Positive selection of self- and alloreactive CD8+ T cells in Tap-1 mutant mice.

Mice with a homozygous deletion in their Tap-1 gene (-/- mice) express very low levels of cell membrane major histocompatibility complex class I molecules and have < 1% peripheral CD8+ T cells. We show that these -/- mice but not their +/- littermates display strong primary syngeneic anti-H-2Kb and -Db-specific responses mediated by CD8+ T cells. These responses are augmented by in vivo priming. Further, -/- mice primed in vivo with H-2d alloantigens generate an anti-H-2d response which appears nearly as strong as that found in +/- littermates. Both -/- anti-H-2b and anti-H-2d T cells do not recognize target cells from Tap-1 -/- animals or Tap-2-deficient RMA-S cells. Thus, some CD8+ anti-self and alloreactive T cells can be selected in the absence of Tap proteins.     

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.     

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.     

Mycobacterial infection in natural killer T cell knockout mice

To gain a better understanding of the pathological role of natural killer (NK) T cells in murine tuberculosis, NKT knockout (KO) mice (J(alpha)281(-/-)mice) were utilized. Eight-week-old NKT KO mice of BALB/c origin and wild-type (WT) mice were infected with Mycobacterium tuberculosis Kurono strain by the airborne route using an airborne infection apparatus, and their capacity to control mycobacterial growth, granuloma formation, and cytokine production were examined. The NKT KO mice developed granulomatous lesions in the lungs; there was no statistically significant difference in pulmonary granuloma size between NKT KO and WT mice (p<0.01). The average CFU values increased 3 weeks post-infection, but decreased 9 and 11 weeks post-infection, in the lungs of NKT KO mice. When stimulated with Kurono strain in vitro, splenic cells from NKT KO mice produced less IFN-gamma than did those from WT mice. Expression of mRNA for IL-2, IL-4, IL-6, IL-10 and IL-12 p40 was slightly lower in NKT KO mice compared with WT mice. Our data indicate that NKT cells play a detrimental role in late-phase mycobacterial infection, although Th1 cells are essential in early-phase mycobacterial infection.     

Adrenocorticotropin stimulates natural killer cell activity.

Central release of CRH has recently been implicated in modulating natural killer cell (NK) activity independent of its role in activation of the hypothalamic-pituitary-adrenal axis. In the present study, NK- and interleukin-2 (IL-2)-activated NK cytotoxicity against K-562 target cells was examined in porcine lymphocytes cultured in vitro with ACTH or pig lymphocytes after iv or im ACTH administration. Physiological concentrations of porcine ACTH (10(-8)-10(-11) M) added to the culture medium had no direct influence on NK- or IL-2-stimulated NK cytotoxicity. In a second experiment four unrestrained pigs with indwelling catheters given an iv bolus of vehicle or ACTH (1 IU/kg BW) at 0800 h showed significantly elevated cortisol levels for 3 h after ACTH. Although serum cortisol had returned to baseline by 4 h after ACTH treatment, NK- and IL-2-stimulated NK cytotoxicity was dramatically elevated (P less than 0.01) compared to that in saline-injected controls. NK cytotoxicity in control pigs followed a diurnal pattern, with low morning and high evening cytotoxicity. Exogenous ACTH, given by bolus in the morning, prevented the normal morning decline in NK cytotoxicity. Because of this unexpected dramatic increase in NK- and IL-2-stimulated NK cytotoxicity in animals given ACTH, the experiment was replicated in two subsequent studies using 16 pigs (8 controls and 8 experimental) in each. Pigs were injected im with either ACTH (1 IU/kg BW) or an equivalent volume of saline at 0600 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and measure the cortisol response. As was observed in the previous study, NK- and IL-2-stimulated NK cytotoxicity was significantly greater (P less than 0.01) than that in saline-injected controls. In the final experiment pigs were given either ACTH (1 IU/kg BW) or an equivalent volume of saline at 1800 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and cortisol response. ACTH administered in the evening increased NK cytotoxicity, but the effect was only marginally significant and far less dramatic than in previous studies. Because ACTH had little effect on NK- and IL-2-stimulated NK activity in vitro, we hypothesize that the stimulatory effect of exogenous ACTH is mediated through an indirect mechanism, possibly through the suppression of central CRH as a result of elevated cortisol. This effect is more pronounced when the stimulatory dose of ACTH is given at a time in the circadian cycle when NK cytotoxicity is normally low.     

Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.

OBJECTIVE. To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. METHODS. The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour 51Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of less than or equal to 3 years disease duration, 11 had diffuse cutaneous SSc of greater than 3 years duration, and 13 had limited cutaneous disease. RESULTS. Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. CONCLUSION. Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.     

KLRL1, a novel killer cell lectinlike receptor, inhibits natural killer cell cytotoxicity

Natural killer (NK) cell inhibitory receptors play important roles in the regulation of target susceptibility to natural killing. Here, we report the molecular cloning and functional characterization of a novel NK cell receptor, KLRL1, from human and mouse dendritic cells. KLRL1 is a type II transmembrane protein with an immunoreceptor tyrosine-based inhibitory motif and a C-type lectinlike domain. The KLRL1 gene is located in the central region of the NK gene complex in both humans and mice, on human chromosome 12p13 and mouse chromosome 6F3, adjacent to the other KLR genes. KLRL1 is preferentially expressed in lymphoid tissues and immune cells, including NK cells, T cells, dendritic cells, and monocytes or macrophages. Western blot and fluorescence confocal microscopy analyses indicated that KLRL1 is a membrane-associated glycoprotein, which forms a heterodimer with an as yet unidentified partner. Human and mouse KLRL1 are both predicted to contain putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs), and immunoprecipitation experiments demonstrated that KLRL1 associates with the tyrosine phosphatases SHP-1 (SH2-domain-containing protein tyrosine phosphatase 1) and SHP-2. Consistent with its potential inhibitory function, pretreatment of target cells with human KLRL1-Fc fusion protein enhances NK-mediated cytotoxicity. Taken together, our results demonstrate that KLRL1 belongs to the KLR family and is a novel inhibitory NK cell receptor.     

Normal development and function of natural killer cells in CD3 epsilon delta 5/delta 5 mutant mice.

The CD3 epsilon polypeptide contributes to the cell surface display as well as to the signal transduction properties of the T-cell antigen receptor complex. Intriguingly, the distribution of CD3 epsilon is not restricted to T cells, since activated mouse, human, and avian natural killer (NK) cells do express intracytoplasmic CD3 epsilon polypeptides. CD3 epsilon is also present in the cytoplasm of fetal thymic T/NK bipotential progenitor cells, suggesting that it constitutes a component of the NK differentiation program. We report here that the genetic disruption of CD3 epsilon exon 5 alters neither NK cell development nor in vitro and in vivo NK functions, although it profoundly blocked T-cell development. These results support the notion that CD3 epsilon is dispensable for mouse NK cell ontogeny and function and further suggest that the common NK/T-cell progenitor cell utilizes CD3 epsilon as a mandatory component only when differentiating toward the T-cell lineage.     

Altered axonal targeting and short-term plasticity in the hippocampus of Disc1 mutant mice

Carefully designed animal models of genetic risk factors are likely to aid our understanding of the pathogenesis of schizophrenia. Here, we study a mouse strain with a truncating lesion in the endogenous Disc1 ortholog designed to model the effects of a schizophrenia-predisposing mutation and offer a detailed account of the consequences that this mutation has on the development and function of a hippocampal circuit. We uncover widespread and cumulative cytoarchitectural alterations in the dentate gyrus during neonatal and adult neurogenesis, which include errors in axonal targeting and are accompanied by changes in short-term plasticity at the mossy fiber/CA3 circuit. We also provide evidence that cAMP levels are elevated as a result of the Disc1 mutation, leading to altered axonal targeting and dendritic growth. The identified structural alterations are, for the most part, not consistent with the growth-promoting and premature maturation effects inferred from previous RNAi-based Disc1 knockdown. Our results provide support to the notion that modest disturbances of neuronal connectivity and accompanying deficits in short-term synaptic dynamics is a general feature of schizophrenia-predisposing mutations.     

Altered behavior and long-term potentiation in type I adenylyl cyclase mutant mice.

The murine Ca(2+)-stimulated adenylyl cyclase (type I) (EC 4.6.1.1), which is expressed predominantly in brain, was inactivated by targeted mutagenesis. Ca(2+)-stimulated adenylyl cyclase activity was reduced 40-60% in the hippocampus, neocortex, and cerebellum. Long-term potentiation in the CA1 region of the hippocampus from mutants was perturbed relative to controls. Both the initial slope and maximum extent of changes in synaptic response were reduced. Although mutant mice learned to find a hidden platform in the Morris water task normally, they did not display a preference for the region where the platform had been when it was removed. These results indicate that disruption of the gene for the type I adenylyl cyclase produces changes in behavior and that the cAMP signal transduction pathway may play an important role in synaptic plasticity.     

Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis

Objective. To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. Methods. The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour ⁵¹Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of ≤3 years disease duration, 11 had diffuse cutaneous SSc of >3 years duration, and 13 had limited cutaneous disease. Results. Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. Conclusion. Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.     

Variable manifestation in natural killer cell leukaemia

Natural killer (NK) cell leukaemias are a relatively rare group of haematological disorders, now entitled in the T/NK lymphoproliferative disorders in the new WHO classification. Recent studies have clarified their biological and clinical manifestation gradually. However, some cases with NK malignancies still remain difficult to diagnose and differentiate into their subtypes in the absence of a distinct diagnostic hallmark, especially at initial presentation. We describe herein five patients with NK leukaemias with respect to the clinical, cytological, immunological and cytogenetic characteristics, varied among each case. Cytologically, two aggressive NK cell leukaemia/lymphoma (ANKL/L) cases were a morphologically hypogranular variant form. Clinically, one with ANKL/L was presented as haemophagocytic syndrome without leukaemic infiltration. Systemic chemotherapy resulted in complete remission in one ANKL/L and two blastic NK cell leukaemia/lymphoma (BNKL/L) patients; however, a good long-term outcome was achieved in only one CD4-positive BNKL/L patient with allogenic bone marrow transplantation. Cytogenetic analysis revealed that recurrent chromosomal aberration was rare; however, two had aberrations at 10p11 and 11q13. From these findings, we conclude that comprehensive individual studies should be carried out in these patients to obtain a correct diagnosis and to design an optimal therapeutic approach.     

Altered sexual and social behaviors in trp2 mutant mice

We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male-female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners.     

Altered expression and function of hepatic natural killer T cells in obese and diabetic KK‐Ay mice

Aim: To evaluate the role of natural killer (NK)T cells in the pathogenesis of non‐alcoholic steatohepatitis (NASH), here we investigated the expression and function of hepatic NKT cells in KK‐A^y mice, an animal model of metabolic syndrome. Methods: Male, 8‐week‐old KK‐A^y and C57Bl/6 mice were fed a high‐fat (HF) diet for 4 weeks. Some mice were given daily intragastric injections of pioglitazone for 5 days prior to or after dietary treatment. Results: In untreated KK‐A^y mice, the percentages of NKT cells in liver mononucleolar cells were nearly one‐third of those in C57Bl/6 controls. Elevations in interleukin (IL)‐4 and interferon (IFN)‐γ mRNA in the liver after a single injection of α‐galactosylceramide (GalCer) were blunted in KK‐A^y mice largely. Percentages of NKT cells, as well as GalCer‐induced increases in IL‐4 mRNA, were blunted significantly in both strains after HF diet feeding for 4 weeks. Interestingly, KK‐A^y mice pretreated with pioglitazone showed significant increases in NKT cell proportion, and GalCer‐induced increases in IL‐4 and IFN‐γ mRNA were also enhanced by pioglitazone. In KK‐A^y mice, the percentages of annexin V positive NKT cells were nearly 2.5‐fold higher than those in C57Bl/6 controls; however, pioglitazone decreased annexin V positive cells significantly. Moreover, pioglitazone increased NKT cell fraction in KK‐A^y mice even after HF diet feeding. Conclusion: KK‐A^y mice exhibit proportional and functional alterations in hepatic NKT cells in close relation with the development of steatohepatitis, and it is postulated that pioglitazone improves steatohepatitis in part through restoration of hepatic NKT cells.     

Thymic selection can compensate for mutations affecting T cell activation and generate a normal T cell repertoire in mutant mice

Thymic selection adjusts the reactivity of the peripheral T cell repertoire to maximize recognition of pathogens and minimize stimulation by innocuous substances and self-antigen. The study of molecules implicated in T cell activation often involves the generation of knockout (-/-) mice. In many instances, knockout animals display revealing phenotypes. But should a lack of phenotype be interpreted as a lack of function? Bcl-xgamma was shown previously to affect T cell activation in vitro, and here we note that overexpression of this molecule increases cell cycling after T cell receptor ligation by antibody. It was therefore surprising that Bcl-xgamma(-/-), Bcl-xgamma transgenic, and WT T cells displayed similar levels of sensitivity to antigen according to ex vivo stimulation. Bcl-xgamma could be demonstrated to influence competitiveness and selection of thymocytes in a manner that counteracted the effects of Bcl-xgamma mutation on T cell activation. These findings suggest that thymic selection can overcome genetic defects in T cell activation to generate a T cell repertoire of normal reactivity.     

PRDM1 is a tumor suppressor gene in natural killer cell malignancies

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that PRDM1 is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of PRDM1 loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in PRDM1 expression--in particular, PRDM1α--in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified MYC and 4-1BBL as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.     

Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation.

Because of the expression of inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I allotypes, a person's natural killer (NK) cells will not recognize and will, therefore, kill cells from individuals lacking his/her KIR epitopes. This study investigated the role of NK cell alloreactivity in human HLA haplotype-mismatched hematopoietic stem cell transplantation and, specifically, the role of the three major NK specificities, ie, those for HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles. In 20 of 60 donor-recipient pairs, KIR epitope incompatibility and functional analyses of donor NK cell clones predicted donor NK cells could cause graft-versus-host (GVH)/graft-versus-leukemia (GVL) reactions. NK cell clones of donor origin were obtained from transplanted recipients and tested for lysis of recipient's cryopreserved pretransplant lymphocytes. Despite the absence of GVH disease, we detected high frequencies of NK clones which killed recipient's target cells. Lysis followed the rules of NK cell alloreactivity, being blocked only by the MHC class I KIR epitope which was missing in the recipient. The alloreactive NK clones also killed the allogeneic leukemia. Transplants from these KIR epitope incompatible donors had higher engraftment rates. Therefore, a GVL effector and engraftment facilitating mechanism, which is independent of T-cell-mediated GVH reactions, may be operational in HLA mismatched hematopoietic cell transplants.