skin biopsy specimens of patients with atopic dermatitis. METHODS: The cytokine production of T(H)2 cells retrovirally transfected with a vector expressing human T-bet was determined by means of intracellular FACS staining and ELISA. The effects of T-bet transfection were analyzed at the mRNA level by means of real-time PCR and DNA microarrays and confirmed by using functional chemokine response assays. RESULTS: Transfection of T-bet into T(H)2 cells induced high levels of IFN-gamma and suppressed IL-5, but IL-2 and IL-4 production remained unchanged. T-bet transfection also induced IL-12Rbeta2 and CXCR3 expression on human T(H)2 cells, whereas the IL-18 receptor was only induced as a consequence of T-bet-mediated increased responsiveness to IL-12. Furthermore, sustained T-bet expression in human T(H)2 cells induced IL-2 production and decreased the secretion of IL-4. In addition, the chemokine receptor repertoire of these cells was changed toward a T(H)1-like profile. CONCLUSION: The combined switch in cytokine pattern and migratory potential of highly polarized human T(H)2 cells mediated by T-bet might provide an additional advantage for the treatment of allergic diseases.

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties.

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.

Targeting CLA/E-selectin interactions prevents CCR4-mediated recruitment of human Th2 memory cells to human skin in vivo.

Naive Th cells, bearing receptors for cutaneous antigens, become activated in skin-draining lymph nodes and express cutaneous lymphocyte antigen (CLA), which confers to these cells the capacity to migrate into the skin to exert their normal effector functions. In the case of atopic dermatitis (AD), allergen-specific Th2 cells generate exacerbated responses and induce skin inflammation. In such a situation, interfering with the specific mechanism of skin homing would provide a therapeutic benefit. Here we report that CLA+ Th2 memory cells, derived from skin lesions of AD patients, selectively migrate to human skin grafts transplanted onto SCID mice in response to CCR4 but not CCR3, CCR8 or CXCR3 ligands. Skin homing of human CCR4+ Th2 memory cells was Pertussis toxin sensitive and restricted to the CLA+ subset. Furthermore, treatment of these mice with anti-E-selectin monoclonal antibody was sufficient to prevent CCL22-mediated Th2 cell migration to human skin, which both, validates the model and highlights the importance of CLA/E-selectin interactions in the homing process of Th2 cells to the skin. Using this mechanistic model we demonstrate that skin homing of human Th2 memory cells can be efficiently suppressed using a low molecular weight E-selectin antagonist, which is of clinical relevance for the treatment of inflammatory skin diseases, including AD.

Regulation of the Cytokine Production of Allergen-Specific Human T-Cell Clones by the Allergen.

Human T-cell clones to bee venom phospholipase A2 (PLA), isolated from allergic, hyposensitized and hyperimmune individuals to bee sting, secrete both IL-4 and IFN-γ after antigen stimulation. However, the production of IL-4 was higher than the production of IFN-γ. The threshold of the antigen dose required for IL-4 secretion was lower than for IFN-γ. Furthermore, clones derived from allergic and hyposensitized individuals generally expressed increasing ratios of IL-4/IFN-γ production with increasing concentrations of PLA. In contrast, most clones isolated from hyperimmune subjects expressed an opposite dose relationship for IL-4/IFN-γ production.