Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells
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ABSTRACT: NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK-cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multi-functional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.
Project description:NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.
Project description:Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H(+) NK cells from Ly49h-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and Ly49h-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the Ifng gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific cis-regulatory mechanism of Ifnγ transcript expression potentially relevant to NK cell function in health and disease.
Project description:One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.
Project description:The similarity of stromal-like Wilms tumor (str-WT) cells with mesenchymal stem cells (MSC), suggests their relevant role in the interplay with immune cells in the tumor microenvironment. We investigated the interaction between str-WT cells and NK cells. We observed that str-WT cells expressed some major ligands for activating and inhibitory NK cell receptors. Moreover, they expressed inhibitory checkpoint molecules involved in the negative regulation of anti-tumor immune response. The analysis of the interaction between str-WT cells and NK lymphocytes revealed that activated NK cells could efficiently degranulate upon interaction with str-WT cells. On the other hand, str-WT cells could exert potent inhibitory effects on cytokine-induced activation of NK cell proliferation and phenotype, which were mediated by the production of IDO and PGE2 inhibitory factors. Our data provide insight into the molecular interactions between str-WT cells and NK lymphocytes that may result in different outcomes possibly occurring in the WT microenvironment.
Project description:Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.
Project description:Long, non-coding RNAs (lncRNAs) are involved in the regulation of many cellular processes. The lncRNA IFNG-AS1 was found to strongly influence the responses to several pathogens in mice by increasing interferon gamma (IFNγ) secretion. Studies have looked at IFNG-AS1 in T cells, yet IFNG-AS1 function in natural killer cells (NKs), an important source of IFNγ, remains unknown. Here, we show a previously undescribed sequence of IFNG-AS1 and report that it may be more abundant in cells than previously thought. Using primary human NKs and an NK line with IFNG-AS1 overexpression, we show that IFNG-AS1 is quickly induced upon NK cell activation, and that IFNG-AS1 overexpression leads to increased IFNγ secretion. Taken together, our work expands IFNG-AS1's activity to the innate arm of the type I immune response, helping to explain its notable effect in animal models of disease.
Project description:Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (CD253/TNFSF10) because of low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of 14 neuroblastoma cell lines to the cytotoxicity of NK cells activated with interleukin (IL)-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in 14 of 17 (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous interferon-? upregulated expression of caspase-8 in 3 of 4 neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against 2 of the 3 lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-interferon-? neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells.
Project description:In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic-stage Plasmodium chabaudi. Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 h postinfection, followed by strong increases in natural killer (NK) cell-associated and major histocompatibility complex class I-related transcripts by 32 h postinfection. We showed by flow cytometry that the observed elevation in NK cell-associated transcripts was the result of a dramatic increase in the proportion of NK cells in the blood during infection. Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection. Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice. These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells.
Project description:Graves' disease (GD) is an autoimmune disorder, driven by the appearance of circulating autoantibodies (Ab) against the thyroid stimulating hormone (TSH) receptor, thus causing hyperthyroidism. While antithyroid drugs, the only available treatment for GD, carry a significant risk of relapse, advances in immunology could pave the way for more effective therapies. Natural killer (NK) cells, divided into cytotoxic CD56dim and cytokine-secreting CD56bright subsets, regulate immune responses through cytokine production and cell lysis and may play a role in the pathogenesis of GD. To investigate their involvement, we conducted flow cytometry on peripheral blood samples from 131 GD patients at various stages (disease onset, on antithyroid drugs, and in remission) and 97 age- and sex-matched healthy controls (HC). We analyzed NK cell subsets, activating (CD16, CD69, NKG2D, NKp30) and inhibitory receptors (CD161, NKG2A), degranulation (CD107a), and intracellular cytokines expression (interferon γ, tumor necrosis factor α). Statistical comparisons were made between GD patients and HC and across disease stages. GD patients had a higher frequency of total NK cells (p < 0.028) and CD56bright NK cells (p < 0.01) but a lower frequency of CD56dim NK cells (p = 0.005) compared to HC. NK cells in GD patients expressed activating receptors more frequently, except for NKG2D, but had decreased cytokine expression and degranulation ability. At GD onset, patients had higher frequencies of total NK cells, CD56bright NK cells, and NK cells expressing activating receptors compared to patients receiving ATD treatment and those in remission. CD161+ NK cells were lower at GD onset and returned to levels of HC following treatment. Correlation analysis revealed that free thyroxine (FT4) levels were inversely correlated with CD107a+ NK cells (p < 0.05) and positively correlated with CD69+ NK cells (p < 0.01). These findings suggest that hyperthyroidism impairs NK cell degranulation, with the increased frequency of NK cells potentially compensating for their reduced function. This dysfunction may contribute to the unregulated immune response in GD, highlighting NK cells as a potential target for novel therapeutic strategies.
Project description:In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic stage Plasmodium chabaudi. Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 hours post-infection, followed by strong increases in natural killer (NK) cell-associated and MHC class I-related transcripts by 32 hours post-infection. We hypothesized that the observed elevation in NK cell-associated transcripts could be the result of a dramatic increase in the proportion of NK cells in the blood during infection, which we confirmed by flow cytometry. Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection. Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice. These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells. Keywords: murine whole blood response to Plasmodium chabaudi infection We analyzed a series of 36 MEEBO arrays on which were hybed RNA amplified from whole blood of C57BL/6 mice either mock-infected or infected with P. chabaudi AS.