The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.     

CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation

Human NK cells can be divided into CD56(dim)CD16(+) killer Ig-like receptors (KIR)(+/-) and CD56(bright)CD16(-) KIR(-) subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56(bright) NK cells mainly gain the signature of CD56(dim) NK cells. Remarkably, KIR can be induced not only on CD56(bright), but also on CD56(dim) KIR(-) NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56(dim) display shorter telomeres than PB- and lymph node (LN)-derived CD56(bright) NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56(bright) NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR(+)CD16(+) NK cells. Altogether, our results suggest that CD56(bright)CD16(-) KIR(-) and CD56(dim)CD16(+)KIR(+/-) NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.     

Cutting edge: engagement of CD160 by its HLA-C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-gamma, TNF-alpha, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Epinephrine-induced mobilization of natural killer (NK) cells and NK-like T cells in HIV-infected patients

HIV infection is known to cause changes in phenotype and function of natural killer (NK) cells. The aim of this study was to characterize the NK cells mobilized from peripheral reservoirs in human immunodeficiency virus (HIV)-infected patients and controls. Seventeen HIV-infected patients and eight age- and sex-matched controls received a 1-h epinephrine infusion. Epinephrine induced mobilization of high numbers of NK-like T cells with no difference between HIV-infected patients and controls. Interestingly, all subjects mobilized NK cells containing increased proportions of perforin, in particular the CD3(-)CD16(+)CD56(+) NK cell subset. The HIV-infected patients mobilized CD3(-)CD16(-)CD56(+) and CD3(-)CD16(+)CD56(+) NK cells to a lesser extent than did controls. In contrast, the HIV-infected patients mobilized relatively more CD3(-)CD16(+)CD56(-) NK cells independent of antiretroviral treatment. It is suggested that these cells represent an immature NK cell subpopulation possibly resulting from an impaired cytokine tissue environment in HIV-infected patients.     

Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance

Normal (noninflamed) human skin contains a network of lymphocytes, but little is known about the homing and function of these cells. The majority of alphabeta T cells in normal skin express CCR8 and produce proinflammatory cytokines. In this study we examined other subsets of cutaneous lymphocytes, focusing on those with potential function in purging healthy tissue of transformed and stressed cells. Human dermal cell suspensions contained significant populations of Vdelta1(+) gammadelta T cells and CD56(+)CD16(-) NK cells, but lacked the subsets of Vdelta2(+) gammadelta T cells and CD56(+)CD16(+) NK cells, which predominate in peripheral blood. The skin-homing receptors CCR8 and CLA were expressed by a large fraction of both cell types, whereas chemokine receptors associated with lymphocyte migration to inflamed skin were absent. Neither cell type expressed CCR7, although gammadelta T cells up-regulated this lymph node-homing receptor upon TCR triggering. Stimulation of cutaneous Vdelta1(+) gammadelta T cell lines induced secretion of large amounts of TNF-alpha, IFN-gamma, and the CCR8 ligand CCL1. In contrast to cutaneous alphabeta T cells, both cell types had the capacity to produce intracellular perforin and displayed strong cytotoxic activity against melanoma cells. We therefore propose that gammadelta T cells and NK cells are regular constituents of normal human skin with potential function in the clearance of tumor and otherwise stressed tissue cells.     

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.     

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.     

Selective reduction of natural killer cells and T cells expressing inhibitory receptors for MHC class I in the livers of patients with hepatic malignancy

Natural killer (NK) and CD56(+) T cells are thought to play a central role in antitumour immunity. Their cytolytic activities are controlled by a variety of receptors including CD94 and killer immunoglobulin-like receptors (KIR), which bind to major histocompatibility complex (MHC) class I molecules on target cells and mediate cell activation or inhibition. We have examined the numbers, phenotypes and antitumour cytotoxic functions of hepatic NK and CD56(+) T cells isolated from 22 patients with hepatic malignancy and 19 healthy donors. Flow cytometry revealed that NK cell numbers were increased among hepatic mononuclear cells in malignancy compared to histologically normal livers (mean: 38% vs 27%; P=0.03), but CD56(+) T cell numbers were not (28% vs 27%). NK cells and CD56(+) T cells from tumour-bearing livers exhibited lymphokine-activated killing of K562 targets and T cell receptor-mediated lysis of P815 cells. The expression of CD94 and the KIR isotypes CD158a, CD158b and KIR3DL1 by CD56(+) T cells and NK cells was significantly and consistently reduced in tumour-bearing livers compared to healthy livers ( P<0.05 in all cases). Simultaneous ligation of CD158a, CD158b and KIR3DL1 caused an overall partial inhibition of CD56(+) T cell cytotoxic activity, suggesting that the observed reductions in KIR(+) cell numbers in malignancy are likely to lead to enhanced cytotoxicity. Our results suggest that, while hepatic CD56(+) T cells are not expanded in malignancy, downregulation of KIR and CD94 expression may be a mechanism by which the hepatic immune system can be activated to facilitate tumour rejection.     

Phenotypic and cytolytic activity of Macaca nemestrina natural killer cells isolated from blood and expanded in vitro

Natural killer (NK) cells from nonhuman primates have not been completely characterized, and methods for expanding nonhuman primates NK cells in vitro have been described only in rhesus species. The purpose of this report was to characterize NK cells in pigtail macaques (Macaca nemestrina), a species that is frequently used in studies of transplantation biology/immunology, virology, vaccine development, and reproductive biology. NK cells from Macaca nemestrina peripheral blood were best defined by the expression of CD16 and CD8alpha, and the absence of CD3. Subsets of these cells express CD56, NKp30, and NKp46. An enhanced ability to kill K562 cells was not present in fluorescence activated cell sorted (FACS)-purified CD16-/CD3+ and CD16-/CD56+ cells isolated from fresh peripheral blood. However, FACS-purified CD16+/CD3- and CD16+/CD56- cells were highly efficient killers of K562 cells. Macaca nemestrina NK cells can be expanded by in vitro culturing of FACS-purified CD16+/CD2-/CD3-/CD56- cells, or from peripheral blood cells depleted of cells expressing CD3, CD4, and HLA-DR. Cells in these cultures expand 70-fold after 21 days of culturing. After culturing, these cells express high levels of natural cytotoxicity receptors (NCRs) NKp30 and NKp46. NK cell populations obtained from FACS-purified CD16+/CD3-, CD16+/CD56- cells and CD3/CD4/HLA-DR-depleted cells were highly efficient killers of K562 cells. These data suggest that a population of highly enriched cytolytic NK cells can be obtained from purified CD16+/CD3- and CD16+/CD56- cells obtained from peripheral blood, as well as from cells that have been cultured and expanded from peripheral blood that is depleted of CD3/CD4/HLA-DR-expressing cells.     

CD56brightCD16+ NK cells: a functional intermediate stage of NK cell differentiation

Human NK cells comprise two main subsets, CD56(bright) and CD56(dim) cells, which differ in function, phenotype, and tissue localization. To further dissect the differentiation from CD56(bright) to CD56(dim) cells, we performed ex vivo and in vitro experiments demonstrating that the CD56(bright)CD16(+) cells are an intermediate stage of NK cell maturation. We observed that the maximal frequency of the CD56(bright)CD16(+) subset among NK cells, following unrelated cord blood transplantation, occurs later than this of the CD56(bright)CD16(-) subset. We next performed an extensive phenotypic and functional analysis of CD56(bright)CD16(+) cells in healthy donors, which displayed a phenotypic intermediary profile between CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells. We also demonstrated that CD56(bright)CD16(+) NK cells were fully able to kill target cells, both by Ab-dependent cell cytotoxicity (ADCC) and direct lysis, as compared with CD56(bright)CD16(-) cells. Importantly, in vitro differentiation experiments revealed that autologous T cells specifically encourage the differentiation from CD56(bright)CD16(-) to CD56(bright)CD16(+) cells. Finally, further investigations performed in elderly patients clearly showed that both CD56(bright)CD16(+) and CD56(dim)CD16(+) mature subsets were substantially increased in older individuals, whereas the CD56(bright)CD16(-) precursor subset was decreased. Altogether, these data provide evidence that the CD56(bright)CD16(+) NK cell subset is a functional intermediate between the CD56(bright) and CD56(dim) cells and is generated in the presence of autologous T CD3(+) cells.     

CD27 defines phenotypically and functionally different human NK cell subsets

The absence of the TNF-receptor family member CD27 marks the stable acquisition of cytolytic effector functions by both CD4(+) and CD8(+) T cells. We found that the majority of circulating human NK cells was CD27(-). These cells were largely CD56(dim), contained high levels of perforin and granzyme B, and were able to exert strong cytotoxic activity. In contrast, circulating CD27(+) NK cells were mostly CD56(dim/bright), had significant lower levels of perforin and granzyme B, and had a low cytolytic potential. Primary and secondary lymphoid organs were markedly enriched for CD27(+) NK cells. When correlating the expression of CD27 to recently defined developmental stages of NK cells in tonsil, we observed that CD27 was exclusively found on mature CD94(+), stage 4 NK cells. On these cells, regulation of CD27 expression appeared to be controlled by the common gamma-chain cytokine IL-15, and down-regulation of CD27 was specifically induced by its ligand, CD70. Thus, the absence of CD27 expression allows the definition of cytotoxic effector cells within the known mature NK cell subsets in humans.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

Identification of resting and type I IFN-activated human NK cell miRNomes reveals microRNA-378 and microRNA-30e as negative regulators of NK cell cytotoxicity

NK cells are important innate immune cells with potent cytotoxicity that can be activated by type I IFN from the host once infected. How NK cell cytotoxicity is activated by type I IFN and then tightly regulated remain to be fully elucidated. MicroRNAs (miRNAs, or miRs) are important regulators of innate immune response, but the full scale of miRNome in human NK cells remains to be determined. In this study, we reported an in-depth analysis of miRNomes in resting and IFN-α-activated human NK cells, found two abundant miRNAs, miR-378 and miR-30e, markedly decreased in activated NK cells by IFN-α, and further proved that miR-378 and miR-30e directly targeted granzyme B and perforin, respectively. Thus, IFN-α activation suppresses miR-378 and miR-30e expression to release cytolytic molecule mRNAs for their protein translation and then augments NK cell cytotoxicity. Importantly, the phenomena are also confirmed in human NK cells activated by other cytokines and even in the sorted CD16(+)CD56(dim)CD69(+) human NK cell subset. Finally, miR-378 and miR-30e were proved to be suppressors of human NK cell cytotoxicity. Taken together, our results reveal that downregulated miR-378 and miR-30e during NK cell activation are negative regulators of human NK cell cytotoxicity, providing a mechanistic explanation for regulation of NK cell function by miRNAs.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.     

Natural killer cell lytic activity and CD56(dim) and CD56(bright) cell distributions during and after intensive training.

The purpose of this study was to examine the impact of intensive training for competitive sports on natural killer (NK) cell lytic activity and subset distribution. Eight female college-level volleyball players undertook 1 mo of heavy preseason training. Volleyball drills were performed 5 h/day, 6 days/wk. Morning resting blood samples were collected before training (Pre), on the 10th day of training (During), 1 day before the end of training (End), and 1 wk after intensive training had ceased (Post). CD3(-)CD16(bright)CD56(dim) (CD56(dim) NK), CD3(-)CD16(dim/-)CD56(bright) NK (CD56(bright) NK), and CD3(+)CD16(-)CD56(dim) (CD56(dim) T) cells in peripheral blood were determined by flow cytometry. The circulating count of CD56(dim) NK cells (the predominant population, with a high cytotoxicity) did not change, nor did the counts for other leukocyte subsets. However, counts for CD56(bright) NK and CD56(dim) T cells (subsets with a lower cytotoxicity) increased significantly (P < 0.01) in response to the heavy training. Overall NK cell cytotoxicity decreased from Pre to End (P = 0.002), with a return to initial values at Post. Lytic units per NK cell followed a similar pattern (P = 0.008). Circulating levels of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha remained unchanged. These results suggest that heavy training can decrease total NK cell cytotoxicity as well as lytic units per NK cell. Such effects may reflect in part an increase in the proportion of circulating NK cells with a low cytotoxicity.