Elisa Montaldo

CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells

Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34+ hematopoietic precursors in human decidua (dCD34+). We show that dCD34+ cells differ from cord blood- or peripheral blood-derived CD34+ precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34+ cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56brightCD16−KIR+/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34+ cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment.

Human RORγt(+)CD34(+) cells are lineage-specified progenitors of group 3 RORγt(+) innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

Human NK cell receptors/markers: A tool to analyze NK cell development, subsets and function

Natural killer (NK) cells are important components of the innate immunity and play a key role in host defense by virtue of their ability to release cytokines and to mediate cytolytic activity against tumor cells and virus‐infected cells. NK cells were first described more than 30 years ago on the basis of their peculiar functional capabilities. Subsequently, thanks to the production of a variety of monoclonal antibodies, it became possible to identify surface receptors and markers expressed by NK cells as well as to characterize their functional properties. Here, we provide a brief historical overview about the discovery of human NK cell receptors and we delineate the main phenotypic features of differentiating and mature NK cells in healthy donors as well as their alterations in certain pathologic conditions.

Group 3 innate lymphoid cells (ILC3s): Origin, differentiation, and plasticity in humans and mice

Since their discovery, innate lymphoid cells (ILCs) have been the subject of intense research. As their name implies, ILCs are innate cells of lymphoid origin, and can be grouped into subsets based on their cytotoxic activity, cytokine profile, and the transcriptional requirements during ILC differentiation. The main ILC groups are “killer” ILCs, comprising NK cells, and “helper‐like” ILCs (including ILC1s, ILC2s, and ILC3s). This review examines the origin, differentiation stages, and plasticity of murine and human ILC3s. ILC3s express the retinoic acid receptor (RAR) related orphan receptor RORγt and the signature cytokines IL‐22 and IL‐17. Fetal ILC3s or lymphoid tissue inducer cells are required for lymphoid organogenesis, while postnatally developing ILC3s are important for the generation of intestinal cryptopatches and isolated lymphoid follicles as well as for the defence against pathogens and epithelial homeostasis. Here, we discuss the transcription factors and exogenous signals (including cytokines, nutrients and cell‐to‐cell interaction) that drive ILC3 lineage commitment and acquisition of their distinctive effector program.

Human Natural Killer Cells: Origin, Receptors, Function, and Clinical Applications

Natural killer (NK) cells are important effectors playing a relevant role in innate immunity, primarily in tumor surveillance and in defenses against viruses. Human NK cells recognize HLA class I molecules through surface receptors (KIR and NKG2A) that inhibit NK cell function and kill target cells that have lost (or underexpress) HLA class I molecules as it occurs in tumors or virus-infected cells. NK cell activation is mediated by an array of activating receptors and co-receptors that recognize ligands expressed primarily on tumors or virus-infected cells. In vivo anti-tumor NK cell activity may be suppressed by tumor or tumor-associated cells. Alloreactive NK cells (i.e. those that are not inhibited by the HLA class I alleles of the patient) derived from HSC of haploidentical donors play a major role in the cure of high-risk leukemia, by killing leukemia blasts and patients DC, thus preventing tumor relapses and graft-versus-host disease. The expression of the HLA-C2-specific activating KIR2DS1 may also contribute to NK alloreactivity in patients expressing C2 alleles. A clear correlation has been proven between the size of the alloreactive NK cell population and the clinical outcome. Recently, haplo-HSCT has been further improved with the direct infusion, together with HSC, of donor-derived, mature alloreactive NK cells and TCRγδ+ T cells - both contributing to a prompt anti-leukemia effect together with an efficient defense against pathogens during the 6- to 8-week interval required for the generation of alloreactive NK cells from HSC.

Identification of diverse innate lymphoid cells in human decidua

Innate lymphoid cells (ILCs) are developmentally related cells that play an important role in innate defenses and tissue remodeling. So far, only natural killer (NK) cells have been identified and functionally characterized in human decidua where they contribute to induction of immune suppression, neo-angiogenesis, and tissue building/remodeling. The presence of other ILC subsets in human decidua has not been yet characterized. Here we identify in human decidua, during early pregnancy, two subsets of decidual group 3 ILC (ILC3), including lymphoid tissue inducer (LTi)-like cells and natural cytotoxicity receptors (NCRs)+ILC3 and interferon-(IFN)γ-producing ILC1, different from NK cells. Decidual LTi-like cells produced interleukin -17 (IL-17) and tumor necrosis factor (TNF), while NCR+ILC3 released IL-22 and IL-8. Importantly, NCR+ILC3 and LTi-like cells established functional interactions with stromal cells. Decidual LTi-like cells differentiated into NCR+ILC3, whereas they marginally contributed to NK cell generation. Our data suggest that decidual ILC3 may play a role in innate defenses and in vessel and tissue building, thus contributing to maintenance of pregnancy.

Human NK Cells: From Surface Receptors to the Therapy of Leukemias and Solid Tumors

Natural Killer (NK) cells are major effector cells of the innate immunity. The discovery, over two decades ago, of major histocompatibility complex-class I-specific inhibitory NK receptors and subsequently of activating receptors, recognizing ligands expressed by tumor or virus-infected cells, paved the way to our understanding of the mechanisms of selective recognition and killing of tumor cells. Although NK cells can efficiently kill tumor cells of different histotypes in vitro, their activity may be limited in vivo by their inefficient trafficking to tumor lesions and by the inhibition of their function induced by tumor cells themselves and by the tumor microenvironment. On the other hand, the important role of NK cells has been clearly demonstrated in the therapy of high risk leukemias in the haploidentical hematopoietic stem cell (HSC) transplantation setting. NK cells derived from donor HSC kill leukemic cells residual after the conditioning regimen, thus preventing leukemia relapses. In addition, they also kill residual dendritic cells and T lymphocytes, thus preventing both GvH disease and graft rejection.

Immune response in the conjunctival epithelium of patients with dry eye

The aim of our project was to test the hypothesis that patients with dry eye have a significant degree of inflammation and lymphocyte infiltration in conjunctival epithelium by using flow cytometry analysis of cells stored in cell culture medium. Impression cytology specimens were collected in 15 normal subjects and 15 dry eye patients. Samples collected from the right eye were placed in Phosphate Buffered Saline containing 0.05% paraformaldehyde (PFA), and samples from the left eye in cell culture medium containing 10% foetal calf serum (FCS). Phenotypic analysis was performed on cells derived from 21 dry eye patients and 16 healthy controls. The cells collected in FCS were stained for the expression of CK19, CD3, CD4, CD8, CD56, CD19, CD20, CD14 and HLA-DR, and analyzed by flow cytometry. FCS samples contained a statistically increased number of cells when compared to PFA samples. No statistically significant differences were present in the number of CD45+CK19- cells, CD3+ and CD4+T cells, B and NK cells in dry eye patients compared to healthy controls. In the dry eye group there was a significant difference in the CD4/CD8 ratio respect to what observed in normal subjects, and an increased number of CD14+ cells. HLA-DR expression was increased only in CK19+ conjunctival epithelial cells of dry eye patients. This study indicates that immune cells isolated from the superficial layer of the conjunctiva may play a pivotal role in the pathogenesis of dry eye, and that a new method of preservation of impression cytology samples can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva.

Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor

Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34(+) umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161(+)CD56(+)LFA-1(-) and more differentiated cytolytic CD161(+)CD56(+)LFA-1(+) NK cells. CD161(+)CD56(+)LFA-1(-) NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161(+)CD56(+)LFA-1(-) NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161(+)CD56(+)LFA-1(-) subset, because it did not induce cytokine release or CD107a expression in CD161(+)CD56(+)LFA-1(+) cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.

Development of human natural killer cells and other innate lymphoid cells.

Innate lymphoid cells (ILC) have recently gained much attention in immunology. They represent a novel developmentally related family. Three distinct subsets have been identified on the basis of phenotypic and functional criteria and termed ILC1, ILC2, and ILC3. The available data suggest that ILC play an important role in innate defenses against different pathogens, in lymphoid organogenesis, and in tissue remodeling. All these aspects are relevant in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, in which donor NK cells are known to play a major therapeutic role, while the involvement of other ILC is still undefined. In this context, it has been postulated that all ILC share a common precursor expressing the ID2 transcription factor. While the differentiation of human NK cells (belonging to ILC1) is now well characterized both in vitro and in vivo, limited information is available on the development of human ILC2 and ILC3 and of their relationships with NK cells. In this review, we will summarize the present knowledge on the developmental relationship among different ILC, with particular focus on early stages of NK cell differentiation, and their features shared with ILC2 and ILC3.