Ornella Patrizi

Inherited DOCK2 Deficiency in Patients with Early-Onset Invasive Infections

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

Background Gain‐of‐function (GOF) mutations affecting the coiled‐coil domain or the DNA‐binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. Objective We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. Methods Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. Results Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN‐&ggr; in response to IL‐15 and reduced proliferation after stimulation with IL‐2 or IL‐15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN‐&agr; is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon‐induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL‐15, whereas STAT5 phosphorylation and DNA binding to IL‐2 receptor &agr; (IL2RA) are reduced or not affected in response to the same cytokine. Conclusion These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities. Graphical abstract Figure. No Caption available.

Effects of opioid therapy on human natural killer cells

Opioid compounds, such as morphine, induce powerful analgesic effects and are extensively used clinically to treat a wide variety of pain. The aim of our study was to evaluate the impact of opioid therapy on phenotype and function peripheral blood NK cells. The patients were referred to three Italian pain therapy centers (Milan, Pavia, Piacenza) for chronic pain in neuropathic or mixed somatic components. The patients were between 18 and 75 years old and were of Caucasian ethnicity. We studied the expression of activating and inhibitory NK receptors to discriminate NK subsets with different CD56 surface expression intensities (CD56(bright) and CD56(dull) NK cells). The flow cytometry analysis of the NK cells was at normal levels in peripheral blood lymphocytes with fewer CD56(bright) compared to the CD56(dull) NK cell subset when compared to blood from drug free donors. Furthermore, the cytolytic activity of in vitro patient NK cells analyzed was not lower, as would be expected from the regular expression of activating NK receptors for both subsets. Taken together, these data indicate that NK cells from opioid treated patients do not show any signs of NK cell immune-suppression.

Occurrence of nodular lymphocyte-predominant hodgkin lymphoma in hermansky-pudlak type 2 syndrome is associated to natural killer and natural killer T cell defects.

Hermansky Pudlak type 2 syndrome (HPS2) is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene). The defect results in the impairment of the adaptor protein 3 (AP-3) complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL) in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf) + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56brightCD16− Natural Killer (NK) cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.

NFKB1 regulates human NK cell maturation and effector functions.

NFKB1, a component of the canonical NF-κB pathway, was recently reported to be mutated in a limited number of CVID patients. CVID-associated mutations in NFKB2 (non-canonical pathway) have previously been shown to impair NK cell cytotoxic activity. Although a biological function of NFKB1 in non-human NK cells has been reported, the role of NFKB1 mutations for human NK cell biology and disease has not been investigated yet. We decided therefore to evaluate the role of monoallelic NFKB1 mutations in human NK cell maturation and functions. We show that NFKB1 mutated NK cells present impaired maturation, defective cytotoxicity and reduced IFN-γ production upon in vitro stimulation. Furthermore, human IL-2 activated NFKB1 mutated NK cells fail to up-regulate the expression of the activating marker NKp44 and show reduced proliferative capacity. These data suggest that NFKB1 plays an essential novel role for human NK cell maturation and effector functions.

Primitive Neuroectodermal Tumor in an Ovarian Cystic Teratoma: Natural Killer and Neuroblastoma Cell Analysis

In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patients peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.

Natural killer cells from patients with recombinase-activating gene and non-homologous end joining gene defects comprise a higher frequency of CD56bright NKG2A+++ cells, and yet display increased degranulation and higher perforin content

Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

B7-H6-mediated downregulation of NKp30 in NK cells contributes to ovarian carcinoma immune escape

In this study the phenotype and function of tumor-associated NK cells from perito- neal fluids of a selected cohort of patients with seropapillary ovarian carcinoma were analyzed. In >50% of these patients the expression of the activating receptor NKp30 (1) in tumor-associated NK cells was substantially reduced as compared to autolo- gous peripheral blood NK cells. The impaired expression of this receptor was associ- ated with the presence of one of its cellular ligands (B7-H6) (2), which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the peri- toneal fluid. NK cells from patients expressing this NKp30low phenotype displayed an impaired IFNγ production and cytolytic function when tested against target cells expressing surface B7-H6. Our data also suggest that in these patients the defective expression and function of NKp30 may be induced by the chronic engagement of this receptor by soluble B7-H6 or by tumor cells expressing this ligand. The impairment of NK cell functions described herein could represent a novel mechanism by which the tumor microenvironment may contribute to the escape from immune surveillance. This work was supported by grants awarded by Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5x1000 no. 9962 and IG 2014 Id. 15704 (Alessandro Moretta); PRIN 2010 (Alessandro Moretta); Progetto di Ricerca Fondazione Carige 2013 (Emanuela Marcenaro); Progetto di Ricerca di Ateneo 2014 (Emanuela Marcenaro). Eric Vivier laboratory is supported by the European Research Council (THINK Advanced Grant), by Equipe Labellisee La Ligue and by institutional grants from INSERM, CNRS, and Aix-Marseille University to CIML.

Reduced NK cell activity and abnormal expression of CCR7 and CXCR1 by NK cells analysis in patients with DOCK8 deficiency

DOCK8-deficiency is an autosomal recessive primary immunodeficiency that is characterized by multiple abnormalities of the immune system, including a defect of NK cell cytotoxicity which could not be restored after IL2 stimulation. Nevertheless, unanswered questions remain regarding how the absence of DOCK8 leads to predisposition for malignancy, viral, fungal, and bacterial infections. To address these questions we have analyzed NK cell phenotype and functions in patients with DOCK8 deficiency. We observed that NK cells derived from five DOCK8-deficient patients displayed dramatically reduced cytotoxicity which was partially restored after IL-2 stimulation. Analysis of activating and inhibitory NK receptors, including KIRs molecules, chemokine receptors and activation markers on gated CD56+cells by cytofluorimetric analysis showed a substantial defect of CCR7 expression by CD56bright NK cells. Noteworthy, we have also detected the expression of NKG2C and of the chemokine receptor CXCR1 on CD56dull NK cells in DOCK8-deficient cells. Because CCR7 expression by NK cells can be induced after cell culture with IL18 we stimulated NK cells from DOCK-8 deficient patients with IL18. Despite unstimulated CD56bright NK cells from DOCK8 patients showed reduced CCR7 expression, we could not detect any increase of CCR7 on CD56 dull and bright NK cells of DOCK8-deficient patients, whereas CCR7 expression on NK cells derived from healthy donors significantly increased from 5% to 17%. Taken together our results suggest that NK cells of DOCK-8 deficient patients show reduced cytotoxicity and abnormal expression of the chemokine receptors CXCR1 and CCR7 suggesting an abnormal recruitment of these cells to secondary lymphoid organs.