Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin.

NKp46+CD3- natural killer lymphocytes isolated from blood, lymphoid organs, lung, liver and uterus can produce granule-dependent cytotoxicity and interferon-gamma. Here we identify in dermis, gut lamina propria and cryptopatches distinct populations of NKp46+CD3- cells with a diminished capacity to degranulate and produce interferon-gamma. In the gut, expression of the transcription factor RORgammat, which is involved in the development of lymphoid tissue-inducer cells, defined a previously unknown subset of NKp46+CD3- lymphocytes. Unlike RORgammat- lamina propria and dermis natural killer cells, gut RORgammat+NKp46+ cells produced interleukin 22. Our data show that lymphoid tissue-inducer cells and natural killer cells shared unanticipated similarities and emphasize the heterogeneity of NKp46+CD3- cells in innate immunity, lymphoid organization and local tissue repair.

Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1.

Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.

Induction of B7-H6, a ligand for the natural killer cell-activating receptor NKp30, in inflammatory conditions.

B7-H6, a member of the B7 family of immunoreceptors, is as a cell-surface ligand for the NKp30-activating receptor expressed on natural killer cells. B7-H6 is not detected in normal human tissues at steady state but is expressed on tumor cells. However, whether B7-H6 can be expressed in other conditions remains unknown. We analyzed here the pathways that lead to the expression of B7-H6 in nontransformed cells. In vitro, B7-H6 was induced at the surface of CD14(+)CD16(+) proinflammatory monocytes and neutrophils upon stimulation by ligands of Toll-like receptors or proinflammatory cytokines such as interleukin-1ß and tumor necrosis factor α. In these conditions, a soluble form of B7-H6 (sB7-H6) was also produced by activated monocytes and neutrophils. In vivo, B7-H6 was expressed on circulating proinflammatory CD14(+)CD16(+) monocytes in a group of patients in sepsis conditions, and was linked to an increased mortality. sB7-H6 was selectively detected in the sera of patients with gram-negative sepsis and was associated with membrane vesicles that co-sedimented with the exosomal fraction. These findings reveal that B7-H6 is not only implicated in tumor immunosurveillance but also participates in the inflammatory response in infectious conditions.

Mechanisms of systemic low-grade inflammation in HIV patients on long-term suppressive antiretroviral therapy: the inflammasome hypothesis.

OBJECTIVE: We aimed to determine the contribution of inflammasome activation in chronic low-grade systemic inflammation observed in patients with HIV (PWH) on long-term suppressive antiretroviral therapy (ART) and to explore mechanisms of such activation. DESIGN: Forty-two PWH on long-term suppressive ART (HIV-RNA < 40 copies/ml) were compared with 10 HIV-negative healthy controls (HC). METHODS: Inflammasome activation was measured by dosing mature interleukin (IL)-1ß and IL-18 cytokines in patient serum. We explored inflammasome pathways through ex vivo stimulation of PWH primary monocytes with inflammasome activators; expression of inflammasome components by transcriptomic analysis; and metabolomics analysis of patient sera. RESULTS: Median (Q1; Q3) age, ART and viral suppression duration in PWH were 54 (48; 60), 15 (9; 20) and 7.5 (5; 12) years, respectively. Higher serum IL-18 was measured in PWH than in HC (61 (42; 77) vs. 36 (27-48 pg/ml), P = 0.009); IL-1ß was detected in 10/42 PWH (0.5 (0.34; 0.80) pg/ml) but not in HC. Monocytes from PWH did not produce more inflammatory cytokines in vitro , but secretion of IL-1ß in response to NOD like receptor family, pyrin domain containing 3 (NLRP3) inflammasome stimulation was higher than in HC. This was not explained at the transcriptional level. We found an oxidative stress molecular profile in PWH sera. CONCLUSION: HIV infection with long-term effective ART is associated with a serum inflammatory signature, including markers of inflammasome activation, and an increased activation of monocytes upon inflammasome stimulation. Other cells should be investigated as sources of inflammatory cytokines in PWH. Oxidative stress might contribute to this chronic low-grade inflammation.

Interferon-γ production by natural killer cells and cytomegalovirus in critically ill patients.

OBJECTIVE: The mechanisms involved in cytomegalovirus reactivation in critically ill patients who were previously immunocompetent are still unknown. The current study was designed to evaluate the possible role of natural killer cells in the reactivation of cytomegalovirus in these patients. DESIGN: Prospective observational. SETTING: : A medical intensive care unit of a university hospital. PATIENTS: Fifty-one subjects, including 15 patients who experienced cytomegalovirus reactivation (cases) during their intensive care unit stay and 15 patients who matched intensive care unit controls, selected from a cohort of consecutive nonimmunocompromised intensive care unit patients, as well as healthy controls. INTERVENTIONS: Tests included weekly systematic immunomonitoring and routine screening for cytomegalovirus infection until discharge from the intensive care unit or death. The immunophenotype and functions of natural killer cells were performed by flow cytometry, and serum levels of pro- and anti-inflammatory cytokines were determined by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: The overall occurrence of cytomegalovirus reactivation in the cohort was 27%. No differences of natural killer cell effector functions were observed at admission between cases and controls. Instead, before cytomegalovirus reactivation, the ability of natural killer cells to secrete interferon-γ was significantly reduced in cases as compared with controls upon stimulation with antibody-coated target cells (p = .029) and with K562 cell stimulation (p = .029). No phenotypic or quantitative differences were observed between cases and controls. Cases exhibited higher levels of interleukin 10 (p = .031) and interleukin 15 (p = .021) than controls before cytomegalovirus reactivation. CONCLUSIONS: Impaired natural killer cell function with reduced interferon-γ secretion precedes the occurrence of cytomegalovirus reactivation among previously immunocompetent critically ill patients.

Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity.

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype, as well as through phenotypic and functional analyses of NK cells, both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether, these results may have important clinical implications for the selection of optimal donors for haplo-HSCT.

Expression of the HLA-C2-specific activating killer-cell Ig-like receptor KIR2DS1 on NK and T cells.

Killer Ig-like receptors (KIRs) are MHC class I-specific receptors expressed by Natural Killer (NK) and T cell subsets. KIRs either inhibit (KIR-L) or activate (KIR-S) lymphocyte functions. Inhibitory KIR2DL1 and activating KIR2DS1 share ligand specificity for the HLA-C2 group, consistent with their almost identical extracytoplasmic domain. This homology hampered the distinction between KIR2DL1 and KIR2DS1. We report here the characterization of the KIR2DS1(+) subsets among primary human NK and T cells. Regardless of the host HLA-C genotype, around 10% of circulating NK cells expressed KIR2DS1 in absence of KIR2DL1. In HLA-C2 individuals, KIR2DS1 was not able to induce NK cell education (i.e., the acquisition of NK cell competence) nor to interfere with KIR2DL1-induced NK cell education. KIR2DS1 was also present on rare oligoclonal TCRalphabeta(+)CD8alpha(+) and TCRalphabeta(+)CD4(-)CD8(-) subsets. As KIR2DS1 has been associated with autoimmunity and hematopoietic stem cell transplantation, these results pave the way to dissect the function of KIR2DS1 in these clinical conditions.

A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo.

Natural killer (NK) cells have been originally defined by their "naturally occurring" effector function. However, only a fraction of human NK cells is reactive toward a panel of prototypical tumor cell targets in vitro, both for the production of interferon-gamma (IFN-gamma) and for their cytotoxic response. In patients with IL12RB1 mutations that lead to a complete IL-12Rbeta1 deficiency, the size of this naturally reactive NK cell subset is diminished, in particular for the IFN-gamma production. Similar data were obtained from a patient with a complete deficit in IL-12p40. In addition, the size of the subset of effector memory T cells expressing CD56 was severely decreased in IL-12Rbeta1- and IL-12p40-deficient patients. Human NK cells thus require in vivo priming with IL-12/23 to acquire their full spectrum of functional reactivity, while T cells are dependent upon IL-12/23 signals for the differentiation and/or the maintenance of CD56(+) effector memory T cells. The susceptibility of IL-12/23 axis-deficient patients to Mycobacterium and Salmonella infections in combination with the absence of mycobacteriosis or salmonellosis in the rare cases of human NK cell deficiencies point to a role for CD56(+) T cells in the control of these infections in humans.

Association of immune-activation and senescence markers with non-AIDS-defining comorbidities in HIV-suppressed patients.

OBJECTIVES: We studied the link between T-cell activation, differentiation and senescence phenotypes and non-AIDS-related comorbidities in HIV-suppressed patients. DESIGN: Patients included in the ANRS CO3 Aquitaine Cohort were consecutively enrolled in this cross-sectional study between October 2011 and May 2013 called Chronic Immune Activation and Senescence (CIADIS) study. METHODS: We summarized immune markers [CD4 and CD8 activation (DR), differentiation (naive and terminally differentiated memory T cells), and senescence (CD57CD28)] in a weighted immune score by principal component analysis called CIADIS. Previously described Veterans Aging Cohort Study (VACS) index and immune risk profile (IRP) scores were calculated. We used adjusted logistic regression to assess the association between the CIADIS score and the presence of at least three non-AIDS-defining comorbidities. RESULTS: Of 876 patients with an undetectable viral load, 73.4% were men and median age was 50.5 years [interquartile range (IQR) 44.7-56.7 years]. Median CD4 T-cell count was 579/µl (IQR 429-759 cells/µl), and median duration of HIV viral suppression was 5.3 years (IQR 2.3-8.7). The weighted CIADIS score was associated with at least three comorbidities (odds ratio 1.3 for 1 SD more, 95% confidence interval 1.0, 1.6) independently of age, sex, AIDS stage, and the Veterans Aging Cohort Study score. The CIADIS and the immune risk profile scores were significantly associated with at least three comorbidities in adjusted models restricted to patients younger than 60 years. None of the tested scores were associated with at least three comorbidities in patients older than 60 years. CONCLUSIONS: The weighted CIADIS score based on activation, senescence, and differentiation markers might help physicians identifying patients at a higher risk for non-AIDS-related comorbidities.

TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Phenotype and functions of natural killer cells in critically-ill septic patients.

RATIONALE: Natural killer cells, as a major source of interferon-γ, contribute to the amplification of the inflammatory response as well as to mortality during severe sepsis in animal models. OBJECTIVE: We studied the phenotype and functions of circulating NK cells in critically-ill septic patients. METHODS: Blood samples were taken <48 hours after admission from 42 ICU patients with severe sepsis (n = 15) or septic shock (n = 14) (Sepsis group), non-septic SIRS (n = 13) (SIRS group), as well as 21 healthy controls. The immuno-phenotype and functions of NK cells were studied by flow cytometry. RESULTS: The absolute number of peripheral blood CD3-CD56(+) NK cells was similarly reduced in all groups of ICU patients, but with a normal percentage of NK cells. When NK cell cytotoxicity was evaluated with degranulation assays (CD107 expression), no difference was observed between Sepsis patients and healthy controls. Under antibody-dependent cell cytotoxicity (ADCC) conditions, SIRS patients exhibited increased CD107 surface expression on NK cells (62.9[61.3-70]%) compared to healthy controls (43.5[32.1-53.1]%) or Sepsis patients (49.2[37.3-62.9]%) (p = 0.002). Compared to healthy (10.2[6.3-13.1]%), reduced interferon-γ production by NK cells (K562 stimulation) was observed in Sepsis group (6.2[2.2-9.9]%, p<0.01), and especially in patients with septic shock. Conversely, SIRS patients exhibited increased interferon-γ production (42.9[30.1-54.7]%) compared to Sepsis patients (18.4[11.7-35.7]%, p<0.01) or healthy controls (26.8[19.3-44.9]%, p = 0.09) in ADCC condition. CONCLUSIONS: Extensive monitoring of the NK-cell phenotype and function in critically-ill septic patients revealed early decreased NK-cell function with impaired interferon-γ production. These results may aid future NK-based immuno-interventions. TRIAL REGISTRATION: NTC00699868.

Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis.

Natural killer (NK) cells are bone marrow (BM)-derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases.

Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency.

Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56(dim) NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients' fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients' growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56(dim) subset in patients was associated with a lower rate of NK CD56(bright) cell proliferation, and the maturation of NK CD56(bright) cells toward an NK CD56(dim) phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.

Intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation.

The spleen is a major homing site for NK cells. How they traffic to and within this site in homeostatic or inflammatory conditions is, however, mostly unknown. Here we show that NK cells enter the spleen through the marginal sinus and home to the red pulp via a pertussis toxin-insensitive mechanism. Upon inflammation induced by poly(I:C) injection or mouse cytomegalovirus infection, many NK cells left the red pulp while others transiently entered the white pulp, predominantly the T cell area. This migration was dependent on both CXCR3 and CCL5, suggesting a synergy between CXCR3 and CCR5, and followed the path lined by fibroblastic reticular cells. Thus, the entry of NK cells in the white pulp is limited by the expression of pro-inflammatory chemokines. This phenomenon ensures the segregation of NK cells outside of the white pulp and might contribute to the control of immunopathology.

Expression of the CD85j (leukocyte Ig-like receptor 1, Ig-like transcript 2) receptor for class I major histocompatibility complex molecules in idiopathic inflammatory myopathies.

OBJECTIVE: Expression of class I major histocompatibility complex (MHC) molecules on the surface of muscle cells is a biologic feature of idiopathic inflammatory myopathies (IIM). Class I MHC-transgenic mouse models support a causative role for class I MHC expression by muscle cells in the pathogenesis of IIM. The muscle lesions are characterized by leukocyte infiltration. We undertook this study to analyze the expression in muscle lesions of various class I MHC-specific receptors on leukocytes and natural killer (NK) cells. METHODS: We generated a panel of cell transfectants to control the immunofluorescence analysis of class I MHC receptor expression. We then analyzed the expression of CD158 (killer cell Ig-like receptors [KIRs]) and CD85j (leukocyte Ig-like receptor 1, Ig-like transcript 2) on muscle sections prepared from 14 patients with IIM (5 with dermatomyositis [DM], 5 with polymyositis [PM], and 4 with sporadic inclusion body myositis [IBM]). RESULTS: We could not detect the presence of NK cells in inflammatory lesions. However, the class I MHC receptor CD85j, but no KIRs, was expressed by inflammatory cells infiltrating muscle lesions in IIM. CONCLUSION: CD85j is expressed in PM and sporadic IBM at the sites of partial invasion and in DM in perivascular inflammation, paving the way for dissecting the role of CD85j in the pathogenesis of inflammatory myopathies.

TLR3 deficiency in patients with herpes simplex encephalitis.

Some Toll and Toll-like receptors (TLRs) provide immunity to experimental infections in animal models, but their contribution to host defense in natural ecosystems is unknown. We report a dominant-negative TLR3 allele in otherwise healthy children with herpes simplex virus 1 (HSV-1) encephalitis. TLR3 is expressed in the central nervous system (CNS), where it is required to control HSV-1, which spreads from the epithelium to the CNS via cranial nerves. TLR3 is also expressed in epithelial and dendritic cells, which apparently use TLR3-independent pathways to prevent further dissemination of HSV-1 and to provide resistance to other pathogens in TLR3-deficient patients. Human TLR3 appears to be redundant in host defense to most microbes but is vital for natural immunity to HSV-1 in the CNS, which suggests that neurotropic viruses have contributed to the evolutionary maintenance of TLR3.

Platelet-endothelial cell adhesion molecule-1 and CD146: soluble levels and in situ expression of cellular adhesion molecules implicated in the cohesion of endothelial cells in idiopathic inflammatory myopathies.

OBJECTIVE: Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases characterized by chronic inflammation of muscles. We investigated the role of cellular adhesion molecules implicated in the cohesion of endothelial cells in IIM. METHODS: In 22 patients with IIM we investigated plasma concentrations of soluble junctional adhesion molecules [platelet-endothelial cell adhesion molecule (sPECAM-1) and sCD146] and cellular adhesion molecules [sP-selectin, sE-selectin, intercellular adhesion molecule (sICAM-1), and vascular cell adhesion molecule (sVCAM-1)] implicated in leukocyte/endothelial cell interactions. Results were compared to a control group. Muscle biopsy samples from 8 out of 22 IIM patients were studied by immunohistochemistry for tissue expression of these molecules and compared to normal muscle samples. PECAM-1 and CD146 expression was also studied using immunoblots from muscle biopsies from 5 patients and 2 controls. RESULTS: We observed distinct patterns of soluble levels and in situ expression between dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (s-IBM). PM samples showed significantly increased levels of sCD146, sPECAM-1, and s-ICAM1 and increased expression of CD146, CD31, and ICAM-1 in endothelial cells, whereas CD146 and ICAM-1 were also recorded in some muscle fibers. In DM, sE-selectin, sP-selectin, and sPECAM-1 were significantly increased, with abnormal expression of ICAM-1 in endothelial cells and perifascicular muscle fibers. In the small group of s-IBM samples, results were similar to PM, but the only significant increase was the level of sPECAM-1. Immunoblots confirmed increased expression of PECAM-1 and CD146 in all IIM muscles in comparison to controls, with the highest expression in PM and IBM samples. CONCLUSION: We observed abnormal increases of soluble levels of adhesion molecules implicated in endothelial cell junctions in PM (sCD146, sPECAM-1) and to a lesser extent in DM and s-IBM (sPECAM-1). We conclude that the distinctly different profiles between PM/s-IBM and DM reflect differences in the pathophysiological background of these diseases.