Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Valpha24+CD4+ NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-alpha cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-alpha killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-alpha mechanisms. TNF-alpha killing requires longer contact between effectors and targets, suggesting that TNF-alpha acts by enhancing perforin killing.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.     

Distinction between IL-13+ and IFN-gamma+ natural killer cells and regulation of their pool size by IL-4

The hypothesis that distinct subsets of NK cells produce type 2 and type 1 cytokines in resting naive lymphocytes was tested analyzing cytokine production at the single-cell level. Two non-overlapping IL-13+ and IFN-gamma+ subsets were identified in adult and neonatal NK cells. IL-2 maintained their relative proportion. Accumulation of the former was induced by IL-4, but not IL-13, and inhibited by IL-12; that of the latter was induced by IL-12 and inhibited by IL-4 and IL-13. IL-4 induced preferential proliferation of the pre-existing peripheral IL-13+ cells, whereas IL-12 had minimal effect on proliferation of the IFN-gamma+ NK cells. The IL-13+ cells (CD161+ only) are phenotypically distinct from the IFN-gamma+ ones (CD56+) before and after culture under any condition analyzed, and produce IL-13 in response to NK-sensitive target cells and PMA+Ca(2+) ionophore, whereas also FcgammaRIIIA and IL-2+IL-12 stimulate IFN-gamma production. These data define the existence and regulation of two distinct resting peripheral NK cell subsets producing type 1 and type 2 cytokines, and suggest possible roles for IL-13+ NK cells in allergy.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

The majority of lamina propria CD4(+) T-cells from scid mice with colitis undergo Fas-mediated apoptosis in vivo

We have previously shown that adoptively transferred CD4(+) T-cells mediate an chronic colitis in severe combined immune deficient (scid) mice. Colitis is accompanied by activation and apoptosis of Fas ligand and TNF-alpha expressing CD4(+) T-cells in the diseased colonic lamina propria (Eur. J. Immunol. 28:3655 (1998)). Here we investigate the apoptosis-inducing mechanism in these lamina propria infiltrating CD4(+) T-cells. We observe that freshly isolated lamina propria CD4(+) T-cells can kill Fas transfected P815 mastocytoma cells in a TCR/CD3 redirected chromium-release assay, but do not express TNF-alpha mediated cytotoxicity. Pre-incubation of the isolated lamina propria CD4(+) T-cells with an anti-FasL antiserum partially blocked killing of the Fas transfected target cells, indicating a role for the Fas-FasL system in the killing process. Treatment of scid mice with colitis with anti-FasL antiserum for 12 h blocked the apoptotic process in lamina propria CD4(+) T-cells by more than 65% compared to mice treated with control antiserum. Together, these results point towards the Fas-FasL and not the TNF-alpha-TNF-alpha receptor system as the primary apoptosis-inducing mechanism of lamina propria CD4(+) T-cells in this model of murine chronic colitis, and suggest an important role for the Fas-FasL system in the maintenance of homeostasis of locally proliferating T-cells.     

IL-2 and IL-7 but not IL-12 protect natural killer cells from death by apoptosis and up-regulate bcl-2 expression.

Human natural killer cells (NK) respond to interleukin-2 (IL-2) with augmented cytolytic activity, cytokine secretion and cell proliferation. Here we show that IL-2 protects NK cells from death by apoptosis (programmed cell death; PCD). Highly purified NK cells (CD3- CD56+) were isolated from peripheral blood lymphocytes (PBL) of either control donors or of an asymptomatic donor with 60% NK cells. Glucocorticosteroids (GCS) induced PCD in NK cells, as shown by nuclear condensation and DNA fragmentation. IL-2 completely prevented GCS-induced PCD in a dose-dependent manner without overcoming GCS-induced inhibition of NK cell proliferation. The IL-2 protective effect was mediated through the p75 beta chain of the IL-2R, as neutralizing monoclonal antibody (mAb) to the p75 beta chain but not to the p55 alpha chain completely abolished the IL-2 anti-apoptotic activity. In addition to IL-2, the cytokines IL-7 and IL-12 have been reported to regulate NK cell functions. Our present data showed that IL-7 but not IL-12 rescued NK cells from apoptosis, but to a lesser extent than IL-2. Although IL-4 had a marginal protective effect, IL-1, IL-3, IL-6, IL-8, interferon-gamma (IFN-gamma) and IFN-alpha, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and granulocyte-macrophage colony-stimulating factor (GM-CSF) displayed no significant activity. Finally, we report that IL-2 and IL-7 enhanced bcl-2 expression in NK cells, suggesting the existence of a bcl-2-dependent survival pathway. In addition to regulating various functions, it is concluded that IL-2 and IL-7 have the ability to prevent PCD in NK cells.     

Age-associated decline in IL-2 and IL-12 induction of LAK cell activity of human PBMC samples

Previously we reported that young and elderly natural killer (NK) cell activity against the standard NK sensitive K562 cell line can be augmented to the same degree by IL-2 and IFN-. We have extended these studies to include IL-12. Similar to IL-2 and IFN-, IL-12 can enhance NK cytotoxicity to the same degree in both young and elderly samples over a wide range of doses and incubation times when K562 cells are used as targets. However, in contrast to our findings with the NK system, we have observed that induction of lymphokine activated killer (LAK) cell activity, as defined by the ability of peripheral blood mononuclear cells (PBMC) samples to lyse the normally NK resistant Daudi cell line, was significantly decreased in the elderly samples compared to young samples. Comparable age-associated differences were observed in LAK activity after induction with IL-2, IL-12, and IFN- at varying doses and incubation times. We hypothesize an age-associated deficiency either in the mechanism of LAK induction or in target cell recognition.     

Recognition of autologous dendritic cells by human NK cells

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK-target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40-transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co-stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL-2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF-alpha of the cultures stimulated the development of a more mature DC phenotype, while addition of IL-10 resulted in a less mature phenotype, with lower expression of CD40 and other co-stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.     

Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16⁺/CD56⁺ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.     

Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation

We previously reported that intracellular oxidation-reduction (redox) regulates NK cell functions and that IL-2-activated NK cells undergo apoptosis upon contact with NK-sensitive target cells. We now report that apoptosis in activated human NK cells is also regulated by redox. Thiol deprivation increased apoptosis in NK cells induced by anti-Fas mAb or Fas ligand-transfected cells, and pretreatment of cells with N- acetyl cysteine, which increased intracellular glutathione, partially inhibited the apoptosis and reversed the effect of thiol-deficient medium, suggesting that Fas-induced apoptosis in NK cells is also redox sensitive. Thiol deprivation did not alter cell surface Fas expression, but did increase ceramide generation following Fas engagement. Although exogenous ceramides induced apoptosis of NK cells, thiol depletion had no effect on this apoptosis. Thiol deprivation increased CPP32 activation induced by Fas engagement, but not by ceramides. These findings suggest that, if ceramide is required for Fas-induced apoptosis, thiol deprivation affects the Fas-mediated signaling pathway at the generation of ceramide and/or upstream thereof. Though tyrosine phosphorylation following Fas engagement was not significantly affected by thiol deprivation, tyrosine dephosphorylation was delayed, suggesting that tyrosine phosphatases may also be redox sensitive. The notion that dephosphorylation is important in the Fas signaling pathway is supported by the finding that tyrosine phosphatase inhibitors significantly enhanced both CPP32 activity and apoptosis following Fas ligation. We conclude that events downstream of tyrosine phosphorylation and upstream of CPP32 activation, including tyrosine dephosphorylation and possibly ceramide generation, are sensitive to regulation by redox in human NK cells, requiring a reducing environment for optimal protection from apoptosis induced by Fas ligation.      

Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12

IL-12 exerts a potent anti-tumor effect, which is possibly mediated by multiple mechanisms including activation of NK and NKT cells, induction of cytotoxic T lymphocytes, and inhibition of angiogenesis. In the present study, we characterized the cytotoxic effector cells and mechanisms responsible for the anti-metastatic effect of IL-12. Administration of IL-12 had a comparable inhibitory effect on experimental lung metastasis of B16 melanoma cells in wild-type C57BL/6 mice and RAG-2-/- mice that lack T and NKT cells, which was abolished by depletion of NK cells. Cytotoxic activity of liver and splenic mononuclear cells against B16 was induced by IL-12 administration in RAG-2-/- mice at a level comparable to that in wild-type mice, which was also abolished by depletion of NK cells. Moreover, the anti-metastatic effect of IL-12 was abrogated by perforin deficiency, but not by Fas ligand deficiency, in association with a lack of IL-12-induced cytotoxic activity of liver and splenic mononuclear cells against B16. These results suggest that perforin-dependent cytotoxicity of IL-12-activated NK cells is sufficient for the anti-metastatic effect of IL-12.     

Stat4-dependent, T-bet-independent regulation of IL-10 in NK cells

Interleukin-10 (IL-10) is intensely studied, yet little is known about the mechanisms that control IL-10 expression. We identified striking similarities between IL-10 and interferon-gamma (IFN-gamma) regulation in mouse natural killer (NK) cells. Like IFN-gamma, IL-10 expression is induced by IL-2 and IL-12 and IL-2+IL-12 stimulation is synergistic. Unlike IFN-gamma, neither IL-18 nor Ly-49D cross-linking induced IL-10 expression however. Additionally, the IL-12 homologs IL-23 and IL-27 also do not regulate NK cell-specific IL-10. We determined that a small population of NK cells accounts for IL-10 production. The induction of IL-10 by IL-2+IL-12 treatment in NK cells appears to be biphasic, with an initial burst of expression which diminishes by 12 h but spikes again at 18 h. We determined that much like IFN-gamma, Stat4 is largely required for IL-12-induced IL-10. Conversely, we observed normal induction of IL-10 in T-bet-deficient NK cells. We identified a Stat4-binding element in the fourth intron of the Il10 gene, which is completely conserved between mouse and human. This intronic Stat4 motif is within a conserved noncoding sequence, which is also a target for cytokine-induced histone acetylation. These findings highlight tissue- and receptor-specific IL-10 regulatory mechanisms, which may be part of an early feedback loop.     

Natural killing of MHC class I(-) lymphoblasts by NK cells from long-term bone marrow culture requires effector cell expression of Ly49 receptors.

NK cells from long-term bone marrow culture (LTBMC) were compared with IL-2-activated splenic NK cells [short-term spleen cell culture (STSC)] with regard to expression of inhibitory Ly49 receptors and cytotoxic function. In the LTBMC, the total number of NK cells expressing either one of the Ly49 molecules A, C/I and G2 was strongly reduced (10-15% of NK1.1(+) cells) compared to the STSC (80-90% of NK1.1(+) cells). With regard to cytotoxic function, we confirmed that LTBMC-derived NK cells efficiently killed the prototype NK target YAC-1. However, against other targets, killing was more variable. First, while STSC-derived NK cells clearly distinguished MHC class I(-) from MHC class I(+) tumor cell targets, LTBMC-derived NK cells did not; they either killed both targets equally well or not at all. Secondly, LTBMC-derived NK cells were largely incapable of killing lymphoblast targets deficient in MHC class I expression. To test whether this cytotoxic defect was due to the low number of Ly49(+) NK cells in the LTBMC, we separated Ly49(+) and Ly49(-) NK cells by cell sorting and tested them individually. This experiment showed that only Ly49(+) NK cells in the LTBMC were able to kill MHC class I(-) lymphoblasts (and to distinguish them from MHC class I(+)), despite good cytotoxicity against YAC-1 cells in both populations. These data suggest that certain modes of NK cell triggering are dependent on Ly49 receptor expression. From our results, we speculate that inhibitory receptors are expressed before triggering receptors for normal self cells during NK cell development, which may be an important mechanism to preserve self tolerance during the early stages of NK cell maturation.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15.

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

In vivo activation of invariant V alpha 14 natural killer T cells by alpha-galactosylceramide sequentially induces Fas-dependent and -independent cytotoxicity

The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.     

Marking IL-4-producing cells by knock-in of the IL-4 gene

IL-4 is a cytokine which can be expressed by a number of cell types including Th2 cells, mast cells and a population of CD4+ NK1.1+ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-secreting Th2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.