In vivo natural killer cell activities revealed by natural killer cell-deficient mice

Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1(+) CD3(-) cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1(+) CD3(-) cells were responsible for acute tumor rejection, establishing the relationship of NK1.1(+) CD3(-) cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNgamma in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.     

The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit(-/-) (W/W) mice. Although c-kit(-/-)NK cells were generated in W/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)(+) cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit(+) NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.     

Differential effects of interleukin-3, interleukin-7, interleukin 15, and granulocyte-macrophage colony-stimulating factor in the generation of natural killer and B cells from primitive human fetal liver progenitors

The regulatory roles of a number of early-acting growth factors on the generation of natural killer (NK) cells and B cells from primitive progenitors were studied. Experiments focused on the contributions of granulocyte-macrophage colony-stimulates factor (GM-CSF) and interleukin-3 (IL-3) to the regulation of the early events of lymphopoiesis.Two progenitor populations isolated from human fetal liver were studied, CD38(-)CD34(++)lineage(-) (Lin(-)) cells (candidate hematopoietic stem cells [HSCs]) and the more mature CD38(+)CD34(++)Lin(-) cells. The effects of different cytokines on the generation of CD56(+)CD3(-) NK cells and CD19(+) B cells were studied in serum-deprived cultures in the absence of stroma.NK cells generated in vitro were able to kill NK-sensitive target cells, expressed NK-associated marker CD161 (NKR-P1A), but exhibited little or no expression of CD2, CD8, CD16, CD94/NKG2A, or killer cell inhibitory receptors (KIRs). Among the cytokine combinations tested, kit ligand (KL) and IL-15 provided the best conditions for generating CD56(+) NK cells from CD38(+)CD34(++)Lin(-) cells. However, either flk-2/flt3 ligand (FL), GM-CSF, IL-3, or IL-7 could partially substitute KL. All of these cytokines also supported the growth of NK-cell progenitors from candidate HSC, with the combination of IL-15, KL, GM-CSF, and FL generating the greatest number of CD56(+) cells. B cells were generated from both progenitor populations in response to the combined effects of KL, FL, and IL-7. Both B and NK cells were generated with the further addition of IL-15 to these cultures. The in vitro generated B cells were CD10(+), CD19(+), HLA-DR(+), HLA-DQ(+), and some were CD20(+), but no cytoplasmic or surface immunoglobulin M expression was observed. In contrast with NK lymphopoiesis, GM-CSF, IL-3, and IL-15 had no effect on the generation of B cells from CD38(-)CD34(++)Lin(-) cells, and GM-CSF inhibited B-cell generation from CD38(+)CD34(++)Lin(-) progenitors. These findings indicate a differential regulation of NK and B lymphopoiesis beginning in the early stages of hematopoiesis as exemplified by the distinctive roles of IL-7, IL-15, GM-CSF, and IL-3.     

Human small-intestinal epithelium contains functional natural killer lymphocytes

BACKGROUND & AIMS: CD3(-) non-T lymphocytes constitute the second most abundant lymphoid subset in the human small-bowel epithelium, and these CD3(-) intraepithelial lymphocytes are virtually absent in active celiac disease. Phenotypically, they resemble natural killer cells and have been termed natural killer-like intraepithelial lymphocytes. Because of the limited availability of appropriate human samples, functional studies have not yet been reported, and it is not yet clear whether these are true natural killer cells. METHODS: We used magnetic bead-based purification and flow cytometry to study several aspects of normal human small-bowel natural killer-like intraepithelial lymphocytes: intracellular cytokine content (basally and after activation); ability to lyse natural killer-sensitive K562 target cells; and expression of perforins, Fas ligand, and other functional markers. RESULTS: CD3(-) intraepithelial lymphocytes cultured in interleukin-2 showed a higher lymphokine-activated killer activity than CD3(+) intraepithelial lymphocytes (48%-83% lysis exerted by CD3(-) intraepithelial lymphocytes at an effector-target cell ratio of 2:1 vs. 8%-18% by CD3(+) intraepithelial lymphocytes). Perforin content correlated with this lytic potential (75% +/- 4% in CD3(-) vs. 5% +/- 4% in CD3(+) intraepithelial lymphocytes). Both CD3(-) and CD3(+) cells displayed a type I cytokine profile (interferon-gamma > tumor necrosis factor-alpha > interleukin-2; undetectable interleukin-4 and interleukin-10). In addition to their activated phenotype, subsets of natural killer-like intraepithelial lymphocytes expressed CD8alphaalpha and intracellular CD3epsilon chain, showing the existence of heterogeneity within this cell lineage. CONCLUSIONS: This is the first demonstration of functional natural killer cells within the human gut epithelium. These cells might play an important role in innate mucosal immunity (host defense and tumor surveillance) and tolerance.     

Skin infiltration of CD56(bright) CD16(-) natural killer cells in a case of X-SCID with Omenn syndrome-like manifestations

We observed a patient with X-linked severe combined immunodeficiency (X-SCID) with Omenn syndrome-like manifestations. X-linked inheritance, absence of CD132 expression and impaired response to interleukin-2 (IL-2) indicated that the case is typical of X-SCID due to gamma(c) defect. However, this case was unusual in that circulating natural killer (NK) cells were increased and nearly half of these NK cells exhibited the CD56(bright) CD16(-) phenotype. A missense mutation was found within exon 5 of the IL2RG gene. The identical mutation was detected within NK, CD4(+) T and B cells. Engraftment of maternally derived NK cells or gene reversion was ruled out. The erythroderma-like skin lesion was characterized by infiltration of the dermis by CD56(bright) NK cells admixed with CD1a(+) dendritic cells (DC). Expression of mRNA for inflammatory cytokines was significantly enhanced within the skin. This may be the first human case to demonstrate that close cell-to-cell contact between DC and NK cells provides an effective alternative pathway for NK cell differentiation/activation in vivo.     

Identification of the earliest prethymic bipotent T/NK progenitor in murine fetal liver.

This article describes the isolation of a novel cell population (B220(lo)c-kit(+)CD19(-)) in the fetal liver that represents 70% of T-cell precursors in this organ. Interestingly, these precursors showed a bipotent T-cell and natural killer cell (NK)- restricted reconstitution potential but completely lacked B and erythromyeloid differentiation capacity both in vivo and in vitro. Moreover, not only mature T-cell receptor (TCR)alphabeta(+) peripheral T cells but also TCRgammadelta(+) and TCRalphabeta(+)CD8alphaalpha(+) intestinal epithelial cells of extrathymic origin were generated in reconstituted mice. The presence of this population in the fetal liver of athymic embryos indicates its prethymic origin. The comparison of the phenotype and differentiation potential of B220(lo)c-kit(+)CD19(-) fetal liver cells with those of thymic T/NK progenitors indicates that this is the most immature common T/NK cell progenitor so far identified. These fetal liver progenitors may represent the immediate developmental step before thymic immigration.     

Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56-negative natural killer cell precursors in the absence of interleukin-15

OBJECTIVE: Human natural killer (NK) cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process. MATERIALS AND METHODS: Human umbilical cord blood (UCB) CD34(+)/Lin(-)/CD38(-) cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development. RESULTS: When human progenitors were cultured on AFT024 with interleukin (IL)-3 and Flt3 ligand (Flt3L) in the absence of interleukin (IL)-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56(-) NK cell precursors (CD34(+)CD7(-), CD34(+)CD7(+), and CD34(-)CD7(+) cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3 + Flt3L to IL-15 increased the absolute number of CD56(+) NK cells from precursors and the acquisition of CD94 and killer immunoglobulin-like receptors (KIR). To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2, which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation. CONCLUSION: Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man.     

Human natural killer cell development and biology

Natural killer cells are important innate immune effector cells with potentially broad applications in the treatment of human malignancy due to their ability to lyse neoplastic cells without the need for tumor-specific antigen recognition. Human NK cells can be divided into two functional subsets based on their surface expression of CD56; CD56(bright) immunoregulatory cells and CD56(dim) cytotoxic cells. In addition to functional differences, these NK cell subsets can be modulated differently by interleukin (IL)-2, which has permitted the development of lower dose, better tolerated IL-2 regimens for the in vivo expansion and activation of NK cells. The importance of early hematopoietic growth factors, such as c-kit ligand and flt-3 ligand, and their synergy with IL-15 in the development of human NK cells in the bone marrow has permitted the investigation of novel cytokine combinations for optimizing in vivo expansion of NK cell in the clinic. The importance of lymph nodes as a site for NK cell development has recently been elucidated. Furthermore, progress in the field of how NK cell recognize target cells via activating and inhibitory receptors, and how the balance of signals from these receptors can modulate NK cell activity has revolutionized our understanding of the selective killing of tumor cells by NK cells while sparing normal cells. In this review, we summarize current understanding of NK cell biology, and highlight how such knowledge may be translated to optimize the efficacy of using autologous or allogeneic NK cell for the immunotherapy of cancer.     

Unique subset of natural killer cells develops from progenitors in lymph node

Natural killer (NK) cells have been thought to develop from committed progenitors in the bone marrow. However, a novel pathway of thymus-dependent NK-cell development that produces a unique subset of NK cells expressing CD127 has recently been reported. We now have identified 2 populations of NK progenitors, one in the thymus and the other in the lymph node (LN). Immature double-negative 2 (CD4(-)CD8(-)CD44(+)CD25(+)) thymocytes have potential to produce NK cells with rearranged T-cell receptor gamma genes (Tcrgamma(+)) in vitro. Tcrgamma(+) NK cells are rare in spleen but relatively abundant in the thymus and LN. Approximately 20% of LN NK cells are Tcrgamma(+), and they are found at similar levels in both CD127(+) and CD127(-) subsets. Moreover, a subpopulation of LN cells resembling immature thymocytes differentiates into Tcrgamma(+) NK cells in vitro and also repopulates the NK compartment in lymphopenic mice. Athymic mice lack the LN NK progenitors expressing CD127 as well as Tcrgamma(+) NK cells. These results suggest that Tcrgamma(+) NK cells may be generated from unique progenitors in the thymus as well as in the LN.     

Altered phenotype of natural killer cell subsets after haploidentical stem cell transplantation

OBJECTIVE: Haplotype-mismatched CD34(+) selected allogeneic stem cell transplantation (HASCT) has been described as a therapeutic option for patients with acute myeloid leukemia. The success of this regimen is based mainly on natural killer (NK) cell-mediated antileukemia effects. MATERIALS AND METHODS: We prospectively investigated NK-cell (CD56(+)/CD3(-)) reconstitution, including expression of antileukemia effector molecules in patients undergoing HASCT. RESULTS: Although absolute NK-cell numbers rapidly increased, their phenotype notably differed compared to healthy controls. In fact, the "effector" CD56(dim) subset was significantly reduced, as was the NKG2D expression on "regulatory" CD56(bright) cells. Perforin was completely absent on NK cells in one-third of patients. The expression of Fas-ligand (Fas-L) on NK cells as well as soluble Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plasma levels were also significantly lower after HASCT. In contrast, expression of TRAIL on CD56(dim) cells and interleukin-15 plasma levels were upregulated. Because the death rate due to relapse or infectious complications was high in the initial phase of the trial, subsequent patients received an adoptive infusion of donor NK cells followed by interleukin-2 in vivo in order to augment NK-cell function. This led to a distinct upregulation of perforin and Fas-L on the CD56(dim) subset accompanied by increased NK-cell cytotoxicity in vitro. CONCLUSION: The phenotype of reconstituting NK cells after HASCT is significantly altered. Whether the clinical outcomes of patients undergoing this regimen can be improved by a cytokine-based modulation of NK-cell activity needs to be determined.     

Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161(+)/CD56(+) NK cells from these cultures contained a tumor necrosis factor alpha (TNF-alpha)(+)/granulocyte macrophage-colony-stimulating factor (GM-CSF)(+) subset, an interferon gamma (IFN-gamma)(+) subset, mostly included within the former, and very few IFN-gamma(-)/IL-13(+) cells. Instead, most immature CD161(+)/CD56(-) NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-alpha, and GM-CSF, but not IFN-gamma, and contained an IL-5(+) subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-gamma. Part of these produced both IFN-gamma and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161(+) NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-gamma.     

A Wilms tumor cell line, HFWT, can greatly stimulate proliferation of CD56+ human natural killer cells and their novel precursors in blood mononuclear cells

OBJECTIVE: A Wilms tumor cell line, HFWT, selectively stimulates expansion of natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). In this study, we attempted to identify NK precursors in PBMC or in cord blood mononuclear cells (CBMC) that preferentially respond to feeder HFWT cells. MATERIALS AND METHODS: Human NK cells or candidate precursor cells were fractionated from PBMC or CBMC by magnetic antibody cell sorting or by flow cytometry and applied to limiting dilution analysis to determine the proportion of NK/NK precursor cells, which are able to proliferate on irradiated HFWT cells. NK and NK precursor cells were cultured in medium containing interleukin-2 (IL-2). Expansion of NK cells from both resting NK cells and NK precursor cells was examined using proliferation from single cells, expression of NK cell markers, and cytotoxic activity. RESULTS: In the limiting dilution analysis, NK cells expanded on irradiated HFWT cells not only from CD3-CD56bright and CD3-CD56dim NK cells, but also from CD16+/-CD122+ cells in the lineage-negative (Lin-, CD3-CD14-CD19-CD56-) cell fraction. The feeder HFWT cells stimulated Lin-CD122+ cell proliferation more strongly than feeder cells from the well-known human NK target cell line K562. CBMC contained significantly higher percentages of Lin-CD122+ cells than PBMC. CONCLUSION: CD3-CD14-CD19-CD56- cells expressing CD122+ (a subunit of the IL-2 receptor) preferentially respond to HFWT feeder cells and are novel precursors of CD3-CD56+ NK cells in human PBMC and CBMC.     

Interleukin-15 activates human natural killer cells to clear the intestinal protozoan cryptosporidium

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3(-)CD16(+)CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.     

Aggressive natural killer cell lymphoproliferative disorder associated with Epstein-Barr viral RNA

Lymphoproliferative disorder of natural killer cells is a heterogeneous disorder, and an association with Epstein-Barr virus (EBV) is suggested in some cases. A Japanese male presenting with recurrent nasopharyngeal problems developed fever, generalized lymphadenopathy, and hepatosplenomegaly. Separated cells from lymph nodes were shown to have a natural killer (NK) cell, CD2(+), CD3(−), CD16(+), CD56(+), HLA-DR(+) phenotype. A progressive abnormality of hepatic function was associated with hepatorenal failure and death. A serologic study suggested reactivated EBV infection. In situ hybridization (ISH) studies showed Epstein-Barr virus-encoded RNA (EBER)-1 in lymph nodes, with lymphocytes infiltrating the liver and tissue from ethmoid sinus surgery 3 years prior to development of obvious lymphoproliferative disease. Polymerase chain reaction performed on lymph node DNA, using oligonucleotide primers specific for the EBV lymphocyte-determined membrane antigen (LYDMA) gene, revealed a single band, suggesting monoclonal proliferation of the tumor. NK activities of the lymphocytes from the lymph node and peripheral blood were markedly decreased. These findings suggest a close relationship between EBV infection and development of NK cell lymphoproliferative disorder. Am. J. Hematol. 54:314–320, 1997. © 1997 Wiley-Liss, Inc.     

Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells

We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)- cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.     

Alterations in natural killer cells and natural killer T cells during acute viral hepatitis E

The mechanism of liver damage in acute hepatitis E is poorly understood. In this study, we assessed the frequency and activation status of natural killer (NK) and natural killer T (NKT) cells and cytotoxic activity of NK cells in the peripheral blood mononuclear cells (PBMCs) obtained from patients with hepatitis E (n = 41) and healthy controls (n = 61). Flow cytometry was used to assess NK (CD3(-)/CD56(+)) and NKT cell (CD3(+)/CD56(+)) fractions (% of PBMCs) and activation status (CD69(+); % of NK, NKT cells). NK cell cytotoxicity was assessed using major histocompatibilities complex-deficient K562 cells as target cells. In 14 patients, the studies were repeated during the convalescence period. Patients had fewer median (range) NK cells [8.9% (2.4-47.0) vs 11.2% (2.6-35.4)] and NKT cells [8.7% (2.8-34.1) vs 13.6% (2.3-36.9)] than controls (P < 0.05 each). Activation markers were present on large proportion of NK cells [43.5% (11.2-58.6) vs 15.5% (3.0-55.8)] and NKT cells [41.5% (17.4-71.1) vs 12.8% (3.3-63.2); P < 0.05 each] from patients. NK cell cytotoxicity was similar in patients and controls. During convalescence, all the parameters normalized. In conclusion, reversible alterations in NK and NKT cell number and activation status during acute hepatitis E suggest a role of these cells in the pathogenesis of this disease.     

Mobilization of natural killer cells inhibits development of collagen-induced arthritis

Although natural killer (NK) cells have been implicated in regulating immune responses, their ability to modulate disease development in autoimmune arthritis has not been analyzed. Here we investigate the contribution of NK cells to regulating collagen-induced arthritis, a well-characterized preclinical model of human rheumatoid arthritis. We find that the disease is induced by the combined action of two CD4(+) T helper (T(H)) subsets: follicular T(H) cells and T(H)17 cells. Both CD4(+) T(H) subsets are highly susceptible to lysis by NK cells after activation. Administration of antibody that activates NK cells through blockade of its inhibitory CD94/NKG2A receptor allows enhanced elimination of pathogenic follicular T(H) and T(H)17 cells and arrest of disease progression. These results suggest that antibody-dependent enhancement of NK activity may yield effective, previously undescribed therapeutic approaches to this autoimmune disorder.