IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo.

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand (CD40LG) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Is there a role for tapered topical dose steroidal treatment for dry eye disease? A randomized, pilot study.

PURPOSE: To evaluate the effect of tapered doses of loteprednol-etabonate in dry eye disease patients. MATERIALS AND METHODS: Dry eye and treatment outcomes were assessed by Schirmer I test, tear BUT, lissamine green conjunctival staining, fluorescein corneal staining, and HLA-DR expression on conjunctival cells. Patients received either loteprednol-etabonate 0.5% twice daily for 14 days tapered to once daily for 14 days, and then twice weekly for 28 days (n = 10), or NaCl 0.9%. RESULTS: A significant decrease of ocular surface inflammation and improvement of symptoms was recorded in the study group compared with controls at days 14 and 56. Change from baseline in HLA-DR expression in CD45+ conjunctival cells was significantly higher in treated patients at day 14. Intraocular pressure and best corrected visual acuity were preserved in all treated eyes. CONCLUSIONS: Tapered doses of loteprednol etabonate 0.5% suspension controlled ocular surface inflammation, improving dry eye symptoms.

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression.

Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

Determinants, mechanisms, and functional outcomes of myeloid cell diversity in cancer.

Most, if not all, aspects of carcinogenesis are influenced by the tumor microenvironment (TME), a complex architecture of cells, matrix components, soluble signals, and their dynamic interactions in the context of physical traits of the tissue. Expanding application of technologies for high-dimensional analyses with single-cell resolution has begun to decipher the contributions of the immune system to cancer progression and its implications for therapy. In this review, we will discuss the multifaceted roles of tumor-associated macrophages and neutrophils, focusing on factors that subvert tissue immune homeostasis and offer therapeutic opportunities for TME reprogramming. By performing a critical analysis of available datasets, we elaborate on diversification mechanisms and unifying principles of myeloid cell heterogeneity in human tumors.

Immune signature drives leukemia escape and relapse after hematopoietic cell transplantation.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-γ or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

Effect of Tyrosin Kinase Inhibitors on NK Cell and ILC3 Development and Function.

Tyrosin kinase inhibitors (TKI) sharply improved the prognosis of Chronic Myeloid Leukemia (CML) and of Philadelphia+ Acute Lymphoblastic Leukemia (Ph+ALL) patients. However, TKI are not curative because of the development of resistance and lack of complete molecular remission in the majority of patients. Clinical evidences would support the notion that patient's immune system may play a key role in preventing relapses. In particular, increased proportions of terminally differentiated CD56+CD16+CD57+ NK cells have been reported to be associated with successful Imatinib therapy discontinuation or with a deep molecular response in Dasatinib-treated patients. In view of the potential role of NK cells in immune-response against CML, it is important to study whether any TKI have an effect on the NK cell development and identify possible molecular mechanism(s) by which continuous exposure to in vitro TKI may influence NK cell development and repertoire. To this end, CD34+ hematopoietic stem cells (HSC) were cultured in the absence or in the presence of Imatinib, Nilotinib, or Dasatinib. We show that all compounds exert an inhibitory effect on CD56+ cell recovery. In addition, Dasatinib sharply skewed the repertoire of CD56+ cell population, leading to an impaired recovery of CD56+CD117-CD16+CD94/NKG2A+EOMES+ mature cytotoxic NK cells, while the recovery of CD56+CD117+CD94/NKG2A-RORγt+ IL-22-producing ILC3 was not affected. This effect appears to involve the Dasatinib-mediated inhibition of Src kinases and, indirectly, of STAT5-signaling activation in CD34+ cells during first days of culture. Our studies, reveal a possible mechanism by which Dasatinib may interfere with the proliferation and maturation of fully competent NK cells, i.e., by targeting signaling pathways required for differentiation and survival of NK cells but not of ILC3.

NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1ß) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

Human innate lymphoid cells.

The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed toward NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development.

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF.

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high-risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft-versus-host disease and could also contribute to anti-microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34(+) cells are enriched in lymphoid-committed precursors, while PB CD34(+) cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56(+) CD161(+) ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56(+) CD161(+) ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34(+) cultures was lower for G-CSF-mobilized HSCs (good mobilizers) than for G-CSF+plerixafor-mobilized HSC (poor mobilizers). Moreover, G-CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G-CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.

Human natural killer cells: news in the therapy of solid tumors and high-risk leukemias.

It is well established that natural killer (NK) cells play an important role in the immunity against cancer, while the involvement of other recently identified, NK-related innate lymphoid cells is still poorly defined. In the haploidentical hematopoietic stem cell transplantation for the therapy of high-risk leukemias, NK cells have been shown to exert a key role in killing leukemic blasts residual after conditioning. While the clinical results in the cure of leukemias are excellent, the exploitation of NK cells in the therapy of solid tumors is still limited and unsatisfactory. In solid tumors, NK cell function may be inhibited via different mechanisms, occurring primarily at the tumor site. The cellular interactions in the tumor microenvironment involve tumor cells, stromal cells and resident or recruited leukocytes and may favor tumor evasion from the host's defenses. In this context, a number of cytokines, growth factors and enzymes synthesized by tumor cells, stromal cells, suppressive/regulatory myeloid and lymphoid cells may substantially impair the function of different tumor-reactive effector cells, including NK cells. The identification and characterization of such mechanisms may offer clues for the development of new immunotherapeutic strategies to restore effective anti-tumor responses. In order to harness NK cell-based immunotherapies, several approaches have been proposed, including reinforcement of NK cell cytotoxicity by means of specific cytokines, antibodies or drugs. These new tools may improve NK cell function and/or increase tumor susceptibility to NK-mediated killing. Hence, the integration of NK-based immunotherapies with conventional anti-tumor therapies may increase chances of successful cancer treatment.

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.

MSC and innate immune cell interactions: A lesson from human decidua.

Both experimental and clinical studies revealed that stromal cells (SC) are present in decidua (DSC) and placenta (PSC) at the early and late phase of pregnancy, respectively, and they may contribute to the induction of an anti-inflammatory/tolerogenic microenvironment crucial for the establishment/maintenance of successful pregnancy. These cells share common features with mesenchymal SC. In the present contribution, we provide an overall view on DSC features and on their ability to recruit NK cells and to regulate both differentiation and function not only of NK cells but also of CD14(+) myeloid cells. NK cells represent the large majority of leukocytes populating decidual tissues during the first trimester of pregnancy. Their cross-talk with DSC is thought to play a key role in the establishment of feto-maternal tolerance. We also discuss recent data suggesting that DSC may contribute to tissue remodeling, placentation, and recruitment of leukocytes also through their interaction with innate lymphoid cells (ILC) such as ILC3, that have recently been shown to be present in decidual tissue.

Unique Eomes(+) NK Cell Subsets Are Present in Uterus and Decidua During Early Pregnancy.

Decidual and uterine natural killer (NK) cells have been shown to contribute to the successful pregnancy both in humans and mice. NK cells represent "cytotoxic" group 1 innate lymphoid cells (ILCs) and are distinct from the recently described "helper" ILC1. Here, we show that both in humans and mice the majority of group 1 ILC in endometrium/uterus and decidua express Eomesodermin (Eomes), thus suggesting that they are developmentally related to conventional NK cells. However, they differ from peripheral NK cells. In humans, Eomes(+) decidual NK (dNK) cells express CD49a and other markers of tissue residency, including CD103, integrin ß7, CD9, and CD69. The expression of CD103 allows the identification of different subsets of IFNγ-producing Eomes(+) NK cells. We show that TGFß can sustain/induce CD103 and CD9 expression in dNK cells and decidual CD34-derived NK cells, indicating that the decidual microenvironment can instruct the phenotype of Eomes(+) NK cells. In murine decidua and uterus, Eomes(+) cells include CD49a(-)CD49b(+) conventional NK cells and CD49a(+) cells. Notably, Eomes(+)CD49a(+) cells are absent in spleen and liver. Decidual and uterine Eomes(+)CD49a(+) cells can be dissected in two peculiar cell subsets according to CD49b expression. CD49a(+)CD49b ((-)) and CD49a(+)CD49b(+) cells are enriched in immature CD11b(low)CD27(high) cells, while CD49a(-)CD49b(+) cells contain higher percentages of mature CD11b(high)CD27(low) cells, both in uterus and decidua. Moreover, Eomes(+)CD49a(+)CD49b(-) cells decrease during gestation, thus suggesting that this peculiar subset may be required in early pregnancy rather than on later phases. Conversely, a minor Eomes(-)CD49a(+) ILC1 population present in decidua and uterus increases during pregnancy. CD49b(-)Eomes(±) cells produce mainly TNF, while CD49a(-)CD49b(+) conventional NK cells and CD49a(+)CD49b(+) cells produce both IFNγ and TNF. Thus, human and murine decidua contains unique subsets of group 1 ILCs, including Eomes(+) and Eomes(-) cells, with peculiar phenotypic and functional features. Our study contributes to re-examination of the complexity of uterine and decidual ILC subsets in humans and mice and highlights the role of the decidual microenvironment in shaping the features of these cells.

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 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 patient's 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.

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.

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.