Progressive lentivirus infection induces natural killer cell receptor-expressing B cells in the gastrointestinal tract.

OBJECTIVE: Recently, a seemingly novel innate immune cell subset bearing features of natural killer and B cells was identified in mice. So-called NKB cells appear as first responders to infections, but whether this cell population is truly novel or is in fact a subpopulation of B cells and exists in higher primates remains unclear. The objective of this study was to identify NKB cells in primates and study the impact of HIV/SIV infections. DESIGN AND METHODS: NKB cells were quantified in both naive and lentivirus infected rhesus macaques and humans by excluding lineage markers (CD3, CD127) and positive Boolean gating for CD20, NKG2A/C and/or NKp46. Additional phenotypic measures were conducted by RNA-probe and traditional flow cytometry. RESULTS: Circulating cytotoxic NKB cells were found at similar frequencies in humans and rhesus macaques (range, 0.01-0.2% of total lymphocytes). NKB cells were notably enriched in spleen (median, 0.4% of lymphocytes), but were otherwise systemically distributed in tonsil, lymph nodes, colon, and jejunum. Expression of immunoglobulin was highly variable, but heavily favoured IgM and IgA rather than IgG. Interestingly, NKB cell frequencies expanded in PBMC and colon during SIV infection, as did IgG expression, but were generally unaltered in HIV-infected humans. CONCLUSION: These results suggest a cell type expressing both natural killer and B-cell features exists in rhesus macaques and humans and are perturbed by HIV/SIV infection. The full functional niche remains unknown, but the unique phenotype and systemic distribution could make NKB cells unique targets for immunotherapeutics or vaccine strategies.

Natural killer cells play an essential role in resolution of antigen-induced inflammation in mice.

This study examined whether NK cells are important for resolution of antigen-induced inflammation. C57BL/6 mice were immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Mice were injected intravenously with anti-asialo GM1 (αASGM1) or a control antibody 24h prior to peritonitis induction and peritoneal exudate collected at different time points. Expression of surface molecules and apoptosis on peritoneal cells was determined by flow cytometry and concentration of chemokines, cytokines, soluble cytokine receptors and lipid mediators by ELISA and LC-MS/MS. Apoptosis in parathymic lymph nodes and spleens was determined by TUNEL staining. Mice administered αASGM1 had lower peritoneal NK cell numbers and a higher number of peritoneal neutrophils 12h after induction of inflammation than control mice. The number of neutrophils was still high in the αASGM1 treated mice when their number had returned to baseline levels in the control mice, 48h after induction of inflammation. Peritoneal concentrations of the neutrophil regulators G-CSF and IL-12p40 were higher at 12h in the αASGM1 treated mice than in the control mice, whereas concentrations of lipid mediators implicated in resolution of inflammation, i.e. LXA4 and PGE2, were lower. Reduced apoptosis was detected in peritoneal neutrophils as well as in draining lymph nodes and spleens from the αASGM1 treated mice compared with that in the control mice. In addition, αASGM1 treated mice had lower number of peritoneal NK cells expressing NKp46 and NKG2D, receptors implicated in NK cell-induced neutrophil apoptosis. Furthermore, αASGM1 treatment completely blocked the increase in CD27+ NK cells that occurred in control mice following induction of inflammation, but CD27+ NK cells have been suggested to have a regulatory role. These results indicate a crucial role for NK cells in resolution of antigen-induced inflammation and suggest their importance in tempering neutrophil recruitment and maintaining neutrophil apoptosis.

Enhanced natural killer cell activity is found in exposed uninfected recipients of hepatitis C-contaminated blood.

A minority of injecting drug users, termed exposed uninfected, are resistant to hepatitis C (HCV) infection despite repeated low-dose exposures. We identify for the first time a cohort of blood recipients who remained uninfected despite large-dose exposure to HCV-contaminated blood and characterize immune factors that may confer protection. Of 1340 blood recipients from the English Look Back database who were transfused HCV-contaminated blood, we identified 8 who remained uninfected. In these 8 exposed uninfecteds, we characterized their natural killer (NK) cell populations and HCV-specific T-cell responses. Findings were compared with 10 spontaneous resolvers of HCV infection, 10 patients with chronic HCV infection and 10 healthy controls. Exposed uninfecteds had significantly greater numbers of NK cells with the activating receptor NKp30+ on CD56bright and CD56dim subsets compared with other groups (P < .05). Following interleukin-2 activation, NK cells of exposed uninfecteds had enhanced cytotoxicity that positively correlated with NKp30 expression (P = .02). Differences in NKp80 and KIR2DL3 expression were also observed. HCV-specific T-cell responses were observed in some exposed uninfecteds but of low amplitude. Exposure without infection following transfusion of HCV-contaminated blood is a very rare phenomenon and suggests a high level of resistance to infection. Enhanced NK cell activation and killing, with weak HCV-specific T-cell responses, were observed many years after exposure in uninfected recipients and may contribute to protection from HCV acquisition, although additional protective factors are being sought in this important cohort.

Natural killer-cell counts are associated with molecular relapse-free survival after imatinib discontinuation in chronic myeloid leukemia: the IMMUNOSTIM study.

Despite persistence of leukemic stem cells, patients with chronic myeloid leukemia who achieve and maintain deep molecular responses may successfully stop the tyrosine kinase inhibitor imatinib. However, questions remain unanswered regarding the biological basis of molecular relapse after imatinib cessation. In IMMUNOSTIM, we monitored 51 patients from the French Stop IMatinib trial for peripheral blood T cells and natural killer cells. Molecular relapse-free survival at 24 months was 45.1% (95% CI: 31.44%-58.75%). At the time of imatinib discontinuation, non-relapsing patients had significantly higher numbers of natural killer cells of the cytotoxic CD56dim subset than had relapsing patients, while CD56bright natural killer cells, T cells and their subsets did not differ significantly. Furthermore, the CD56dim natural killer-cell count was an independent prognostic factor of molecular-relapse free survival in a multivariate analysis. However, expression of natural killer-cell activating receptors, BCR-ABL1+ leukemia cell line K562-specific degranulation and cytokine-induced interferon-gamma secretion were decreased in non-relapsing and relapsing patients as compared with healthy individuals. After imatinib cessation, the natural killer-cell count increased significantly and stayed higher in non-relapsing patients than in relapsing patients, while receptor expression and functional properties remained unchanged. Altogether, our results suggest that natural killer cells may play a role in controlling leukemia-initiating cells at the origin of relapse after imatinib cessation, provided that these cells are numerous enough to compensate for their functional defects. Further research will decipher mechanisms underlying functional differences between natural killer cells from patients and healthy individuals and evaluate the potential interest of immunostimulatory approaches in tyrosine kinase inhibitor discontinuation strategies. (ClinicalTrial.gov Identifier NCT00478985).

The microenvironment of visceral adipose tissue and liver alter natural killer cell viability and function.

The role of NK cells in visceral adipose tissue (VAT) and liver inflammation in obesity is not fully understood. This study investigated the frequency, cytokine expression, chemokine receptor, and cytotoxicity receptor profile of NK cells in the blood, omentum, and liver of patients with the obesity-associated cancer, oesophageal adenocarcinoma (OAC). The effect of chronically inflamed tissue microenvironments on NK cell viability and function was also examined. We identified significantly lower NK cell frequencies in the liver of OAC patients compared with healthy controls and within the omentum and liver of OAC patients compared with blood, whereas IL-10-producing populations were significantly higher. Interestingly, our data suggest that reduced frequencies of NK cells in omentum and liver of OAC patients are not a result of impaired NK cell chemotaxis to these tissues. In fact, our functional data revealed that secreted factors from omentum and liver of OAC patients induce significant levels of NK cell death and lead to reduced percentages of TNF-α+ and NKP46+ NK cells and higher frequencies of IL-10-producing NK cells. Together, these data suggest that the omental and hepatic microenvironments of OAC patients alter the NK cell phenotype to a more anti-inflammatory homeostatic role.

Specific phenotype and function of CD56-expressing innate immune cell subsets in human thymus.

Whereas innate immune cells, such as NK and innate lymphoid cells (ILCs), have been characterized in different human tissues, knowledge on the thymic CD56-expressing cell subsets is limited. In this study, the rare subpopulations of thymic CD56+CD3- cells from samples of >100 patients have been successfully analyzed. The results revealed fundamental differences between thymic and peripheral blood (PB) CD56+CD3- cells. Thymic tissues lacked immunoregulatory CD56highCD16dim NK cells but showed two Eomes+CD56dim subsets on which common NK cell markers were significantly altered. CD56dimCD16high cells expressed high amounts of NKG2A, NKG2D, and CD27 with low CD57. Conversely, CD56dimCD16dim cells displayed high CD127 but low expression of KIR, NKG2D, and natural cytotoxicity receptors (NCRs). Thymic CD56+CD3- cells were able to gain cytotoxicity but were especially immunoregulatory cells, producing a broad range of cytokines. Finally, one population of thymic CD56+ cells resembled conventional NK cells, whereas the other represented a novel, noncanonical NK subset.

Overexpression of CD158 and NKG2A Inhibitory Receptors and Underexpression of NKG2D and NKp46 Activating Receptors on NK Cells in Acute Myeloid Leukemia.

BACKGROUND AND AIMS: Natural killer (NK) cells are innate immune system cells that are actively involved in immune-surveillance of tumor cells. Recognition of tumors by NK cells occurred via natural cytotoxicity receptors and killer cell immunoglobulin-like receptors. Some ligands of the activating receptors seem to be present on malignant cells from patients with acute myeloid leukemia. The aim of the study was to evaluate the expression of activating receptors such as NKG2D, DNAM-1, NKp30, and NKp46, and inhibitory receptors such as NKG2A, CD158b, CD158a, and CD158e1 on NK cells from patients with newly diagnosed acute myeloid leukemia before and after stimulation with IL-2 and IL-12. METHODS: Patients were divided into two groups: group 1 AML M3, and group 2 non-M3 AML. Flow cytometry was performed on whole PBMC to evaluate NK cell receptors. RESULTS: Twenty one AML patients, aged 26-78 years, and 11 matched healthy individuals were studied. NKG2D, and NKp46 expression was decreased in group 1 (p <0.019). Patients in Group 2 showed underexpression of the activating receptors NKp46. Differences after stimulation of NK cells with IL-2 and IL-12 were observed only in Group 2, in which a significant decrease in the expression of NKp46 receptor was found (p <0.0016). Patients in groups 1 and 2 showed overexpression of the inhibitory receptors CD158b (p <0.007) and NKG2A (p <0.01). CONCLUSIONS: NKG2D receptor expression is decreased in patients with AML M3. In addition, patients with all FAB types of AML have overexpression of inhibitory receptors such as CD158b and NKG2A and decreased expression of the activating receptor NKp46.

[Expression and significance of the NK cell receptors in primary hepatocellular carcinoma and paracancerous tissues].

AIM: To investigate the expression of the activating and inhibitory receptors on the surface of NK cells of primary hepatocellular carcinoma and its adjacent tissues, and the relationship between these two receptors and occurrence and development of primary liver cancer was analyzed. METHODS: The number and activity of the NK cells, the expression of the activating and the inhibitory receptors on the surface of those cells were detected flow cytometry and immunohistochemistry, which were obtained from 52 cases of primary hepatocellular carcinoma and its adjacent tissues. The relative analysis was done between those results and clinical relative factors. RESULTS: In the tissues of primary hepacellular carcinoma, the number of NK cells is lower than that in the adjacent tissues obviously (P<0.01); the expression of activating receptors, NKG2D and NKP44, is also lower than that in the adjacent tissues obviously (P<0.05); the expression of inhibitory receptors, CD158b and CD159a, is significantly higher than that in the adjacent tissues (P<0.05). A negative correlation was found between the expression of NKG2D, NKP30 and NKP44 and the clinical stage of the liver cancer. The expression of NKG2D, NKP30 and NKP44 was higher in patients with early and middle stages (P<0.05). The content of the inhibitory receptors of NK cells, CD158b and CD159a, is higher in tissues from patients with advanced cancer stage (P<0.05). That's also correlated with the level of AFP and the HBsAg. There is no significant statistical difference between the expression of NK receptors and the distant metastasis, tumor differentiation as well as the tumor size (P>0.05). CONCLUSION: The decrease of NK cell numbers and the activating NK cell receptors and the increase of the inhibitory receptors would be relevant to the incidence of primary hepacellular carcinoma.

Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity.

Chikungunya virus (CHIKV) is a worldwide emerging pathogen. In humans it causes a syndrome characterized by high fever, polyarthritis, and in some cases lethal encephalitis. Growing evidence indicates that the innate immune response plays a role in controlling CHIKV infection. We show here that CHIKV induces major but transient modifications in NK-cell phenotype and function soon after the onset of acute infection. We report a transient clonal expansion of NK cells that coexpress CD94/NKG2C and inhibitory receptors for HLA-C1 alleles and are correlated with the viral load. Functional tests reveal cytolytic capacity driven by NK cells in the absence of exogenous signals and severely impaired IFN-γ production. Collectively these data provide insight into the role of this unique subset of NK cells in controlling CHIKV infection by subset-specific expansion in response to acute infection, followed by a contraction phase after viral clearance.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection.

HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

Sequential recovery of NK cell receptor repertoire after allogeneic hematopoietic SCT.

Alloreactivity of natural killer (NK) cells contributes to the GVL reaction after allogeneic hematopoietic SCT (allo-HSCT). However, various procedure-related factors may affect NK cell maturation and their ability to recognize and kill leukemic cells. In this study, we prospectively evaluated expression of NK cell inhibitory receptors in 83 adults treated with myeloablative, killer cell Ig-like receptor (KIR)-ligand-matched allo-HSCT. NK cell maturation was evaluated by comparing the phenotypic patterns after allo-HSCT with the donor ones. The frequencies of KIR3DL1 were comparable to the donor ones on day +28, while they decreased significantly starting from day +100. The expression of KIR2DL2/3 was significantly lower in patients compared with donors up to day +100. The expression of KIR2DL1, despite continues growth, remained significantly decreased for 1 year after allo-HSCT. NKG2A was over-expressed up to day +180. Within 1 year after allo-HSCT, the NK cell phenotypic pattern tended to recapitulate the donor type. The process was disturbed by the use of steroids with significant differences observed on days +56 (P=0.01) and +100 (P=0.04). Up to day +100, reconstitution of NK cell receptor repertoire correlated with the absolute numbers of circulating CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells. Our observations should be taken into account when trying to predict potential benefit from NK cell alloreactivity.

Methods to identify and characterize different NK cell receptors and their ligands.

Different cellular immune responses are modulated by the cross talk between activating and inhibitory signaling pathways initiated via different cell surface receptors. Similarly, the killing of NK cells is controlled by multiple activating and inhibitory surface receptors. In humans, the major NK triggering receptors, identified so far, include NKp80, 2B4 NKG2D, and CD16 and the natural cytotoxic receptors (collectively named NCRs) include NKp46, NKp44, and NKp30. The two major families of MHC-specific inhibitory receptors identified in humans are the Ig superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily. The different inhibitory receptors show diverse specificity and discriminate between different class I MHC proteins. Much is known about the function and expression patterns of the different NK cell receptors, but the ligand identity of several of the activating NK cell receptors is yet to be discovered. This chapter introduces several research tools that can be used to uncover the identities of different ligands for NK cell receptors.