CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

Peripheral natural killer cell maturation depends on the transcription factor Aiolos

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b^highCD27⁻ NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27⁻ NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.     

NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation

NK T cells are a lymphocyte lineage that is selected by CD1d and is characterized by the ability to rapidly secrete large amounts of both IFN-gamma and IL-4 after TCR stimulation. Using reactivity to CD1d tetramers to define presumptive NK T cells, several NK T cell progenitor populations were characterized based upon NK marker expression and CD4 vs CD8 expression. The earliest populations were found to be negative for NK markers and could proliferate to IL-7, while mature NK T cells did not. The NK1.1(-) NK T cell progenitors were capable of up-regulating NK1.1 when transferred in vivo. Upon stimulation, the NK1.1(-) populations secrete IL-4, but little IFN-gamma. As the cells mature and up-regulate NK1.1, they acquire the ability to secrete IFN-gamma. Finally, the Tec family tyrosine kinase Itk is necessary for optimal NK1.1 up-regulation and hence final maturation of NK T cells. The itk(-/-) mice also display a progressive decrease in NK T cells in older animals, suggesting a further role in peripheral maintenance.     

Participation of the CD27 antigen in the regulation of IL-2-activated human natural killer cells.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.     

Age-associated rapid and Stat6-independent IL-4 production by NK1-CD4+8- thymus T lymphocytes.

The source of IL-4 required for priming naive T cells into IL-4-secreting effectors has not been clearly identified. Here we show that upon TCR stimulation, thymus NK1-CD4+8- T cells produced IL-4, the magnitude of which was inversely correlated with age. This IL-4 production response by Th2-prone BALB/c mice was approximately 9-fold that of Th1-prone C57BL/10 mice. More than 90% of activated NK1-CD4+8- thymocytes did not use the invariant V alpha 14-J alpha 281 chain characteristic of typical CD1-restricted NK1+CD4+ T cells. Stat6-null NK1-CD4+8- thymocytes produced bioactive IL-4, with induction of IL-4 mRNA expression within 1 h of stimulation. Our results support the possibility that TCR repertoire-diverse conventional NK1-CD4+ T cells are a potential IL-4 source for directing naive T cells toward Th2/type 2 CD8+ T cell (Tc2) effector development.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.     

Overexpression of Bcl-2 differentially restores development of thymus-derived CD4-8+ T cells and intestinal intraepithelial T cells in IFN-regulatory factor-1-deficient mice

Mice lacking IFN-regulatory factor (IRF)-1 have reduced numbers of mature CD8+ T cells within the thymus and peripheral lymphoid organs, suggesting a critical role of IRF-1 in CD8(+) T cell differentiation. Here we show that endogenous Bcl-2 expression is substantially reduced in IRF-1(-/-)CD8+ thymocytes and that introduction of a human Bcl-2 transgene driven by Emu or lck promoter in IRF-1(-/-) mice restores the CD8(+) T cell development. Restored CD8+ T cells are functionally mature in terms of allogeneic MLR and cytokine production. In contrast to thymus-derived CD8+ T cells, other lymphocyte subsets including NK, NK T, and TCR-gammadelta(+) intestinal intraepithelial lymphocytes, which are also impaired in IRF-1(-/-) mice, are not rescued by expressing human Bcl-2. Our results indicate that IRF-1 differentially regulates the development of these lymphocyte subsets and that survival signals involving Bcl-2 are critical for the development of thymus-dependent CD8+ T cells.     

Down-regulation of p27Kip1 expression is required for development and function of T cells

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.     

Monocyte-induced down-modulation of CD16 and CD56 antigens on human natural killer cells and its regulation by histamine H2-receptors.

Human natural killer (NK) cells carry CD16/FcR and CD56 cell-surface Ag but lack the T-cell marker CD3. Here we show that incubation of resting human NK cells with CD3-/16+/56+ phenotype with autologous monocytes induced the disappearance of CD16 and CD56 cell-surface Ag on NK-cells but did not affect CD2 or CD3 Ag expression on T-cells. Monocyte-induced down-modulation of NK-cell-surface Ag was cell-contact dependent and induced only by freshly isolated monocytes, recovered from peripheral blood by counter-current centrifugal elutriation. Adherence of monocytes abrogated the capacity to induce down-modulation of NK-cell-surface Ag. The biogenic amine histamine dose-dependently reversed the monocyte-induced down-modulation of CD16 and CD56 on CD3- NK-cells. The effect of histamine was mediated by H2-type receptors on monocytes. The data presented are suggestive of a cell-cell-mediated interaction between monocytes and NK-cells which modulates surface expression of NK-cell Ag and its histaminergic regulation.     

Differential roles for IL-15R alpha-chain in NK cell development and Ly-49 induction

IL-15Ralpha-deficient (IL-15Ralpha(-/-)) mice lack NK cells. However, when bone marrow (BM) progenitors from IL-15Ralpha(-/-) mice were cultured with IL-7, stem cell factor and flt3 ligand, followed by IL-15, they were able to differentiate into functional NK cells, indicating that IL-15Ralpha is not critical for NK cell development. Whereas NK cells generated in vitro from IL-15Ralpha(-/-) BM progenitors expressed CD94/NKG2, they failed to express Ly-49 receptors. In keeping with this, when IL-15Ralpha(-/-) BM cells were transferred into wild type recipients, they gave rise to NK cells in vivo, but with greatly reduced expression of Ly-49 receptors. Furthermore, the small numbers of NK cells found in IL-15(-/-) as well as IL-15Ralpha(-/-) but not flt3 ligand(-/-) mice expressed much lower levels of Ly-49 receptors than those from wild type mice. These results indicate a novel role for IL-15Ralpha-chain in Ly-49 induction on developing NK cells.     

CD11b?CD27? NK Cells Are Associated with the Progression of Lung Carcinoma

NK cells are a major component of the antitumour immune response that limits tumour progression. However, it has been reported that tumour-infiltrating NK (TINK) cells from patients with non-small-cell lung carcinoma (NSCLC) exhibit profound defects in degranulation and IFN-γ production. In support of this notion, we report a novel mechanism associated with tumour escape from NK cell-mediated antitumour immunity in lung carcinoma. In this study, we investigated the phenotypic profile of TINK cells based on the expression of the NK-cell maturation markers CD11b and CD27. Interestingly, we found a substantial CD11b⁻CD27⁻ (DN) NK-cell population harboured within the tumour tissues. The presence of this CD11b⁻CD27⁻ NK subset indicated that the TINK cells were of an immature and inactive phenotype. Remarkably, we determined that the presence of DN NK cells had an impact on the clinical outcomes of patients with NSCLC, as the frequency of tumour-infiltrating DN NK cells was positively correlated with the tumour stage and tumour size. We further used a murine Lewis lung cancer (LLC) model to confirm the correlation between the frequency of tumour-infiltrating DN NK cells and the progression of lung carcinoma. Together, our findings demonstrate that the tumour microenvironment may render TINK cells less tumouricidal and thereby contribute to cancer progression.     

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably alpha-galactosylceramide (alpha-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver alpha-GalCer/CD1d tetramer-reactive (alpha-GalCer/CD1d(+)) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas alpha-GalCer/CD1d(+)NK1.1(+) T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.     

Natural killer cells in free-living Mus musculus have a primed phenotype

Recent reports have shown that natural killer (NK) cells may be long-lived, possess memory-like features and may need microbial priming to become fully reactive. Thus, the notion that these cells are typically innate, nonadaptive lymphocytes has been challenged. If microbial priming is essential for functional maturity, it is necessary to raise the question whether NK cells of laboratory mice, kept under strict hygienic conditions, represent these cells adequately. In their natural habitat, mice will encounter microbes to a greater extent, and we here investigated whether NK cells of feral mice showed signs of being primed. In comparison with C57BL/6 mice raised under specific pathogen-free conditions, NK cells from feral mice had high expression of CD69, KLRG1, granzyme B and NKp46 and a higher proportion of CD27+ cells, mostly CD11b-, as well as a higher presence in peripheral lymph nodes. Following cytokine stimulation, feral mouse NK cells had quickly inducible CD25 expression and a stronger interferon-gamma response. These findings indicate a high degree of pre-activation of NK cells of free-living mice, indicating a strong environmental impact on NK cells, which may be highly relevant for interpretation of studies in the mouse model.     

The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts

Human NK cells are divided into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. We tested the hypothesis that CD56(bright) NK cells can differentiate into CD56(dim) cells by prospectively isolating and culturing each NK subset in vitro and in vivo. Our results show that CD56(bright) cells can differentiate into CD56(dim) both in vitro, in the presence of synovial fibroblasts, and in vivo, upon transfer into NOD-SCID mice. In vitro, this differentiation was inhibited by fibroblast growth factor receptor-1 Ab, demonstrating a role of the CD56 and fibroblast growth factor receptor-1 interaction in this process. Differentiated CD56(dim) cells had reduced IFN-gamma production but increased perforin expression and cytolysis of cell line K562 targets. Flow cytometric fluorescent in situ hybridization demonstrated that CD56(bright) NK cells had longer telomere length compared with CD56(dim) NK cells, implying the former are less mature. Our data support a linear differentiation model of human NK development in which immature CD56(bright) NK cells can differentiate into CD56(dim) cells.     

NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation

NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1(+) NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1(-) NK cells are precursors of KLRG1(+) NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1(+) NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45(-/-) mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.     

Positive and negative regulation of the IL-27 receptor during lymphoid cell activation

Previous reports have focused on the ability of IL-27 to promote naive T cell responses but the present study reveals that surface expression of WSX-1, the ligand-specific component of the IL-27R, is low on these cells and that highest levels are found on effector and memory CD4(+) and CD8(+) T cells. Accordingly, during infection with Toxoplasma gondii, in vivo T cell activation is associated with enhanced expression of WSX-1, and, in vitro, TCR ligation can induce expression of WSX-1 regardless of the polarizing (Th1/Th2) environment present at the time of priming. However, while these data establish that mitogenic stimulation promotes expression of WSX-1 by T cells, activation of NK cells and NKT cells prompts a reduction in WSX-1 levels during acute toxoplasmosis. Together, with the finding that IL-2 can suppress expression of WSX-1 by activated CD4(+) T cells, these studies indicate that surface levels of the IL-27R can be regulated by positive and negative signals associated with lymphoid cell activation. Additionally, since high levels of WSX-1 are evident on resting NK cells, resting NKT cells, effector T cells, regulatory T cells, and memory T cells, the current work demonstrates that IL-27 can influence multiple effector cells of innate and adaptive immunity.