Pertussis toxin-sensitive GTP-binding proteins regulate activation-induced apoptotic cell death of human natural killer cells

Apoptosis of natural killer (NK) cells can be induced by non-specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD 16 and the interleukin-2 receptor (IL-2R) molecules, but not with either anti-CD 16 or IL-2 alone. Whereas blockade of GTP-binding protein (G protein)-mediated signal transduction using ADP-ribosylating bacterial toxins or the GTPase-resistant GTP analog guanosine 5′-0-(3-thiotriphosphate (GTPγS) does not affect non-specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti-CD16/IL-2. The G proteins involved in the regulation of activation-induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non-specific GTP analog GTPγS but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin. A pertussis toxin mutant that lacks ADP-ribosylating activity, but conserves the membrane translocating and T cell-mitogenic effects of the native molecule, fails to inhibit NK apoptosis. To exert their apoptosis-inhibitory effect, PTX and GTPγS must be employed before cells are activated. Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis-inducing signals, but not in the effector stage of apoptosis. Pre-incubation with PTX or GTPγS does not affect the activation of NK cells by CD 16 cross-linking, IL-2 stimulation - or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization. Moreover, neither PTX nor GTPγS compromise the effector function of NK cells or the susceptibility of target cells to NK-mediated lysis. These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down-regulation of NK responses.     

Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.     

The role of activation-induced cell death in the differentiation of T-helper-cell subsets

Activation-induced cell death (AICD) has been demonstrated in T-cell hybridomas, immature thymocytes, and activated mature T cells. However, the molecular mechanisms of AICD and its physiological role in T-helper-cell differentiation remain uncertain. Recently, we have shown that Th1 and Th2 cells have distinct mechanisms of AICD. Our findings suggest that signaling from cytokines initiates the differentiation program, but that the selective action of death effectors determines the fate of differentiating T-helper cells, and thus, the ultimate balance between T-helper subpopulations. Among T cells, activation-induced expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is observed exclusively in Th2 clones and primary T-helper cells differentiated under Th2 conditions, while the expression of CD95L (Fas ligand) occurs mainly in Th1 cells. Furthermore, Th1 cells are more susceptible than Th2 cells to apoptosis induced through either TRAIL or CD95L, and radiolabeled Th1 cells can be induced into apoptosis via fratricide by both Th1 and Th2 cells, while Th2 cells are spared. The pan-caspase inhibitor, z-VAD, prevents AICD in Th1 cells, but not Th2 cells, indicating different mechanisms of AICD in each T-helper subtype. Antibody blockade of TRAIL and CD95L significantly boosts interferon-γ (IFN-γ) production in vitro. Also, young mice with mutant CD95 (MRL/MpJ-lpr/lpr) have a stronger Th1 response to ovalbumin immunization than do controls. We conclude that apoptosis mediated by CD95L and TRAIL is critical in the selective removal of differentiating T helper cells.     

Primary bone natural killer/T cell lymphoma,nasal type without EBV infection: a case report

Primary bone NK/T cell lymphoma is very rare. We report a case of 52-year-old man of primary bone NK/T cell lymphoma and then progressed to NK leukemia. The patient had low-grade fever for 4-month, and Ultrasonic B revealed a diffuse hepatosplenomegaly without lymphadenopathy. PET scanning showed increased FDG uptake in many bones of the whole body. The diagnosis was established by bone specimen. These neoplastic cells demonstrated a typical immunophenotype of CD56, CD3, CD2 and MPO positive, and CD5, CD20, CD30, PAX-5, CD4 and CD8 negative. Primary bone ENKTL is very rare; it should be made with the combination of clinical feature, PET-CT image, and pathological characteristics, and should be distinguished from other lymphomas or leukemia involved in bone.     

The molecular mechanism in activation-induced cell death of an Ag-reactive B cell clone

TPA-1 is a subclone of B cell hybridomas established by somatic hybridization using B cells of A/J mice immunized with TNP-LPS, and expresses a receptor for TNP on the cell membrane. The present study showed that TPA-1 was induced to apoptotic cell death upon treatment with TNP-BSA. Therefore, TPA-1 is considered to provide a good model for the study on activation-induced cell death of mature B cells induced by soluble antigen. TNP-BSA treatment caused the generation of a large amount of intracellular reactive oxygen species (ROS) of TPA-1, and the addition of the monovalent thiol-reactive compound: monochlorobimane (MCB) rescued it from apoptosis as well as the antioxidant reagent: N-acetyl-L-cysteine. Furthermore, MCB markedly inhibited the generation of ROS and prevented the disruption of mitochondrial membrane potential that was induced by TNP-BSA treatment. In addition, it counteracted the effect of TNP-BSA on the expression of the Bcl-2 family, resulting in down-regulation of Bax and Bad and up-regulation of Bcl-XL. Taken together, these results suggest strongly that oxidative stress of mitochondria may be involved directly in apoptotic cell death by engagement of antigen receptors on mature B cells with soluble antigen.     

Activation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase 8 activation

Defects in Fas receptor signaling lead to compromised maintenance of lymphocyte homeostasis and peripheral immune tolerance, leading in turn to autoimmune disorders. Therefore, agents that can enhance Fas-mediated apoptosis may be therapeutically useful in management of such disorders. In this study, we focused on the effect of cAMP on Fas-mediated apoptosis in human T cells. We show that elevation of intracellular cAMP levels by forskolin, an activator of adenylyl cyclase, 3-isobutyl-1-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterases, or prostaglandin E(2) potentiates Fas-induced apoptosis in Jurkat cells. Accordingly, cAMP was found to enhance the cleavage of caspase 8 at death-inducing signaling complex and lead to augmentation of the processing of Fas effector proteins. We also demonstrate that cAMP enahnaces Fas-induced apoptosis in normal human T cells and activation-induced cell death in Jurkat cells. These findings provide a rationale for investigating the feasibility of using cAMP-elevating agents to potentiate apoptosis in T cells with aberrant Fas signaling.     

Granzyme B leakage-induced apoptosis is a crucial mechanism of cell death in nasal-type NK/T-cell lymphoma

This study aims to investigate the role of granzyme B in the apoptosis of nasal-type NK/T-cell lymphoma. Twenty-four nasal-type NK/T-cell lymphomas were examined by TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) assay and immunohistochemical staining for active caspase 3, poly(ADP-ribose) polymerase (PARP-1/p85)/p85, and Bcl-2. In addition, HANK-1 and NKL cell lines were analyzed using Western blot analysis. Immunoprecipitation was performed to identify the binding of granzyme B and intrinsic serpin proteinase inhibitor 9 (PI-9). To localize granzyme B, immunogold labeling and immunofluorescence staining were performed. The expression level of granzyme B in tumor tissue was correlated with the apoptosis rate (P=0.015), degree of necrosis (P=0.002), and the levels of active caspase 3 (P=0.036) and poly ADP-ribose polymerase (PARP)-1/p85 (P=0.040). The granzyme B-positive HANK-1 cell line showed increased spontaneous cell death compared to the granzyme B-negative NKL cell line. The untreated HANK-1 cells released cytochrome c into the cytosol with cleavage of caspase 3 and PARP-1. Treatment with granzyme B inhibitor and caspase inhibitor decreased the cleavage of PARP-1. By performing immunogold labeling, granzyme B was identified within the cytolytic granules as well as in the cytosol. Confocal microscopy and immunoprecipitation assays confirmed the colocalization of PI-9 and granzyme B, which formed an SDS-resistant complex. These results suggested that granzyme B leakage induces cell death in NK/T-cell lymphomas via both caspase-dependent and -independent mechanisms, and this leads to the extensive necrosis that is commonly seen in NK/T-cell lymphoma.     

Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity

A major goal in immunodiagnostics has been the development of assay systems that can measure CD8⁺ T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to this end. Using grB transfected and untransfected Chinese hamster ovary (CHO) cells and T cell lines, we show that the antibody pair utilized was grB-specific and that only activated T cells secrete grB. GrB release began within 4 h after antigen stimulation and stopped within 40 h. Side-by-side comparison showed grB ELISPOT assays to have a higher resolution than classic chromium-release assays in terms of signal-to-noise ratio. The linearity of the relation of the number of CD8⁺ effector T cells plated to grB spots detected suggests that grB ELISPOT assays measure the frequencies of grB-secreting cells directly. Reactivity to HIV peptides was seen in grB ELISPOT assays of freshly isolated PBMC from HIV patients, consistent with the detection of peptide-specific memory cells. The higher resolution and lower labor and material investment should make grB ELISPOT assays an attractive alternative to chromium-release assays in monitoring the clonal sizes of specific CD8 memory cells in vivo.     

Evidence that the KIR2DS5 gene codes for a surface receptor triggering natural killer cell function

In this study, after immunization with NK cells from a KIR2DS5(+) donor and screening on cell transfectants expressing different members of the killer immunoglobulin-like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb-mediated engagement, can induce both cytotoxicity and cytokine release. Using co-transfection and co-immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5-Fc fusion protein does not bind to cell transfectants expressing different HLA-C alleles, suggesting that, if KIR2DS5 does recognize HLA-C molecules, this may only occur in the presence of certain peptides.     

B cell superantigens: a microbe's answer to innate-like B cells and natural antibodies

Marginal zone B cells and B-1 cells have been termed innate-like B cells as they express limited repertoires that play special roles in immune defenses against common infections. These B cells are the sources of natural antibodies and are capable of highly accelerated clonal responses that help counter blood-borne infections. We have characterized a class of microbial product with highly adapted binding interactions with host immunoglobulins/B cell receptors (BCRs), which enable the targeting of large supra-clonal sets of B cells for activation-associated apoptotic death. In recent studies, we have shown that all B cells with V region-targeted BCRs are susceptible. However, compared to follicular B cells, in vivo exposure preferentially causes innate-like B cells to undergo induced death with subsequent long-lasting supra-clonal depletion and immune tolerance. Based on these properties, it is likely that B cell superantigens influence the pathogenesis of some common infections, but also may provide novel therapeutic opportunities to treat B cell neoplastic and autoimmune diseases.     

Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment

It has been suggested that vitamin B12 (vit.B12) plays an important role in immune system regulation, but the details are still obscure. In order to examine the action of vit.B12 on cells of the human immune system, lymphocyte subpopulations and NK cell activity were evaluated in 11 patients with vit.B12 deficiency anaemia and in 13 control subjects. Decreases in the number of lymphocytes and CD8+ cells and in the proportion of CD4+ cells, an abnormally high CD4/CD8 ratio, and suppressed NK cell activity were noted in patients compared with control subjects. In all 11 patients and eight control subjects, these immune parameters were evaluated before and after methyl-B12 injection. The lymphocyte counts and number of CD8+ cells increased both in patients and in control subjects. The high CD4/CD8 ratio and suppressed NK cell activity were improved by methyl-B12 treatment. Augmentation of CD3-CD16+ cells occurred in patients after methyl-B12 treatment. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, lectin-stimulated lymphocyte blast formation, and serum levels of immunoglobulins were not changed by methyl-B12 treatment. These results indicate that vit.B12 might play an important role in cellular immunity, especially relativing to CD8+ cells and the NK cell system, which suggests effects on cytotoxic cells. We conclude that vit.B12 acts as an immunomodulator for cellular immunity.     

Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death

Stimulation of previously activated T cells results in apoptosis, termed activation-induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin-specific T cell receptor (TCR)-transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas-FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti-FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR-transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas-mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine-producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas-mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL-independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL-mediated as well as Fas/FasL-independent AICD in the regulation of helper T cell responses.     

Interleukin-12 is involved in the enhancement of human natural killer cell activity by Lactobacillus casei Shirota

We conducted a placebo-controlled, cross-over trial to examine the effect of Lactobacillus casei Shirota (LcS) on natural killer (NK) cell activity in humans. NK cell activity exhibited a declining trend during the period of placebo ingestion, but NK cell activity increased after intake for 3 weeks of fermented milk containing 4 x 10(10) live LcS. When human peripheral blood mononuclear cells were cultured in the presence of heat-killed LcS, NK cell activity was enhanced. The ability of LcS to enhance NK cell activity and induce interleukin (IL)-12 production was correlated, and the addition of anti-IL-12 monoclonal antibody reduced the enhancement of NK cell activity triggered by LcS. In addition, separation of NK cells from LcS-stimulated monocytes with membrane filter reduced NK cell activity to the intermediate level and almost deprived monocytes of the ability to produce IL-12. These results demonstrate that LcS can enhance NK cell activity in vivo and in vitro in humans, and IL-12 may be responsible for enhancement of NK cell activity triggered by LcS.     

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.     

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A,but lacking inhibitory killer Ig-like receptors

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Bone marrow versus thymic pathways of natural killer cell development

Summary:  Understanding natural killer (NK) cell developmental pathways is crucial for harnessing the potential therapeutic benefits of this specialized lymphocyte subset. The bone marrow (BM) plays a major role in NK cell development, providing the appropriate environmental cues for NK cell commitment and subsequent NK cell differentiation. Nevertheless, the molecular signals provided in this context remain enigmatic. It is widely assumed that BM seeds the periphery with NK cells. However, the precise origins of NK cells found in lymphoid organs and tissues are not defined. Recently, we found that thymic NK cells bear molecular markers and functional attributes that distinguish them from most peripheral NK cells. We find that NK cells are actively exported from the thymus to the periphery, suggesting that thymus-derived NK cells may have unique roles both intrathymically and in secondary lymphoid organs. Here we compare the properties of thymic NK cells with properties of other NK cell subsets that have been identified in the mouse. We propose that heterogeneity in NK cell function can be achieved through distinct thymic and bone marrow pathways of NK cell development.     

Mouse adenovirus type 1 infection of natural killer cell-deficient mice

Natural killer (NK) cells contribute to the initial nonspecific response to viral infection, and viruses exhibit a range of sensitivities to NK cells in vivo. We investigated the role of NK cells in infection of mice by mouse adenovirus type 1 (MAV-1) using antibody-mediated depletion and knockout mice. MAV-1 causes encephalomyelitis and replicates to highest levels in brains. NK cell-depleted mice infected with MAV-1 showed brain viral loads 8-20 days p.i. that were similar to wild-type control non-depleted mice. Mice genetically deficient for NK cells behaved similarly to wild-type control mice with respect to brain viral loads and survival. We conclude that NK cells are not required to control virus replication in the brains of MAV-1-infected mice.